U.S. patent application number 17/579517 was filed with the patent office on 2022-07-21 for anti-pd-l1 antibody.
The applicant listed for this patent is Fuso Pharmaceutical Industries, Ltd., National University Corporation Hokkaido University. Invention is credited to Satoru Konnai, Naoya Maekawa, Shiro Murata, Chie Nakajima, Asami Nishimori, Kazuhiko Ohashi, Tomohiro Okagawa, Yasuhiko Suzuki.
Application Number | 20220227871 17/579517 |
Document ID | / |
Family ID | 1000006238816 |
Filed Date | 2022-07-21 |
United States Patent
Application |
20220227871 |
Kind Code |
A1 |
Konnai; Satoru ; et
al. |
July 21, 2022 |
ANTI-PD-L1 ANTIBODY
Abstract
The present invention provides an anti-PD-L1 antibody capable of
repeated administration even to animals other than rat. An
anti-PD-L1 antibody comprising (a) a light chain comprising a light
chain variable region containing CDR1 having the amino acid
sequence of QSLLYSENQKDY (SEQ ID NO: 37), CDR2 having the amino
acid sequence of WAT and CDR3 having the amino acid sequence of
GQYLVYPFT (SEQ ID NO: 38) and the light chain constant region of an
antibody of an animal other than rat; and (b) a heavy chain
comprising a heavy chain variable region containing CDR1 having the
amino acid sequence of GYTFTSNF (SEQ ID NO: 39), CDR2 having the
amino acid sequence of IYPEYGNT (SEQ ID NO: 40) and CDR3 having the
amino acid sequence of ASEEAVISLVY (SEQ ID NO: 41) and the heavy
chain constant region of an antibody of an animal other than rat. A
pharmaceutical composition comprising the above anti-PD-L1 antibody
as an active ingredient. A method for preparing the above
anti-PD-L1 antibody is also provided.
Inventors: |
Konnai; Satoru; (Sapporoshi,
JP) ; Ohashi; Kazuhiko; (Sapporoshi, JP) ;
Murata; Shiro; (Sapporoshi, JP) ; Okagawa;
Tomohiro; (Sapporoshi, JP) ; Nishimori; Asami;
(Sapporoshi, JP) ; Maekawa; Naoya; (Sapporoshi,
JP) ; Suzuki; Yasuhiko; (Sapporoshi, JP) ;
Nakajima; Chie; (Sapporoshi, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Fuso Pharmaceutical Industries, Ltd.
National University Corporation Hokkaido University |
Osaka
Sapporoshi |
|
JP
JP |
|
|
Family ID: |
1000006238816 |
Appl. No.: |
17/579517 |
Filed: |
January 19, 2022 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
16325040 |
Feb 12, 2019 |
11312773 |
|
|
PCT/JP2017/029055 |
Aug 10, 2017 |
|
|
|
17579517 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 2317/92 20130101;
C07K 2317/565 20130101; C07K 2317/76 20130101; C07K 16/2827
20130101; C07K 2317/24 20130101 |
International
Class: |
C07K 16/28 20060101
C07K016/28 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 15, 2016 |
JP |
2016-159088 |
Aug 15, 2016 |
JP |
2016-159089 |
Mar 27, 2017 |
JP |
2017-061454 |
Jun 5, 2017 |
JP |
2017-110723 |
Claims
1. An anti-PD-L1 antibody comprising (a) a light chain comprising a
light chain variable region containing CDR1 having the amino acid
sequence of QSLLYSENQKDY (SEQ ID NO: 37), CDR2 having the amino
acid sequence of WAT and CDR3 having the amino acid sequence of
GQYLVYPFT (SEQ ID NO: 38) and the light chain constant region of a
canine antibody; and (b) a heavy chain comprising a heavy chain
variable region containing CDR1 having the amino acid sequence of
GYTFTSNF (SEQ ID NO: 39), CDR2 having the amino acid sequence of
IYPEYGNT (SEQ ID NO: 40) and CDR3 having the amino acid sequence of
ASEEAVISLVY (SEQ ID NO: 41) and the heavy chain constant region of
a canine antibody.
2. The antibody of claim 1, wherein the light chain variable region
and the heavy chain variable region are derived from rat.
3. The antibody of claim 2, wherein the light chain variable region
is the light chain variable region of a rat anti-bovine PD-L1
antibody and the heavy chain variable region is the heavy chain
variable region of a rat anti-bovine PD-L1 antibody.
4. The antibody of claim 3, wherein the light chain variable region
has the amino acid sequence as shown in SEQ ID NO. 1 and the heavy
chain variable region has the amino acid sequence as shown in SEQ
ID NO: 2.
5. The antibody of claim 1, wherein the light chain constant region
of a canine antibody has the amino acid sequence of the constant
region of lambda chain or kappa chain.
6. The antibody of claim 1, wherein the heavy chain constant region
of a canine antibody has the amino acid sequence of the constant
region of an immunoglobulin equivalent to human IgG4.
7. The antibody of claim 6, wherein the animal other than rat is
canine; the light chain constant region of the canine antibody has
the amino acid sequence of the constant region of lambda chain; and
the heavy chain constant region of the canine antibody has the
amino acid sequence of the constant region of an immunoglobulin
equivalent to human IgG4.
8. The antibody of claim 7, wherein the light chain constant region
of the canine antibody has the amino acid sequence as shown in SEQ
ID NO: 3 and the heavy chain constant region of the canine antibody
has the amino acid sequence as shown in SEQ ID NO: 4.
9. The antibody of claim 1 which has a four-chain structure
comprising two light chains and two heavy chains.
10. A pharmaceutical composition comprising the antibody of claim 1
as an active ingredient.
11. The pharmaceutical composition of claim 10 for treatment of
cancers and/or infections.
12. The pharmaceutical composition of claim 11, wherein the cancers
and/or infections are selected from the group consisting of
neoplastic diseases, leukemia, Johne's disease, anaplasmosis,
bacterial mastitis, mycotic mastitis, mycoplasma infections,
tuberculosis, Theileria orientalis infection, cryptosporidiosis,
coccidiosis, trypanosomiasis and leishmaniasis.
13. An artificial genetic DNA comprising (a') a DNA encoding a
light chain comprising a light chain variable region containing
CDR1 having the amino acid sequence of QSLLYSENQKDY (SEQ ID NO:
37), CDR2 having the amino acid sequence of WAT and CDR3 having the
amino acid sequence of GQYLVYPFT (SEQ ID NO: 38) and the light
chain constant region of a canine antibody and (b') a DNA encoding
a heavy chain comprising a heavy chain variable region containing
CDR1 having the amino acid sequence of GYTFTSNF (SEQ ID NO: 39),
CDR2 having the amino acid sequence of IYPEYGNT (SEQ ID NO: 40) and
CDR3 having the amino acid sequence of ASEEAVISLVY (SEQ ID NO: 41)
and the heavy chain constant region of a canine antibody.
14. A vector comprising the artificial genetic DNA of claim 13.
15. A host cell transformed with the vector of claim 14.
16. A method of preparing an antibody, comprising culturing the
host cell of claim 15 and collecting an anti-PD-L1 antibody from
the resultant culture.
17. A DNA encoding a light chain comprising a light chain variable
region containing CDR1 having the amino acid sequence of
QSLLYSENQKDY (SEQ ID NO: 37), CDR2 having the amino acid sequence
of WAT and CDR3 having the amino acid sequence of GQYLVYPFT (SEQ ID
NO: 38) and the light chain constant region of a canine
antibody.
18. A DNA encoding a heavy chain comprising a heavy chain variable
region containing CDR1 having the amino acid sequence of GYTFTSNF
(SEQ ID NO: 39), CDR2 having the amino acid sequence of IYPEYGNT
(SEQ ID NO: 40) and CDR3 having the amino acid sequence of
ASEEAVISLVY (SEQ ID NO: 41) and the heavy chain constant region of
a canine antibody.
19. A method of treating cancers and/or infections in a subject,
comprising administering to a subject in need thereof a
pharmaceutically effective amount the antibody of claim 1.
Description
PRIORITY APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 16/325,040, Filed Feb. 12, 2019, which is a U.S. National Stage
Filing under 35 U.S.C. 371 from International Application No.
PCT/JP2017/029055, filed on Aug. 10, 2017, and published as
WO2018/034225 on Feb. 22, 2018, which claims the benefit of
priority to Japanese Application No. 2017-110723, filed on Jun. 5,
2017 and to Japanese Application No. 2017-061454, filed on Mar. 27,
2017 and to Japanese Application No. 2016-159089, filed on Aug. 15,
2016 and to Japanese Application No. 2016-159088, filed on Aug. 15,
2016; the benefit of priority of each of which is hereby claimed
herein, and which applications and publication are hereby
incorporated herein by reference in their entireties.
TECHNICAL FIELD
[0002] The present invention relates to an anti-PD-L1 antibody.
More specifically, the present invention relates to an anti-PD-L1
antibody comprising a variable region containing
complementarity-determining regions (CDR) of a rat anti-bovine
PD-L1 antibody and a constant region of an antibody of an animal
other than rat.
BACKGROUND ART
[0003] Programmed cell death 1 (PD-1), an immunoinhibitory
receptor, and its ligand programmed cell death ligand 1 (PD-L1) are
molecules identified by Prof. Tasuku Honjo et al., Kyoto
University, as factors which inhibit excessive immune response and
are deeply involved in immunotolerance (Non-Patent Document No. 1:
Ishida Y, Agata Y, Shibahara K, Honjo T The EMBO Journal. 1992
November; 11(11):3887-3895). Recently, it has been elucidated that
these molecules are also involved in immunosuppression in tumors.
In the field of human medical care, an antibody drug that inhibits
the effect of PD-1 has been developed and put into practical use
(Opdivo.TM., Ono Pharmaceutical Co., Ltd.)
[0004] To date, the present inventors have been developing an
immunotherapy for animal refractory diseases targeting PD-1 or
PD-L1, and have revealed that this novel immunotherapy is
applicable to multiple-diseases and multiple-animals. (Non-Patent
Document No. 2: Ikebuchi R, Konnai S, Okagawa T, Yokoyama K,
Nakajima C, Suzuki Y, Murata S, Ohashi K. Immunology. 2014 August;
142(4):551-61: Non-Patent Document No. 3: Maekawa N. Konnai S,
Ikebuchi R, Okagawa T. Adachi M, Takagi S. Kagawa Y, Nakajima C.
Suzuki Y, Murata S, Ohashi K. PLoS One. 2014 Jun. 10; 9(6):e98415;
Non-Patent Document No. 4: Mingala C N, Konnai S, Ikebuchi R.
Ohashi K. Comp. Immunol. Microbiol. Infect. Dis. 2011 January;
34(1):55-63.)
[0005] However, the antibodies which the present inventors have
prepared to date are rat antibodies, and therefore it is impossible
to administer those antibodies repeatedly to animals other than
rat.
PRIOR ART LITERATURE
Non-Patent Documents
[0006] Non-Patent Document No. 1: Ishida Y. Agata Y, Shibahara K,
Honjo T The EMBO Journal. 1992 November; 11(11):3887-3895. [0007]
Non-Patent Document No. 2: Ikebuchi R, Konnai S, Okagawa T,
Yokoyama K, Nakajima C, Suzuki Y. Murata S, Ohashi K. Immunology.
2014 August; 142(4):551-61. [0008] Non-Patent Document No. 3:
Maekawa N, Konnai S, Ikebuchi R. Okagawa T, Adachi M, Takagi S.
Kagawa Y. Nakajima C, Suzuki Y, Murata S. Ohashi K. PLoS One. 2014
Jun. 10; 9(6):e98415. [0009] Non-Patent Document No. 4: Mingala C
N, Konnai S, Ikebuchi R, Ohashi K. Comp. Immunol. Microbiol.
Infect. Dis. 2011 January; 34(1):55-63.
DISCLOSURE OF THE INVENTION
Problem for Solution by the Invention
[0010] It is an object of the present invention to provide an
anti-PD-L1 antibody capable of repeated administration even to
animals other than rat.
Means to Solve the Problem
[0011] The present inventors have determined the variable regions
of a rat anti-bovine PD-L1 monoclonal antibody (4G12) capable of
inhibiting the binding of canine PD-1 to PD-L1, and then combined
genes encoding the resultant variable regions with genes encoding
the constant regions of a canine immunoglobulin (IgG-D equivalent
to human IgG4) to thereby obtain a chimeric antibody gene, which
was introduced into Chinese hamster ovary cells (CHO cells). By
culturing/proliferating the resultant CHO cells, the present
inventors have succeeded in preparing a rat-canine chimeric
anti-PD-L1 antibody. Further, the present inventors have determined
the CDRs of the variable region of the rat anti-bovine PD-L1
monoclonal antibody 4G12.
[0012] Furthermore, the present inventors have determined the
variable regions of the rat anti-bovine PD-L1 monoclonal antibody
4G12 capable of inhibiting the binding of bovine PD-1 to PD-L1, and
then combined genes encoding the resultant variable regions with
genes encoding the constant regions of a bovine immunoglobulin
(bovine IgG1, with mutations having been introduced into the
putative binding sites of Fc.gamma. receptors in CH2 domain in
order to inhibit ADCC activity; see FIG. 19 for amino acid numbers
and mutations: 250 E.fwdarw.P, 251 L.fwdarw.V, 252 P.fwdarw.A, 253
G.fwdarw.deletion, 347 A.fwdarw.S, 348 P.fwdarw.S; Ikebuchi R,
Konnai S, Okagawa T, Yokoyama K, Nakajima C, Suzuki Y, Murata S,
Ohashi K. Immunology 2014 August; 142(4):551-561) to thereby obtain
a chimeric antibody gene. This gene was introduced into Chinese
hamster ovary cells (CHO cells). By culturing/proliferating the
resultant cells, the present inventors have succeeded in preparing
a rat-bovine chimeric anti-PD-L1 antibody. The present invention
has been achieved based on these findings.
[0013] A summary of the present invention is as described below.
[0014] (1) An anti-PD-L1 antibody comprising (a) a light chain
comprising a light chain variable region containing CDR1 having the
amino acid sequence of QSLLYSENQKDY (SEQ ID NO: 37), CDR2 having
the amino acid sequence of WAT and CDR3 having the amino acid
sequence of GQYLVYPFT (SEQ ID NO: 38) and the light chain constant
region of an antibody of an animal other than rat; and (b) a heavy
chain comprising a heavy chain variable region containing CDR1
having the amino acid sequence of GYTFTSNF (SEQ ID NO: 39), CDR2
having the amino acid sequence of IYPEYGNT (SEQ ID NO: 40) and CDR3
having the amino acid sequence of ASEEAVISLVY (SEQ ID NO: 41) and
the heavy chain constant region of an antibody of an animal other
than rat. [0015] (2) The antibody of (1) above, wherein the light
chain variable region and the heavy chain variable region are
derived from rat. [0016] (3) The antibody of (2) above, wherein the
light chain variable region is the light chain variable region of a
rat anti-bovine PD-L1 antibody and the heavy chain variable region
is the heavy chain variable region of a rat anti-bovine PD-L1
antibody. [0017] (4) The antibody of (3) above, wherein the light
chain variable region has the amino acid sequence as shown in SEQ
ID NO. 1 and the heavy chain variable region has the amino acid
sequence as shown in SEQ ID NO: 2. [0018] (5) The antibody of any
one of (1) to (4) above, wherein the light chain constant region of
an antibody of an animal other than rat has the amino acid sequence
of the constant region of lambda chain or kappa chain. [0019] (6)
The antibody of any one of (1) to (5) above, wherein the heavy
chain constant region of an antibody of an animal other than rat
has the amino acid sequence of the constant region of an
immunoglobulin equivalent to human IgG4. [0020] (7) The antibody of
any one of (1) to (5) above, wherein the animal other than rat is
bovine and the heavy chain constant region of the bovine antibody
has mutations introduced thereinto that reduce ADCC activity and/or
CDC activity. [0021] (8) The antibody of (6) above, wherein the
animal other than rat is canine; the light chain constant region of
the canine antibody has the amino acid sequence of the constant
region of lambda chain; and the heavy chain constant region of the
canine antibody has the amino acid sequence of the constant region
of an immunoglobulin equivalent to human IgG4. [0022] (9) The
antibody of (7) above, wherein the light chain constant region of
the bovine antibody has the amino acid sequence of the constant
region of lambda chain and the heavy chain constant region of the
bovine antibody has mutations introduced thereinto that reduce ADCC
activity and/or CDC activity. [0023] (10) The antibody of (8)
above, wherein the light chain constant region of the canine
antibody has the amino acid sequence as shown in SEQ ID NO: 3 and
the heavy chain constant region of the canine antibody has the
amino acid sequence as shown in SEQ ID NO: 4. [0024] (11) The
antibody of (9) above, wherein the light chain constant region of
the bovine antibody has the amino acid sequence as shown in SEQ ID
NO: 100 and the heavy chain constant region of the bovine antibody
has the amino acid sequence as shown in SEQ ID NO: 102. [0025] (12)
The antibody of any one of (1) to (11) above which has a four-chain
structure comprising two light chains and two heavy chains. [0026]
(13) A pharmaceutical composition comprising the antibody of any
one of (1) to (12) above as an active ingredient. [0027] (14) The
composition of (13) above for prevention and/or treatment of
cancers and/or inflammations. [0028] (15) The composition of (14)
above, wherein the cancers and/or inflammations are selected from
the group consisting of neoplastic diseases, leukemia, Johne's
disease, anaplasmosis, bacterial mastitis, mycotic mastitis,
mycoplasma infections (such as mycoplasma mastitis, mycoplasma
pneumonia or the like), tuberculosis, Theileria orientalis
infection, cryptosporidiosis, coccidiosis, trypanosomiasis and
leishmaniasis. [0029] (16) An artificial genetic DNA comprising
(a') a DNA encoding a light chain comprising a light chain variable
region containing CDR1 having the amino acid sequence of
QSLLYSENQKDY (SEQ ID NO: 37), CDR2 having the amino acid sequence
of WAT and CDR3 having the amino acid sequence of GQYLVYPFT (SEQ ID
NO: 38) and the light chain constant region of an antibody of an
animal other than rat and (b') a DNA encoding a heavy chain
comprising a heavy chain variable region containing CDR1 having the
amino acid sequence of GYTFTSNF (SEQ ID NO: 39), CDR2 having the
amino acid sequence of IYPEYGNT (SEQ ID NO: 40) and CDR3 having the
amino acid sequence of ASEEAVISLVY (SEQ ID NO: 41) and the heavy
chain constant region of an antibody of an animal other than rat.
[0030] (17) A vector comprising the artificial genetic DNA of (16)
above. [0031] (18) A host cell transformed with the vector of (17)
above. [0032] (19) A method of preparing an antibody, comprising
culturing the host cell of (18) above and collecting an anti-PD-L1
antibody from the resultant culture. [0033] (20) A DNA encoding a
light chain comprising a light chain variable region containing
CDR1 having the amino acid sequence of QSLLYSENQKDY (SEQ ID NO:
37), CDR2 having the amino acid sequence of WAT and CDR3 having the
amino acid sequence of GQYLVYPFT (SEQ ID NO: 38) and the light
chain constant region of an antibody of an animal other than rat.
[0034] (21) A DNA encoding a heavy chain comprising a heavy chain
variable region containing CDR1 having the amino acid sequence of
GYTFTSNF (SEQ ID NO: 39), CDR2 having the amino acid sequence of
IYPEYGNT (SEQ ID NO: 40) and CDR3 having the amino acid sequence of
ASEEAVISLVY (SEQ ID NO: 41) and the heavy chain constant region of
an antibody of an animal other than rat.
[0035] The present specification encompasses the contents disclosed
in the specifications and/or drawings of Japanese Patent
Applications No. 2016-159088, No. 2016-159089, No. 2017-110723 and
No. 2017-61454 based on which the present patent application claims
priority.
Effect of the Invention
[0036] According to the present invention, a novel anti-PD-L1
antibody has been obtained. This antibody is applicable even to
those animals other than rat.
BRIEF DESCRIPTION OF THE DRAWINGS
[0037] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of the necessary fee.
[0038] FIG. 1 Inhibition of the binding of recombinant canine PD-L1
to recombinant canine PD-1. The binding of canine PD-L1-Ig to
canine PD-1-Ig was detected on ELISA plates. The optical density
(O.D.) without addition of antibody was taken as 100%. O.D. at each
antibody concentration was shown as relative value. Among rat
anti-bovine PD-L1 monoclonal antibodies 4G12 (Rat IgG2a (.kappa.)),
5A2 (Rat IgG1 (.kappa.)) and 6G7 (Rat IgM (.kappa.)) which showed
cross-reaction with canine PD-L1, clones 4G12 and 6G7 exhibited a
high binding inhibition capacity.
[0039] FIG. 2 Schematic drawings of pDC6 vector and a rat-canine
chimeric anti-PD-L1 antibody.
[0040] FIG. 3 Expression and purification of rat-canine chimeric
anti-PD-L1 antibodies c4G12 and c6G7. SDS-PAGE was performed under
non-reducing conditions, followed by visualization of bands by CBB
staining. a: purification with protein A alone. b: a +gel
filtration chromatography.
[0041] FIG. 4 PD-1/PD-L1 binding inhibition activities of
rat-canine chimeric anti-PD-L1 antibodies c4G12 and c6G7.
[0042] FIG. 5 Establishment of cell clones capable of high
expression of rat-canine chimeric anti-PD-L1 antibody c4G12.
[0043] FIG. 6 SDS-PAGE images of rat-canine chimeric anti-PD-L1
antibody c4G12. Rat anti-bovine PD-L1 antibody 4G12 and rat-canine
chimeric anti-PD-L1 antibody c4G12 were electrophoresed under
reducing conditions and non-reducing conditions, followed by
visualization of bands by CBB staining. Under reducing conditions,
a band of antibody's heavy chain was detected at around 50 kDa and
a band of antibody's light chain at around 25 kDa. No bands other
than the bands of interest were detected.
[0044] FIG. 7 Inhibitory activities of rat anti-bovine PD-L1
antibody 4G12 and rat-canine chimeric anti-PD-L1 antibody c4G12
against canine PD-1/PD-L1 binding and CD80/PD-L1 binding. Rat
anti-bovine PD-L1 monoclonal antibody 4G12 and rat-canine chimeric
anti-PD-L1 antibody c4G12 reduced the amounts of binding of
PD-L1-Ig to canine PD-1-Ig and CD80-Ig. No change due to
chimerization of the antibody was observed in binding inhibition
activity
[0045] FIG. 8 Canine immune cell activation effect by rat-canine
chimeric anti-PD-L1 antibody c4G12. Canine PBMCs were cultured
under stimulation for 3 days, followed by determination of IL-2 and
IFN-.gamma. concentrations in the supernatant by ELISA. Further,
nucleic acid analogue EdU was added to the culture medium at day 2
of the culture under stimulation, followed by determination of the
EdU uptake by flow cytometry. Rat-canine chimeric anti-PD-L1
antibody c4G12 increased the production of IL-2 and IFN-.gamma.
from canine PBMCs and enhanced proliferation of CD4+ and CD8.sup.+
lymphocytes.
[0046] FIG. 9 Expression of PD-L1 in oral melanoma (A) and
undifferentiated sarcoma (B)
[0047] FIG. 10 CT images and appearances of tumor in a test of
treatment by administering rat-canine chimeric anti-PD-L1 antibody
c4G12 to a dog with oral melanoma. (a,d) Before the start of the
treatment, (b,e) at week 10 of the treatment, and (cf) at week 34
of the treatment. A remarkable anti-tumor effect was recognized
upon five administrations of the antibody (at week 10 from the
start of the treatment). At week 34, a further reduction of tumor
was confirmed.
[0048] FIG. 11 Time-dependent changes in the longest diameter of
the tumor in the dog with oral melanoma shown in FIG. 10. Reduction
by 30% or more compared to the baseline longest diameter was
regarded as partial response (PR).
[0049] FIG. 12 CT images in a test of treatment by administering
rat-canine chimeric anti-PD-L1 antibody c4G12 to a dog with
undifferentiated sarcoma. (a,c) Before the start of the treatment,
(b,d) at week 3 of the treatment. A remarkable reduction of tumor
was recognized upon two administrations of the antibody.
[0050] FIG. 13 CT images in a test of treatment by administering
rat-canine chimeric anti-PD-L1 antibody c4G12 to dogs with oral
melanoma (pulmonary metastatic cases). (a,d,g) Before the start of
the treatment, (b,e,h) at week 6 of the treatment, and (c,f,i) at
week 18 of the treatment. A plurality of pulmonary metastatic
lesions disappeared upon nine administrations of the antibody.
[0051] FIG. 14 Time-dependent changes in the proportion survival of
dogs with oral melanoma after the occurrence of pulmonary
metastasis. In the antibody administration group, the survival
duration may have been prolonged compared to the control group.
[0052] FIG. 15 CDR1, CDR2 and CDR3 regions in the light chain
variable region and the heavy chain variable region of rat
anti-bovine PD-L1 antibody 4G12 are illustrated.
[0053] FIG. 16 Cross-reactivity of rat anti-bovine PD-L1 antibody
4012. It was confirmed that rat anti-bovine PD-L1 antibody 4G12
binds to ovine PD-L1 and porcine PD-L1.
[0054] FIG. 17 Reactivity of rat anti-bovine PD-L1 antibody 4G12
with water buffalo leukocytes. Rat anti-bovine PD-L1 antibody 4G12
strongly bound to blood macrophages (CD14.sup.+ CD11b.sup.+ cells)
of water buffalo, whereas rat anti-bovine PD-L1 antibody 4G12 bound
weakly to lymphocytes (CD14.sup.-CD11b.sup.- cells) of water
buffalo. It is believed that this difference in binding reflects
the expression levels of PD-L1 in macrophages and lymphocytes.
[0055] FIG. 18 Inhibition test on rat anti-bovine PD-L1 antibody
4G12 against ovine or porcine PD-1/PD-L1 binding. It was
demonstrated that rat anti-bovine PD-L1 antibody 4G12 is capable of
inhibiting ovine and porcine PD-1/PD-L1 binding in a concentration
dependent manner.
[0056] FIG. 19 The amino acid sequence of rat-bovine chimeric
anti-bovine PD-L1 antibody ch4G12. CDR1, CDR2 and CDR3 regions in
the light chain variable region and the heavy chain variable region
of rat anti-bovine PD-L1 antibody 4G12 are shown. Further, amino
acids introduced as mutations to bovine IgG1 (CH2 domain) are also
shown (amino acid numbers and mutations: 250 E.fwdarw.P, 251
L.fwdarw.V, 252 P.fwdarw.A, 253 G.fwdarw.deletion, 347 A.fwdarw.S,
348 P.fwdarw.S).
[0057] FIG. 20 Schematic drawings of pDC6 vector and rat-bovine
chimeric anti-bovine PD-L1 antibody ch4G12.
[0058] FIG. 21 Confirmation of the purity of purified rat-bovine
chimeric anti-bovine PD-L1 antibody ch4G12.
[0059] FIG. 22 Binding specificity of rat-bovine chimeric
anti-bovine PD-L1 antibody ch4G12.
[0060] FIG. 23 Inhibitory activity of rat-bovine chimeric
anti-bovine PD-L1 antibody ch4G12 against bovine PD-1/PD-L1 binding
(the test results of inhibition against binding of bovine PD-L1
expressing cells and soluble bovine PD-1).
[0061] FIG. 24 Inhibitory activity of rat-bovine chimeric
anti-bovine PD-L1 antibody ch4G12 against bovine PD-1/PD-L1 binding
(the test results of inhibition against binding of bovine PD-1
expressing cells and soluble bovine PD-L1).
[0062] FIG. 25 Activation effect of rat-bovine chimeric anti-bovine
PD-L1 antibody ch4G12 on bovine lymphocyte response (in terms of
cell proliferation).
[0063] FIG. 26 Activation effect of rat-bovine chimeric anti-bovine
PD-L1 antibody ch4G12 on bovine lymphocyte response to BLV antigen
(in terms of IFN-.gamma. production).
[0064] FIG. 27 The proliferation response of T cells against BLV
antigen in a calf experimentally infected with BLV through
administration of rat-bovine chimeric anti-bovine PD-L1 antibody
ch4G12.
[0065] FIG. 28 Changes in BLV provirus loads in the calf
experimentally infected with BLV through administration of
rat-bovine chimeric anti-bovine PD-L1 antibody ch4G12.
BEST MODES FOR CARRYING OUT THE INVENTION
[0066] Hereinbelow, the present invention will be described in
detail.
[0067] The present invention provides an anti-PD-L1 antibody
comprising (a) a light chain comprising a light chain variable
region containing CDR1 having the amino acid sequence of
QSLLYSENQKDY (SEQ ID NO: 37), CDR2 having the amino acid sequence
of WAT and CDR3 having the amino acid sequence of GQYLVYPFT (SEQ ID
NO: 38) and a light chain constant region of an antibody of an
animal other than rat; and (b) a heavy chain comprising a heavy
chain variable region containing CDR1 having the amino acid
sequence of GYTFTSNF (SEQ ID NO: 39), CDR2 having the amino acid
sequence of IYPEYGNT (SEQ ID NO: 40) and CDR3 having the amino acid
sequence of ASEEAVISLVY (SEQ ID NO: 41) and a heavy chain constant
region of an antibody of an animal other than rat.
[0068] CDR1, CDR2 and CDR3 in the light chain variable region (VL)
of rat anti-bovine PD-L1 antibody 4G12 are a region consisting of
the amino acid sequence of QSLLYSENQKDY (SEQ ID NO: 37), a region
consisting of the amino acid sequence of WAT and a region
consisting of the amino acid sequence of GQYLVYPFT (SEQ ID NO: 38),
respectively (see FIG. 15).
[0069] Further, CDR1, CDR2 and CDR3 in the heavy chain variable
region (VH) of rat anti-bovine PD-L1 antibody 4G12 are a region
consisting of the amino acid sequence of GYTFTSNF (SEQ ID NO: 39),
a region consisting of the amino acid sequence of IYPEYGNT (SEQ ID
NO: 40) and a region consisting of the amino acid sequence of
ASEEAVISLVY (SEQ ID NO: 41), respectively (see FIG. 15).
[0070] In the amino acid sequences of QSLLYSENQKDY (SEQ ID NO: 37),
WAT and GQYLVYPFT (SEQ ID NO: 38), as well as the amino acid
sequences of GYTFTSNF (SEQ ID NO: 39), IYPEYGNT (SEQ ID NO: 40) and
ASEEAVISLVY (SEQ ID NO: 41), one, two, three, four or five amino
acids may be deleted, substituted or added.
[0071] As used herein, the term "antibody" is a concept
encompassing not only full-length antibodies but also antibodies of
smaller molecular sizes such as Fab, F(ab)'.sub.2, ScFv, Diabody,
V.sub.H, V.sub.L, Sc(Fv).sub.2, Bispecific sc(Fv).sub.2, Minibody,
scFv-Fc monomer and scFv-Fc dimer.
[0072] In the anti-PD-L1 antibody of the present invention, VL and
VH thereof may be derived from rat. For example, VL thereof may be
the VL of a rat anti-bovine PD-L1 antibody, and VH thereof may be
the VH of the rat anti-bovine PD-L1 antibody.
[0073] The amino acid sequence of the VL and the amino acid
sequence of the VH of the rat anti-bovine PD-L1 antibody are shown
in SEQ ID NOS: 1 and 2, respectively. The amino acid sequences as
shown in SEQ ID NOS: 1 and 2 may have deletion(s), substitution(s)
or addition(s) of one or several (e.g., up to five, about 10 at the
most) amino acids. Even when such mutations have been introduced,
the resulting amino acid sequences are capable of having the
function as VL or VH of the PD-L1 antibody.
[0074] The VL and VH of an antibody of an animal other than rat may
be derived from an animal which produces a PD-L1 that cross-reacts
with rat anti-bovine PD-L1 antibody 4G12.
[0075] There are two types of immunoglobulin light chain, which are
called Kappa chain (.kappa.) and Lambda chain (.lamda.). In the
anti-PD-L1 antibody of the present invention, the light chain
constant region (CL) of an antibody of an animal other than rat may
have the amino acid sequence of the constant region of either Kappa
chain or Lambda chain. However, the relative abundance of Lambda
chain is higher in ovine, feline, canine, equine and bovine, and
that of Kappa chain is higher in mouse, rat, human and porcine.
Since a chain with a higher relative abundance is considered to be
preferable, an ovine, feline, canine, equine or bovine antibody
preferably has the amino acid sequence of the constant region of
Lambda chain whereas a mouse, rat, human or porcine antibody
preferably has the amino acid sequence of the constant region of
Kappa chain.
[0076] The heavy chain constant region (CH) of an antibody of an
animal other than rat may have the amino acid sequence of the
constant region of an immunoglobulin equivalent to human IgG4.
Immunoglobulin heavy chain is classified into .gamma. chain, .mu.
chain, .alpha. chain, .delta. chain and .epsilon. chain depending
on the difference in constant region. According to the type of
heavy chain present, five classes (isotypes) of immunoglobulin are
formed, they are IgG, IgM, IgA, IgD and IgE.
[0077] Immunoglobulin G (IgG) accounts for 70-75% of human
immunoglobulins and is the most abundantly found monomeric antibody
in plasma IgG has a four-chain structure consisting of two light
chains and two heavy chains. Human IgG1, IgG2 and IgG4 have
molecular weights of about 146,000, whereas human IgG3 has a long
hinge region that connects Fab region and Fc region and has a
larger molecular weight of 170,000. Human IgG1 accounts for about
65%, human IgG2 about 25%, human IgG3 about 7%, and human IgG4
about 3% of human IgG. They are uniformly distributed inside and
outside of blood vessels. Having a strong affinity for Fc receptors
and complement factors on effector cell surfaces, human IgG1
induces antibody-dependent cell cytotoxicity (ADCC) and also
activates complements to induce complement-dependent cell
cytotoxicity (CDC). Human IgG2 and IgG4 are low at ADCC and CDC
activities because their affinity for Fc receptors and complement
factors is low.
[0078] Immunoglobulin M (IgM), which accounts for about 10% of
human immunoglobulins, is a pentameric antibody consisting of five
basic four-chain structures joined together. It has a molecular
weight of 970,000. Usually occurring only in blood, IgM is produced
against infectious microorganisms and takes charge of early stage
immunity.
[0079] Immunoglobulin A (IgA) accounts for 10-15% of human
immunoglobulins. It has a molecular weight of 160,000. Secreted IgA
is a dimeric antibody consisting of two IgA molecules joined
together. IgA1 is found in serum, nasal discharge, saliva and
breast milk. In intestinal juice, IgA2 is found abundantly.
[0080] Immunoglobulin D (IgD) is a monomeric antibody accounting
for no more than 1% of human immunoglobulins. IgD is found on B
cell surfaces and involved in induction of antibody production.
[0081] Immunoglobulin E (IgE) is a monomeric antibody that occurs
in an extremely small amount, accounting for only 0.001% or less of
human immunoglobulins. Immunoglobulin E is considered to be
involved in immune response to parasites but in advanced countries
where parasites are rare. IgE is largely involved in bronchial
asthma and allergy among other things.
[0082] With respect to canine, sequences of IgG-A (equivalent to
human IgG2), IgG-B (equivalent to human IgG1), IgG-C (equivalent to
human IgG3) and IgG-D (equivalent to human IgG4) have been
identified as the heavy chain of IgG. In the antibody of the
present invention, an IgG's heavy chain constant region with
neither ADCC activity nor CDC activity is preferable (IgG4 in
human). In the case where the constant region of an immunoglobulin
equivalent to human IgG4 has not been identified, one may use a
constant region that has lost both ADCC activity and CDC activity
as a result of introducing mutations into the relevant region of an
immunoglobulin equivalent to human IgG4.
[0083] With respect to bovine, sequences of IgG1, IgG2 and IgG3
have been identified as the heavy chain of IgG. In the antibody of
the present invention, an IgG's heavy chain constant region with
neither ADCC activity nor CDC activity is preferable (IgG4 in
human). Although the constant region of wild-type human IgG1 has
ADCC activity and CDC activity, it is known that these activities
can be reduced by introducing amino acid substitutions or deletions
into specific sites. In bovine, the constant region of an
immunoglobulin equivalent to human IgG4 has not been identified, so
mutations may be added at the relevant region of an immunoglobulin
equivalent to human IgG1 and the resultant constant region then
used. As one example, the amino acid sequence of the CH of a bovine
antibody (IgG1 chain, GenBank: X62916) having mutations introduced
into CH2 domain and a nucleotide sequence for such amino acid
sequence (after codon optimization) are shown in SEQ ID NOS: 102
and 102, respectively.
[0084] When an animal other than rat is canine, an anti-PD-L1
antibody is more preferable in which (i) the CL of a canine
antibody has the amino acid sequence of the constant region of
Lambda chain and (ii) the CH of the canine antibody has the amino
acid sequence of the constant region of an immunoglobulin
equivalent to human IgG4.
[0085] When an animal other than rat is bovine, an anti-PD-L1
antibody is more preferable in which (i) the CL of a bovine
antibody has the amino acid sequence of the constant region of
Lambda chain and (ii) the CH of the bovine antibody has mutations
introduced thereinto that reduce ADCC activity and/or CDC
activity.
[0086] The anti-PD-L1 antibody of the present invention encompasses
rat-canine chimeric antibodies, caninized antibodies, complete
canine-type antibodies, rat-bovine chimeric antibodies, bovinized
antibodies and complete bovine-type antibodies. However, animals
are not limited to canine and bovine and may be exemplified by
human, porcine, simian, mouse, feline, equine, goat, sheep, water
buffalo, rabbit, hamster, guinea pig and the like.
[0087] For example, the anti-PD-L1 antibody of the present
invention may be an anti-PD-L1 antibody in which the CL of a canine
antibody has the amino acid sequence as shown in SEQ ID NO: 3 and
the CH of the canine antibody has the amino acid sequence as shown
in SEQ ID NO: 4.
[0088] As a further example, the anti-PD-L1 antibody of the present
invention may be an anti-PD-L1 antibody in which the CL of a bovine
antibody has the amino acid sequence as shown in SEQ ID NO: 100 and
the CH of the bovine antibody has the amino acid sequence as shown
in SEQ ID NO: 102.
[0089] The amino acid sequences as shown in SEQ ID NOS: 3 and 4 as
well as SEQ ID NOS: 100 and 102 may have deletion(s),
substitution(s) or addition(s) of one or several (e.g., up to five,
about 10 at the most) amino acids. Even when such mutations have
been introduced, the resulting amino acid sequences are capable of
having the function as CL or CH of the PD-L1 antibody.
[0090] The anti-PD-L1 antibody of the present invention may have a
four-chain structure comprising two light chains and two heavy
chains.
[0091] The anti-PD-L1 antibody of the present invention may be
prepared as described below. Briefly, an artificial gene is
synthesized which comprises (i) the identified variable region
sequences of a rat anti-bovine PD-L1 antibody and (ii) the constant
region sequences of an antibody of an animal other than rat (e.g.,
canine or bovine) (preferably, human IgG4 antibody; antibody
equivalent to human IgG4 antibody; or an immunoglobulin equivalent
to human IgG1, in which mutations have been introduced into the
relevant region to reduce ADCC activity and/or CDC activity). The
resultant gene is inserted into a vector (e.g., plasmid), which is
then introduced into a host cell (e.g., mammal cell such as CHO
cell). The host cell is cultured, and the antibody of interest is
collected from the resultant culture.
[0092] The amino acid sequence and the nucleotide sequence of the
VL of the rat anti-bovine PD-L1 antibody identified by the present
inventors are shown in SEQ ID NOS: 1 and 5, respectively. Further,
the nucleotide sequence after codon optimization is shown in SEQ ID
NO: 15.
[0093] The amino acid sequence and the nucleotide sequence of the
VH of the rat anti-bovine PD-L1 antibody identified by the present
inventors are shown in SEQ ID NOS: 2 and 6, respectively. Further,
the nucleotide sequence after codon optimization is shown in SEQ ID
NO: 16.
[0094] The amino acid sequence and the nucleotide sequence of the
CL (Lambda chain, GenBank: E02824.1) of a canine antibody are shown
in SEQ ID NOS: 3 and 7, respectively. Further, the nucleotide
sequence after codon optimization is shown in SEQ ID NO: 17.
[0095] The amino acid sequence and the nucleotide sequence of the
CL (Lambda chain, GenBank: X62917) of a bovine antibody are shown
in SEQ ID NOS: 100 and 101, respectively. Further, the nucleotide
sequence after codon optimization is shown in SEQ ID NO: 104.
[0096] The amino acid sequence and the nucleotide sequence of the
CH (IgG-D chain, GenBank: AF354267.1) of the canine antibody are
shown in SEQ ID NOS: 4 and 8, respectively. Further, the nucleotide
sequence after codon optimization is shown in SEQ ID NO: 18.
[0097] The amino acid sequence and the nucleotide sequence (after
codon optimization) of the CH (IgG1 chain, modified from GenBank:
X62916) of the bovine antibody are shown in SEQ ID NOS: 102 and
103, respectively.
[0098] Further, SEQ ID NO: 9 shows the amino acid sequence of a
chimeric light chain consisting of the VL of the rat anti-bovine
PD-L1 antibody and the CL (Lambda chain, GenBank: E02824.1) of the
canine antibody. The nucleotide sequence (after codon optimization)
of the chimeric light chain consisting of the VL of the rat
anti-bovine PD-L1 antibody and the CL (Lambda chain, GenBank:
E02824.1) of the canine antibody is shown in SEQ ID NO: 19.
[0099] Further, SEQ ID NO: 105 shows the amino acid sequence of a
chimeric light chain consisting of the VL of the rat anti-bovine
PD-L1 antibody and the CL (Lambda chain, GenBank: X62917) of the
bovine antibody. The nucleotide sequence (after codon optimization)
of the chimeric light chain consisting of the VL of the rat
anti-bovine PD-L1 antibody and the CL (Lambda chain, GenBank:
X62917) of the bovine antibody is shown in SEQ ID NO: 107.
[0100] SEQ ID NO; 10 shows the amino acid sequence of a chimeric
heavy chain consisting of the VH of the rat anti-bovine PD-L1
antibody and the CH (IgG-D chain, GenBank: AF354267.1) of the
canine antibody. The nucleotide sequence (after codon optimization)
of the chimeric heavy chain consisting of the VH of the rat
anti-bovine PD-L1 antibody and the CH (IgG-D chain, GenBank:
AF354267.1) of the canine antibody is shown in SEQ ID NO: 20.
[0101] SEQ ID NO: 106 shows the amino acid sequence of a chimeric
heavy chain consisting of the VH of the rat anti-bovine PD-L1
antibody and the CH (IgG1 chain, modified from GenBank: X62916) of
the bovine antibody. The nucleotide sequence (after codon
optimization) of the chimeric heavy chain consisting of the VH of
the rat anti-bovine PD-L1 antibody and the CH (IgG1 chain, modified
from GenBank: X62916) of the bovine antibody is shown in SEQ ID NO:
108.
[0102] Amino acid sequences and nucleotide sequences of CLs and CHs
for various animals other than the above may be obtained from known
databases for use in the present invention.
[0103] Amino acid sequences and nucleotide sequences of CLs and CHs
for canine, ovine, porcine, water buffalo, human and bovine are
summarized in the table below.
Table.
TABLE-US-00001 [0104] TABLE GenBank Accession IMGT Species Ig
Domain Nucleotide Sequence Amino Acid Sequence No. Database
Reference Canine Canine IgG-D GCCTCCACCACGGCCCCCTCG
ASTTAPSVFPLAPSCGSTS AF354267 http://ww Tang L, et al., (Scienti- Ig
GTTTTCCCACTGGCCCCCAGC GSTVALACLVSGYFPEPVT w.imgt.or Vet. Immunol.
fic heavy TGCGGGTCCACTTCCGGCTCC VSWNSGSLTSGVHTFPSV g/IMGTre
Immunopathol, Name: chain ACGGTGGCCCTGGCCTGCCTG LQSSGLYSLSSTVTVPSSR
pertoire/i 80 (3-4), 259-270 Canis constant GTGTCAGGCTACTTCCCCGAG
WPSETFTCNVVHPASNTK ndex.php (2001), PMID: lupus region
CCTGTAACTGTGTCCTGGAAT VDKPVPKESTCKCISPCPV ?section = 11457479
familiaris) (CH1- TCCGGCTCCTTGACCAGCGGT PESLGGPSVFIFPPKPKDIL
LocusGe CH3) GTGCACACCTTCCCGTCCGTC RITRTPEITCVVLDLGRED nes&repe
CTGCAGTCCTCAGGGCTCTAC PEVQISWFVDGKEVHTAK rtoire = ge
TCCCTCAGCAGCACGGTGAC TQPREQQFNSTYRVVSVL netable & s
AGTGCCCTCCAGCAGGTGGC PIEHQDWLTGKEFKCRVN pecies = do
CCAGCGAGACCTTCACCTGCA HIGLPSPIERTISKARGQAH g & group =
ACGTGGTCCACCCGGCCAGC QPSVYVLPPSPKELSSSDT IGHC AACACTAAAGTAGACAAGCC
VTLTCLIKDFFPPEIDVEW AGTGCCCAAAGAGTCCACCT QSNGQPEPESKYHTTAPQ
GCAAGTGTATATCCCCATGCC LDEDGSYFLYSKLSVDKS CAGTCCCTGAATCACTGGGAG
RWQQGDTFTCAVMHEAL GGCCTTCGGTCTTCATCTTTCC QNHYTDLSLSHSPGK*
CCCGAAACCCAAGGACATCCT (SEQ ID NO: 4) CAGGATTACCCGAACACCCGA
GATCACCTGTGTGGTGTTAGA TCTGGGCCGTGAGGACCCTG AGGTGCAGATCAGCTGGTTCG
TGGATGGTAAGGAGGTGCAC ACAGCCAAGACGCAGCCTCG TGAGCAGCAGTTCAACAGCA
CCTACCGTGTGGTCAGCGTCC TCCCCATTGAGCACCAGGACT GGCTCACCGGAAAGGAGTTC
AAGTGCAGAGTCAACCACAT AGGCCTCCCGTCCCCCATCGA GAGGACTATCTCCAAAGCCAG
AGGGCAAGCCCATCAGCCCA GTGTGTATGTCCTGCCACCAT CCCCAAAGGAGTTGTCATCCA
GTGACACGGTCACCCTGACCT GCCTGATCAAAGACTTCTTCC CACCTGAGATTGATGTGGAGT
GGCAGAGCAATGGACAGCCG GAGCCCGAGAGCAAGTACCA CACGACTGCGCCCCAGCTGG
ACGAGGACGGGTCCTACTTCC TGTACAGCAAGCTCTCTGTGG ACAAGAGCCGCTGGCAGCAG
GGAGACACCTTCACATGTGCG GTGATGCATGAAGCTCTACAG AACCACTACACAGATCTATCC
CTCTCCCATTCTCCGGGTAAA TGA (SEQ ID NO: 8) Canine Ig
CAGCCCAAGGCCTCCCCCT QPKASPSVTLFPPSSEE E02824 Not None Ig lambda
CGGTCACACTCTTCCCGCC LGANKATLVCLISDFYP registered light (CL)
CTCCTCTGAGGAGCTCGGC SGVTVAWKASGSPVTQ chain GCCAACAAGGCCACCCTGG
GVETTKPSKQSNNKYA constant TGTGCCTCATCAGCGACTTC ASSYLSLTPDKWKSHSS
region TACCCCAGCGGCGTGACGG FSCLVTHEGSTVEKKVA TGGCCTGGAAGGCAAGCGG
PAECS* (SEQ ID NO: 3) CAGCCCCGTCACCCAGGGC GTGGAGACCACCAAGCCCT
CCAAGCAGAGCAACAACAA GTACGCGGCCAGCAGCTAC CTGAGCCTGACGCCTGACA
AGTGGAAATCTCACAGCAG CTTCAGCTGCCTGGTCACG CACGAGGGGAGCACCGTGG
AGAAGAAGGTGGCCCCCGC AGAGTGCTCTTAG (SEQ ID NO: 7) Ovine Ovine `IgG1
GCCTCAACAACACCCCCGAA ASTTPPKVYPLTSCCGDTS X69797 http://ww Dufour V.
et al., (Scienti- heavy AGTCTACCCTCTGACTTCTTG SSIVTLGCLVSSYMPEPVT
w.imgt.or J. Immunol., 156. tic chain CTGCGGGGACACGTCCAGCT
VTWNSGALTSGVHTFPAI g/IMGTre 2163-2170 Name: constant
CCATCGTGACCCTGGGCTGCC LQSSGLYSLSSVVTVPAST pertoir/li (1996). PMID:
Ovis region TGGTCTCCAGCTATATGCCCG SGAQTFICNVAHPASSTKV ndex.php
8690905 aries) (CH1.sup.~ AGCCGGTGACCGTGACCTGG DKRVEPGCPDPCKHCRCP
?section = CH3) AACTCTGGTGCCCTGACCAGC PPELPGGPSVFIFPPKPKDT LocusGe
GGCGTGCACACCTTCCCGGCC LTISGTPEVTCVVVDVGQ nes & repe
ATCCTGCAGTCCTCCGGGCTC DDPEVQFSWFVDNVEVRT rtoire = ge
TACTCTCTCAGCAGCGTGGTG ARTKPREEQFNSTFRVVSA netable & s
ACCGTGCCGGCCAGCACCTC LPIQHQDWTGGKEFKCKV pecies = sh
AGGAGCCCAGACCTTCATCTG HNEALPAPIVRTISRTKGQ eep & grou
CAACGTAGCCCACCCGGCCA AREPQVYVLAPPQEELSK p = IGHC
GCAGCACCAAGGTGGACAAG STLSVTCLVTGFYPDYIAV CGTGTTGAGCCCGGATGCCCG
EWQKNGQPESEDKYGTT GACCCATGCAAACATTGCCGA TSQLDADGSYFLYSRLRV
TGCCCACCCCCTGAGCTCCCC DKNSWQEGDTYACVVMH GGAGGACCGTCTGTCTTCATC
EALHNHYTQKSISKPPGK* TTCCCACCGAAACCCAAGGA (SEQ ID NO: 42)
CACCCTTACAATCTCTGGAAC GCCCGAGGTCACGTGTGTGGT GGTGGACGTGGGCCAGGATG
ACCCCGAGGTGCAGTTCTCCT GGTTCGTGGACAACGTGGAG GTGCGCACGGCCAGGACAAA
GCCGAGAGAGGAGCAGTTCA ACAGCACCTTCCGCGTGGTCA GCGCCCTGCCCATCCAGCACC
AAGACTGGACTGGAGGAAAG GAGTTCAAGTGCAAGGTCCA CAACGAAGCCCTCCCGGCCC
CCATCGTGAGGACCATCTCCA GGACCAAAGGGCAGGCCCGG GAGCCGCAGGTGTACGTCCTG
GCCCCACCCCAGGAAGAGCT CAGCAAAAGCACGCTCAGCG TCACCTGCCTGGTCACCGGCT
TCTACCCAGACTACATCGCCG TGGAGTGGCAGAAAAATGGG CAGCCTGAGTCGGAGGACAA
GTACGGCACGACCACATCCCA GCTGGACGCCGACGGCTCCTA CTTCCTGTACAGCAGGCTCAG
GGTGGACAAGAACAGCTGGC AAGAAGGAGACACCTACGCG TGTGTGGTGATGCACGAGGCT
CTGCACAACCACTACACACA GAAGTCGATCTCTAAGCCTCC GGGTAAATGA (SEQ ID NO:
43) IgG2 GCCTCCACCACAGCCCCGAA ASTTAPKVYPLTSCCGDTS X70983 Clarkson
C.A. et AGTCTACCCTCTGACTTCTTG SSSSIVTLGCLVSSYMPEP al., Mol.
CTGCGGGGACCACGTCCAGCT VTVTWNSGALTSGVHTFP Immunol., 30,
CCAGCTCCATCGTGACCCTGG AILQSSGLYSLSSVVTVPA 1195-1204
GCTGCCTGGTCTCCAGCTATAT STSGAQTFICNVAHPASSA (1993). PMID:
GCCCGAGCCGGTGACCGTGA KVDKRVGISSDYSKCSKP 8413324
CCTGGAACTCTGGTGCCCTGA PCVSRPSVFIFPPKPKDSL CCAGCGGCGTGCACACCTTCC
MITGTPEVTCVVVDVGQG CGGCCATCCTGCAGTCCTCCG DPEVQFSWFVDNVEVRTA
GGCTCTACTCTCTCAGCAGCG RTKPREEQFNSTFRVVSAL TGGTGACCGTGCCGGCCAGC
PIQHDHWTGGKEFKCKV ACCTCAGGAGCCCAGACCTTC HSKGLPAPIVRTISRAKGQ
ATCTGCAACGTAGCCCACCCG AREPQVYVLAPPQEELSK GCCAGCAGCGCCAAGGTGGA
STLSVTCLVTGFYPDYIAV CAAGCGTGTTGGGATCTCCAG EWQRARQPESEDKYGTTT
TGACTACTCCAAGTGTTCTAA SQLDADGSYFLYSRLRVD ACCGCCTTGCGTGAGCCGACC
KSSWQRGDTYACVVMHE GTCTGTCTTCATCTTCCCCCCG ALHNHYTQKSISKPPGK*
AAACCCAAGGACAGCCTCAT (SEQ ID NO: 44) GATCACAGGAACGCCCGAGG
TCACGTGTGTGGTGGTGGACG TGGGCCAGGGTGACCCCGAG GTGCAGTTCTCCTGGTTCGTG
GACAACGTGGAGGTGCGCAC GGCCAGGACAAAGCCGAGAG AGGAGCAGTTCAACAGCACC
TTCCGCGTGGTCAGCGCCCTG CCCATCCAGCACGACCACTGG ACTGGAGGAAAGGAGTTCAA
GTGCAAGGTCCACAGCAAAG GCCTCCCGGCCCCCATCGTGA GGACCATCTCCAGGGCCAAA
GGGCAGGCCCGGGAGCCGCA GGTGTACGTCCTGGCCCCACC CCAGGAAGAGCTCAGCAAAA
GCACGCTCAGCGICACCTGCC TGGTCACCGGCTTCTACCCAG ACTACATCCICCGTGGAGTGGC
AGAGAGCGCGGCAGCCTGAG TCGGAGGACAAGTACGGCAC GACCACATCCCAGCTGGACGC
CGACGGCTCCTACTTCCTGTA CAGCAGGCTCAGGGTGGACA AGAGCAGCTGGCAAAGAGGA
GACACCTACGCGTGTGTGGTG ATGCACGAGGCTCTGCACAAC CACTACACACAGAACITCGATC
TCTAAGCCTCCGGGTAAATGA (SEQ ID NO: 45) Ovine Ig
CCATCCGTCTTCCTCTTCAAA PSVFLFKPSEEQLRTGTVS X54110 Not Jenne C.N. et
al., light kappa CCATCTGAGGAACAGCTGAG VVCLVNDFYPKDINVKVK registered
Dev. Comp. chain (CK) GACCGGAACTGTCTCTGTCGT VDGVTQNSNFQNSFTDQD
Immunol. 30 (1- constant GTGCTTGGTGAATGATTTCTA SKKSTYSLSSTLTLSSSEY
2), 165-174 region CCCCAAAGATATCAATGTCAA QSHNAYACEVSHKSLPTA (2006).
PMID: GGTGAAAGTGGATGGGGTTA LVKSFNKNEC* (SEQ ID 16083958
CCCAGAACAGCAACTTCCAG NO: 46) AACAGCTTCACAGACCAGGA
CAGCAAGAAAAGCACCTACA GCCTCAGCAGCACCCTGACA CTGTCCAGCTCAGAGTACCAG
AGCCATAACGCCTATGCGTGT GAGGTCAGCCACAAGAGCCT GCCCACCGCCCTCGTCAAGA
GCTTCAATAAGAATGAATGTT AG (SEQ ID NO: 47) Ig GGTCAGCCCAAGTCCGCACC
GQPKSAPSVTLFPPSTEEL AY734681 lambda CTCGGTCACCCTGTTCCCGCC
STNKATVVCLINDFYPGS (CL) TTCCACGGAGGAGCTCAGTAC VNVVWKADGSTINQNVK
CAACAAGGCCACCGTGGTGT TTQASKQSNSKYAASSYL GTCTCATCAACGACTTCTACC
TLTGSEWKSKSSYTCEVT CGGGTAGCGTGAACGTGGTCT HEGSTVTKTVKPSECS*
GGAAGGCAGATGGCAGCACC (SEQ ID NO: 48) ATCAATCAGAACGTGAAGACC
ACCCAGGCCTCCAAACAGAG CAACAGCAAGTACGCGGCCA GCAGCTACCTGACCCTGACGG
GCAGCGAGTGGAAGTCTAAG AGCAGTTACACCTGCGAGGTC ACGCACGAGGGGAGCACCGT
GACGAAGACAGTGAAGCCCT CAGAGTGTTCTTAG (SEQ ID NO: 49) Porcine Porcine
IgG1.sup.a GCCCCCAAGACGGCCCCATCG APKTAPSVYPLAPCGRDT U03781
http://ww Butler J.E. et al., (Scienti- Ig GTCTACCCTCTGGCCCCCTGC
SGPNVALGCASSYFPEPV w.imgt.or Immunogenetics fic heavy
GGCAGGGACACGTCTGGCCC TMTWNSGALTSGVHTFPS g/IMGTre 61(3): 209-230
Name: chain TAACGTGGCCTTGGGCTGCCT VLQPSGLYSLSSMVTVPAS pertoire/i
(2009). PMID: Sus constant GGCCTCAAGCTACTFCCCCGA
SLSSKSYTCNVNHPATTTK ndex.php 19048248 scrofa) region
GCCAGTGACCATGACCTGGA VDKRVGTKTKPPCPICPGC ?section = Kacskovics I.
et (CH1- ACTCGGGCGCCCTGACCAGT EVAGPSVFIFPPKPKDTLM LocusGe al., J.
Immunol. CH3) GGCGTGCATACCTTCCCATCC ISQTPEVTCVVVDVSKEH nes &
repe 153(8): 3565- GTCCTGCAGCCGTCAGGGCTC AEVQFSWYVDGVEVHTA rtoire =
ge 3573 (1994). TACTCCCTCAGCAGCATGGTG ETRPKEEQFNSTYRVVSV netable
& s PMID: 7930579 ACCGTGCCGGCCAGCAGCCT LPIQHQDWLKGKEFKCKV
pecies = Pi GTCCAGCAAGAGCTACACCT NNVDLPAPITRTISKAIGQS g & group
= GCAATGTCAACCACCCGGCCA REPQVYTLPPPAEELSRSK IGHC
CCACCACCAAGGTGGACAAG VTVTCLVIGFYPPDIHVEW CGTGTTGGAACAAAGACCAA
KSNGQPEPEGNYRTTPPQ ACCACCATGTCCCATATGCCC QDVDGTFFLYSKLAVDKA
AGGCTGTGAAGTGGCCGGGC RWDHGETFECAVMHEAL CCTCGGTCTTCATCTTCCCTCC
HNHYTQKSISKTQGK* AAAACCCAAGGACACCCTCA (SEQ ID NO: 50)
TGATCTCCCAGACCCCCGAGG
TCACGTGCGTGGTGGTGGAC GTCAGCAAGGAGCACGCCGA GGTCCAGTTCTCCTGGTACGT
GGACGGCGTAGAGGTGCACA CGGCCGAGACGAGACCAAAG GAGGAGCAGTTCAACAGCAC
CTACCGTGTGGTCAGCGICCT GCCCATCCAGCACCAGGACTG GCTGAAGGGGAAGGAGTTCA
AGTGCAAGGTCAACAACGTA GACCTCCCAGCCCCCATCACG AGGACCATCTCCAAGGCTATA
GGGCAGAGCCGGGAGCCGCA GGTGTACACCCTGCCCCCACC CGCCGAGGAGCTGTCCAGGA
GCAAAGTCACCGTAACCTGCC TGGTCATTGGCTTCTACCCAC CTGACATCCATGTTGAGTGGA
AGAGCAACGGACAGCCGGAG CCAGAGGGCAAFTACCGCACC ACCCCGCCCCAGCAGGACGT
GGACGGGACCTTCTTCCTGTA CAGCAAGCTCGCGGTGGACA AGGCAAGATGGGACCATGGA
GAAACATTTGAGTGTGCGGTG ATGCACGAGGCTCTGCACAAC CACTACACCCAGAAGTCCATC
TCCAAGACTCAGGGTAAATGA (SEQ ID NO: 51) IgG1.sup.b
GCCCCCAAGACGGCCCCATCG APKTAPSVYPLAPCGRDV U03778
GTCTACCCTCTGGCCCCCTGC SGPNVALGCLASSYFPEPV GGCAGGGACGTGTCTGGCCCT
TVTWNSGALTSGVHTFPS AACGTGGCCTTGGGCTGCCTG VLQPSGLYSLSSMVTVPAS
GCCTCAAGCTACTTCCCCGAG SLSSKSYTCNVNHPATTTK CCAGTGACCGTGACCTGGAA
VDKRVGIHQPQTCPICPGC CTCGGGCGCCCTGACCAGTG EVAGPSVFIFPPKPKDTLM
GCGTGCACACCTTCCCATCCG ISQTEVTCVVVDVSKEH TCCTGCAGCCGTCAGGGCTCT
AEVQFSWYVDGVEVHTA ACTCCCTCAGCAGCATGGTGA ETRPKEEQFNSTYRVVSC
CCGTGCCGGCCAGCAGCCTGT LPIQHQDWLKGKEFKCKV CCAGCAAGAGCTACACCTGC
NNVDLPAPITRTISKAIGQS AATGTCAACCACCCGGCCACC REPQVYTLPPPAEELSRSK
ACCACCAAGGTGGACAAGCG VTLTCLVIGFYPPDIHVEW TGTTGGAATACACCAGCCGCA
KSNGQPEPENTYRTTPPQ AACATGTCCCATATGCCCAGG QDVDGTFFLYSKLAVDKA
CTGTGAAGTGGCCGGGCCCTC RWDHGDKFECAVMHEAL GGTCTTCATCTTCCCTCCAAA
HNHYTQKSISKTQGK* ACCCAAGGACACCCTCATGAT (SEQ ID NO: 52)
CTCCCAGACCCCCGAGGTCAC GTGCGTGGTGGTGGACGTCA GCAAGGAGCACGCCGAGGTC
CAGTTCTCCTGGTACGTGGAC GGCGTAGAGGTGCACACGGC CGAGACGAGACCAAAGGAGG
AGCAGTTCAACAGCACCTACC GTGTGGTCAGCGTCCTGCCCA TCCAGCACCAGGACTGGCTG
AAGGGGAAGGAGTTCAAGTG CAAGGTCAACAACGTAGACC TCCCAGCCCCCATCACGAGGA
CCATCTCCAAGGCTATAGGGC AGAGCCGGGAGCCGCAGGTG TACACCCTGCCCCCACCCGCC
GAGGAGCTGTCCAGGAGCAA AGTCACGCTAACCTGCCTGGT CATTGGCTTCTACCCACCTGA
CATCCATGTTGAGTGGAAGAG CAACGGACAGCCGGAGCCAG AGAACACATACCGCACCACCC
CGCCCCAGCAGGACGTGGAC GGGACCTTCTTCCTGTACAGC AAACTCGCGGTGGACAAGGC
AAGATGGGACCATGGAGACA AATTTGAGTGTGCGGTGATGC ACGAGGCTCTGCACAACCACT
ACACCCAGAAGTCCATCTCCA AGACTCAGGGTAAATGA (SEQ ID NO: 53) IgG2.sup.a
GCCCCCAAGACGGCCCCATCG APKTAPSVYPLAPCSRDTS U03779
GTCTACCCTCTGGCCCCCTGC GPNVALGCLASSYFPEPVT AGCAGGGACACGTCTGGCCC
VTWNSGALSSGVHTFPSV TAACGTGGCCTTGGGCTGCCT LQPSGLYSLSSMVTVPASS
GGCCTCAAGCTACTTCCCCGA LSSKSYTCNVNHPATTTK GCCAGTGACCGTGACCTGGA
VDKRVGTKTKPPCPICPAC ACTCGGGCGCCCTGTCCAGTG ESPGPSVFIFPPKPKDTLMI
GCGTGCATACCTTCCCATCCG SRTPQVTCVVVDVSQENP TCCTGCAGCCGTCAGGGCTCT
EVQFSWYVDGVEVHTAQ ACTCCCTCAGCAGCATGGTGA TRPKEEQFNSTYRVVSVLP
CCGTGCCGGCCAGCAGCCTGT IQHQDWLNGKEFKCKVN CCAGCAAGAGCTACACCTGC
NKDLPAPITRIISKAKGQT AATGTCAACCACCCGGCCACC REPQVYTLPPHAEELSRSK
ACCACCAAGGTGGACAAGCG VSITCLVIGFYPPDIDVEW TGTTGGAACAAAGACCAAAC
QRNGQPEPEGNYRTTPPQ CACCATGTCCCATATGCCCAG QDVDGTYFLYSKFSVDKA
CCTGTGAATCCCAGGGCCCT SWQGGGIFQCAVMHEAL CGGTCTTCATCTTCCCTCCAA
HNHYTQKSISKTPGK* AACCCAAGGACACCCTCATGA (SEQ ID NO: 54)
TCTCCCGGACACCCCAGGTCA CGTGCGTGGTGGTTGATGTGA GCCAGGAGAACCCGGAGGTC
CAGTTCTCCTGGTACGTGGAC GGCGTAGAGGTGCACACGGC CCAGACGAGGCCAAAGGAGG
AGCAGTTCAACAGCACCTACC GCGTGGTCAGCGTCCTACCCA TCCAGCACCAGGACTGGCTG
AACGGGAAGGAGTTCAAGTG CAAGGTCAACAACAAAGACC TCCCAGCCCCCATCACAAGGA
TCATCTCCAAGGCCAAAGGGC AGACCCGGGAGCCGCAGGTG TACACCCTGCCCCCACACGCC
GAGGAGCTGTCCAGGAGCAA AGTCAGCATAACCTGCCTGGT CATTGGCTTCTACCCACCTGA
CATCGATGTCGAGTGGCAAAG AAACGGACAGCCGGAGCCAG AGGGCAATTACCGCACCACCC
CGCCCCAGCAGGACGTGGAC GGGACCTACTTCCTGTACAGC AAGTTCTCGGTGGACAAGGC
CAGCTGGCAGGGTGGAGGCA TATTCCAGTGTGCGGTGATGC ACGAGGCTCTGCACAACCACT
ACACCCAGAAGTCTATCTCCA AGACTCCGGGTAAATGA (SEQ ID NO: 55) IgG2.sup.b
GCCCCCAAGACGGCCCCATTG APKTAPLVYPLAPCGRDT U03780
GTCTACCCTCTGGCCCCCTGC SGPNVALGCLASSYFPEPV GGCAGGGACACGTCTGGCCC
TVTWNSGALTSGVHTFPS TAACGTGGCCTTGGGCTGCCT VLQPSGLYSLSSMVTVPAS
GGCCTCAAGCTACTTCCCCGA SLSSKSYTCNVNHPATTTK GCCAGTGACCGTGACCTGGA
VDKRVGTKTKPPCPICPAC ACTCGGGCGCCCTGACCAGT ESPGPSVFIFPPKPKDTLMI
GGCGTGCATACCTTCCCATCC SRTPQVTCVVVDVSQENP GTCCTGCAGCCGTCAGGGCTC
EVQFSWYVDGVEVHTAQ TACTCCCTCAGCAGCATGGTG TRPKEFQFNSTYRVVSVLP
ACCGTGCCGGCCAGCAGCCT IQHQDWLNGKEFKCKVN GTCCAGCAAGAGCTACACCT
NKDLPAPITRIISKAKGQT GCAATGTCAACCACCCGGCCA REPQVYTLPPHAEELSRSK
CCACCACCAAGGTGGACAAG VSITCLVIGFYPPDIDVEW CGTGTTGGAACAAAGACCAA
QRNGQPEPEGNYRTTPPQ ACCACCATGTCCCATATGCCC QDVDGTYFLYSKFSVDKA
AGCCTGTGAATCGCCAGGGCC SWQGGGIFQCAVMHEAL CTCGGTCTTCATCTTCCCTCCA
HNHYTQKSISKTPGK* AAACCCAAGGACACCCTCAT (SEQ ID NO: 56)
GATCTCCCGGACACCCCAGGT CACGTGCGTGGTAGTTGATGT GAGCCAGGAGAACCCGGAGG
TCCAGTTCTCCTGGTACGTGG ACGGCGTAGAGGTGCACACG GCCCAGACGAGGCCAAAGGA
GGAGCAGTTCAACAGCACCT ACCGCGTGGTCAGCGTCCTGC CCATCCAGCACCAGGACTGGC
TGAACGGGAAGGAGTTCAAG TGCAAGGTCAACAACAAAGA CCTCCCAGCCCCCATCACAAG
GATCATCTCCAAGGCCAAAGG GCAGACCCGGGAGCCGCAGG TGTACACCCTGCCCCCACACG
CCGAGGAGCTGTCCAGGAGC AAAGTCAGCATAACCTGCCTG GTCATTGGCTTCTACCCACCT
GACATCGATGTCGAGTGGCAA AGAAACGGACAGCCGGAGCC AGAGGGCAATTACCGCACCA
CCCCGCCCCAGCAGGACGTG GACGGGACCTACTTCCTGTAC AGCAAGTTCTCGGTGGACAA
GGCCAGCTGGCAGGGTGGAG GCATATTCCAGTGTGCGGTGA TGCACGAGGCTCTGCACAAC
CACTACACCCAGAAGTCTATC TCCAAGACTCCGGGTAAATGA (SEQ ID NO: 57) IgG3
GCCTACAACACAGCTCCATCG AYNTAPSVYPLAPCGRDV EU372658
GTCTACCCTCTGGCCCCCTGT SDHNVALGCLVSSYFPEPV GGCAGGGACGTGTCTGATCAT
TVTWNSGALSRVVHTFPS AACGTGGCCTTGGGCTGCCTT VLQPSGLYSLSSMVIVAAS
GTCTCAAGCTACTTCCCCGAG SLSTLSYTCNVYHPATNTK CCAGTGACCGTGACCTGGAA
VDKRVDIEPPTPICPEICSC CTCGGGTGCCCTGTCCAGAGT PAAEVLGAPSVFLFPPKPK
CGTGCATACCTTCCCATCCGT DILMISRTPKVTCVVVDVS CCTGCAGCCGTCAGGGCTCTA
QEEAEVQFSWYVDGVQL CTCCCTCAGCAGCATGGTGAT YTAQTRPMEEQFNSTYRV
CGTGGCGGCCAGCAGCCTGT VSVLPIQHQDWLKGKEFK CCACCCTGAGCTACACGTGCA
CKVNNKDLLSPITRTISKA ACGTCTACCACCCGGCCACCA TGPSRVPQVYTLPPAWEEL
ACACCAAGGTGGACAAGCGT SKSKVSITCLCTGFYPPDI GTTGACATCGAACCCCCCACA
DVEWQSNGQQEPEGNYR CCCATCTGTCCCGAAATTTGC TTPPQQDVDGTYFLYSKL
TCATGCCCAGCTGCAGAGGTC AVDKVRWQRGDLFQCAV CTGGGAGCACCGTCGGTCTTC
MHEALHNHYTQKSISKTQ CTCTTCCCTCCAAAACCCAAG GK (SEQ ID NO: 58)
GACATCCTCATGATCTCCCGG ACACCCAAGGTCACGTGCGT GGTGGTGGACGTGAGCCAGG
AGGAGGCTGAAGTCCAGTTC TCCTGGTACGTGGACCCCGTA CAGTTGTACACGGCCCAGAC
GAGGCCAATGGAGGAGCAGT TCAACAGCACCTACCGCGTGG TCAGCGTCCTGCCCATCCAGC
ACCAGGACTGGCTGAAGGGG AAGGAGTTCAAGTGCAAGGT CAACAACAAAGACCTCCTTTC
CCCCATCACGAGGACCATCTC CAAGGCTACAGGGCCGAGCC GGGTGCCGCAGGTGTACACC
CTGCCCCCAGCCTGGGAAGA GCTGTCCAAGAGCAAAGTCA GCATAACCTGCCTGGTCACTG
GCTTCTACCCACCTGACATCG ATGTCGAGTGGCAGAGCAAC GGACAACAAGAGCCAGAGGG
CAATTACCGCACCACCCCGCC CCAGCAGGACGTGGATGGGA CCTACTTCCTGTACAGCAAGC
TCGCGGTGGACAAGGTCAGG TGGCAGCGTGGAGACCTATTC CAGTGTGCGGTGATGCACGA
GGCTCTGCACAACCACTACAC CCAGAAGTCCATCTCCAAGAC TCAGGGTAAATGA (SEQ ID
NO: 59) IgG4.sup.a ACCTTCCCATCCGTCCTGCAG TFPSVLQPSGLYSLSSMVT U03782
CCGTCAGGGCTCTACTCCCTC VPASSLSSKSYTCNVNHPA AGCAGCATGGTGACCGTGCC
TTTKVDKRVGTKTKPPCPI GGCCAGCAGCCTGTCCAGCA CPACEGPGPSAFIFPPKPK
AGAGCTACACCTGCAATGTCA DTLMISRTPKVTCVVVDV ACCACCCGGCCACCACCACC
SQENPEVQFSWYVDGVE AAGGTGGACAAGCGTGTTGG VHTAQTRPKEEQFNSTYR
AACAAAGACCAAACCACCAT VVSVLPIQHQDWLNGKEF GTCCCATATGCCCAGCCTGTG
KCKVNNKDLPAPITRIISK AAGGGCCCGGGCCCTCGGCC AKGQTREPQVYTLPPPTE
TTCATCTTCCCTCCAAAACCC ELSRSKVTLTCLVTGFYPP AAGGACACCCTCATGATCTCC
DIDVEWQRNGQPEPEGNY CGGACCCCCAAGGTCACGTG RTTPPQQDVDGTYFLYSK
CGTGGTGGTAGATGTGAGCCA LAVDKASWQRGDTFQCA GGAGAACCCGGAGGTCCAGT
VMHEALHNHYTQKSIFKT TCTCCTGGTACGTGGACGGCG PGK* (SEQ ID NO: 60)
TAGAGGTGCACACGGCCCAG ACGAGGCCAAAGGAGGAGCA GTTCAACAGCACCTACCGCGT
GGTCAGCGTCCTGCCCATCCA
GCACCAGGACTGGCTGAACG GGAAGGAGTTCAAGTGCAAG GTCAACAACAAAGACCTCCC
AGCCCCCATCACAAGGATCAT CTCCAAGGCCAAAGGGCAGA CCCGGGAGCCGCAGGTGTAC
ACCCTGCCCCCACCCACCGAG GAGCTGTCCAGGAGCAAAGT CACGCTAACCTGCCTGGTCAC
TGGCTTCTACCCACCTGACAT CGATGTCGAGTGGCAAAGAA ACGGACAGCCGGAGCCAGAG
GGCAATTACCGCACCACCCCG CCCCAGCAGGACGTGGACGG GACCTACTTCCTGTACAGCAA
GCTCGCGGTGGACAAGGCCA GCTGGCAGCGTGGAGACACA TTCCAGTGTGCGGTGATGCAC
GAGGCTCTGCACAACCACTAC ACCCAGAAGTCCATCTTCAAG ACTCCGGGTAAATGA (SEQ ID
NO: 61) IgG4.sup.b GCCCCCAAGACGGCCCCATCG APKTAPSVYPLAPCGRDV
EU372654 GTCTACCCTCTGGCCCCCTGC SGPNVALGCLASSYFPEPV
GGCAGGGACGTGTCTGGCCCT TVTWNSGALTSGVHTFPS AACGTGGCCTTGGGCTGCCTG
VLQPSGLYSLSSMVTVPAS GCCTCAAGCTACTTCCCCGAG SLSSKSYTCNVNHPATTTK
CCAGTGACCGTGACCTGGAA VDKRVGIHQPQTCPICPAC CTCGGGCGCCCTGACCAGTG
EGPGPSAFIFPPKPKDTLM GCGTGCACACCTTCCCATCCG ISRTPKVTCVVVDVSQEN
TCCTGCAGCCGTCAGGGCTCT PEVQFSWYVDGVEVHTA ACTCCCTCAGCAGCATGGTGA
QTRPKEEQFNSTYRVVSV CCGTGCCGGCCAGCAGCCTGT LLIQHQDWLNGKEFKCK
CCAGCAAGAGCTACACCTGC VNNKDLPAPITRIISKAKG AATGTCAACCACCCGGCCACC
QTREPQVYTLPPPTEELSR ACCACCAAGGTGGACAAGCG SKVTLTCLVTGFYPPDIDV
TGTTGGAATACACCAGCCGCA EWQRNGQPEPEGNYRTTP AACATGTCCCATAIGCCCAGC
PQQDVDGTYFLYSKLAVD CTGTGAAGGGCCCGGGCCCT KASWQRGDTFQCAVMHE
CGGCCTTCATCTTCCCTCCAA ALHNGYT (SEQ ID NO: AACCCAAGGACACCCTCATGA 62)
TCTCCCGGACCCCCAAGGTCA CGTGCGTGGTGGTTGATGTGA GCCAGGAGAACCCGGAGGTC
CAGTTCTCCTGGTACGTGGAC GGCGTAGAGGTGCACACGGC CCAGACGAGGCCAAAGGAGG
AGCAGTTCAACAGCACCTACC GCGTGGTCAGCGTCCTGCTCA TCCAGCACCAGGACTGGCTG
AACGGGAAGGAGTTCAAGTG CAAGGTCAACAACAAAGACC TCCCAGCCCCCATCACAAGGA
TCATCTCCAAGGCCAAAGGGC AGACCCGGGAGCCGCAGGTG TACACCCTGCCCCCACCCACC
GAGGAGCTGTCCAGGAGCAA AGTCACGCTAACCTGCCTGGT CACTGGCTTCTACCCACCTGA
CATCGATGTCGAGTGGCAAAG AAACGGACAGCCGGAGCCAG AGGGCAATTACCGCACCACCC
CGCCCCAGCAGGACGTGGAC GGGACCTACTTCCTGTACAGC AAGCTCGCGGTGGACAAGGC
CAGCTGGCAGCGTGGAGACA CATTCCAGTGTGCGGTGATGC ACGAGGCTCTGCACAACCACT
ACACCC (SEQ ID NO: 63) IgG5.sup.a GCCCCCAAGACGGCCCCATCG
APKTAPSVYPLAPCSRDTS EU372657 GTCTACCCTCTGGCCCCCTGC
GPNVALGCLVSSYFPEPVT AGCAGGGACACGTCTGGCCC VTWNSGALTSGVHTFPSV
TAACGTGGCCTTGGGCTGCCT LQPSGLYSLSSMVTVPAHS GGTCTCAAGCTACTTCCCCGA
LSSKRYTCNVNHPATKTK GCCAGTGACCGTGACCTGGA VDLCVGRPCPICPGCEVA
ACTCGGGCGCCCTGACCAGT GPSVFIFPPKPKDILMISRT GGCGTGCACACCTTCCCATCC
PEVTCVVVDVSKEHAEV GTCCTGCAGCCGTCAGGGCTC QFSWYVDGEEVHTAEFRP
TACTCCCTCAGCAGCATGGTG KEEQFNSTYRVVSVLPIQH ACCGTGCCGGCCCACAGCTTG
EDWLKGKEFECKVNNED TCCAGCAAGCGCTATACGTGC LPGPITRTISKAKGVVRSP
AATGTCAACCACCCAGCCACC EVYTLPPPAEELSKSIVTLT AAAACCAAGGTGGACCTGTG
CLVKSIFP?FIHVEWKING TGTTGGACGACCATGTCCCAT KPEPENAYRTTPPQEDEDR
ATGCCCAGGCTGTGAAGTGGC TYFLYSKLAVDKARWDH CGGGCCCTCGGTCTTCATCTT
GETFECAVMHEALHNHY CCCTCCAAAACCCAAGGACAT TQKSISKTQGK* (SEQ ID
CCTCATGATCTCCCGGACCCC NO: 64) CGAGGTCACGTGCGTGGTGG
TGGACGTCAGCAAGGAGCAC GCCGAGGTCCAGTTCTCCTGG TACGTGGACGGCGAAGAGGT
GCACACGGCCGAGACGAGGC CAAAGGAGGAGCAGTTCAAC AGCACCTACCGCGTGGTCAGC
GTCCTGCCCATCCAGCACGAG GACTGGCTGAAGGGGAAGGA GTTCGAGTGCAAGGTCAACA
ACGAAGACCTCCCAGGCCCC ATCACGAGGACCATCTCCAAG GCCAAAGGGGTGGTACGGAG
CCCGGAGGTGTACACCCTGCC CCCACCCGCCGAGGAGCTGT CCAAGAGCATAGTCACGCTAA
CCTGCCTGGTCAAAAGCATCT TCCCGNCTTTCATCCATGTTG AGTGGAAAATCAACGGAAAA
CCAGAGCCAGAGAACGCATAT CGCACCACCCCGCCTCAGGA GGACGAGGACAGGACCTACT
TCCTGTACAGCAAGCTCGCGG TGGACAAGGCAAGATGGGAC CATGGAGAAACATTTGAGTGT
GCGGTGATGCACGAGGCTCTG CACAACCACTACACCCAGAA GTCCATCTCCAAGACTCAGGG
TAAATGA (SEQ ID NO: 65) IgG5.sup.b GCCTACAACACAGCTCCATCG
AYNTAPSVYPLAPCGRDV EU372656 GTCTACCCTCTGGCCCCCTGT
SDHNVALGCLVSSYFPEPV GGCAGGGACGTGTCTGATCAT TVTWNWGAQTSGVHTFP
AACGTGGCCTTGGGCTGCCTG SVLQPSGLYSLSSTVTVPA GTCTCAAGCTACTTCCCCGAG
HSLSSKCFTCNVNHPATTT CCAGTGACCGTGACCTGGAA KVDLCVGKKTKPRCPICP
CTGGGGCGCCCAGACCAGTG GCEVAGPSVFIFPPKPKDIL GCGTGCACACCTTCCCATCCG
MISRTPEVTCVVVDVSKE TCCTGCAGCCGTCAGGGCTCT HAEVQFSWYVDGEEVHT
ACTCCCTCAGCAGCACGGTG AETRPKEEQFNSTYRVVS ACCGTGCCGGCCCACAGCTTG
VLPIQHEDWLKGKEFECK TCCAGCAAGTGCTTCACGTGC VNNEDLPGPITRTISKAKG
AATGTCAACCACCCGGCCACC VVRSPEVYTLPPPAEELSK ACCACCAAGGTGGACCTGTG
SIVTLTCLVKSFFPPFIHVE TGTTGGAAAAAAGACCAAGC WKINGKPEPENAYRTTPP
CTCGATGTCCCATATGCCCAG QEDEDGTYFLYSKFSVEK GCTGTGAAGTGGCCGGGCCC
FRWHSGGIHCAVMHEAL TCGGTCTTCATCTTCCCTCCA HNHYT (SEQ ID NO: 66)
AAACCCAAGGACATCCTCATG ATCTCCCGGACCCCCGAGGTC ACGTGCGTGGTGGTGGACGT
CAGCAAGGAGCACGCCGAGG TCCAGTTCTCCTGGTACGTGG ACGGCGAAGAGGTGCACACG
GCCGAGACGAGACCAAAGGA GGAGCAGTTCAACAGCACTT ACCGCGTGGTCAGCGTCCTGC
CCATCCAGCACGAGGACTGG CTGAAGGGGAAGGAGTTCGA GTGCAAGGTCAACAACGAAG
ACCTCCCAGGCCCCATCACGA GGACCATCTCCAAGGCCAAA GGGGTGGTACGGAGCCCGGA
GGTGTACACCCTGCCCCCACC CGCCGAGGAGCTGTCCAAGA GCATAGTCACGCTAACCTGCC
TGGTCAAAAGCTTCTTCCCGC CTTTCATCCATGTTGAGTGGA AAATCAACGGAAAACCAGAG
CCAGAGAACGCATACCGCAC CACCCCGCCCCAGGAGGACG AGGACGGGACCTACTTCCTGT
ACAGCAAGTTCTCGGTGGAA AAGTTCAGGTGGCACAGTGG AGGCATCCACTGTGCGGTGAT
GCACGAGGCTCTGCACAACC ACTACACCC (SEQ ID NO: 67) IgG6
GCCCCCAAGACGGCCCCATCG APKTAPSVYPLAPCGRDT EU372655
GTCTACCCTCTGGCCCCCTGC SGPNVALGCLASSYFPEPV GGCAGGGACACGTCTGGCCC
TLTWNSGALTSGVHTFPS TAACGTGGCCTTGGGCTGCCT VLQPSGLYSLSSMVTVPAS
GGCCTCAAGCTACTTCCCCGA SLSSKSYTCNVNHPATTTK GCCAGTGACCCTGACCTGGA
VDLCVGRPCPICPACEGPG ACTCGGGCGCCCTGACCAGT PSVFIFPPKPKDTLMISRTP
GGCGTGCATACCTTCCCATCC QVTCVVVDVSQENPEVQF GTCCTGCAGCCGTCAGGGCTC
SWYVDGVEVHTAQTRPK TACTCCCTCAGCAGCATGGTG EAQFNSTYRVVSVLPIQHE
ACCGTGCCGGCCAGCAGCCT DWLKGKEFECKVNNKDL GTCCAGCAAGAGCTACACCT
PAPITRIISKAKGPSREPQV GCAATGTCAACCACCCGGCCA YTLSPSAEELSRSKVSITCL
CCACCACCAAGGTGGACCTG VTGFYPPDIDVEWKSNGQ TGTGTTGGACGACCATGTCCC
PEPEGNYRTTPPQQDVDG ATATGCCCAGCCTGTGAAGGG TYFLYSKLAVDKASWQRG
CCCGGGCCCTCGGTCTTCATC DPFQCAVMHEALHNHYT TTCCCTCCAAAACCCAAGGAC (SEQ
ID NO: 68) ACCCTCATGATCTCCCGGACA CCCCAGGTCACGTGCGTGGTG
GTAGATGTGAGCCAGGAAAA CCCGGAGGTCCAGTTCTCCTG GTATGTGGACGGTGTAGAGGT
GCACACGGCCCAGACGAGGC CAAAGGAGGCGCAGTTCAAC AGCACCTACCGTGTGGTCAGC
GTCCTGCCCATCCAGCACGAG GACTGGCTGAAGGGGAAGGA GTTCGAGTGCAAGGTCAACA
ACAAAGACCTCCAGCCCCCA TCACAAGGATCATCTCCAAGG CCAAAGGGCCGAGCCGGGAG
CCGCAGGTGTACACCCTGTCC CCATCCGCCGAGGAGCTGTCC AGGAGCAAAGTCAGCATAAC
CTGCCTGGTCACTGGCTTCTA CCCACCTGACATCGATGTCGA GTGGAAGAGCAACGGACAGC
CGGAGCCAGAGGGCAATTAC CGCACCACCCCGCCCCAGCA GGACGTGGACGGGACCTACT
TCCTGTACAGCAAGCTCGCGG TGGACAAGGCCAGCTGGCAG CGTGGAGACCCATTCCAGTGT
GCGGTGATGCACGAGGCTCTG CACAACCACTACACCC (SEQ ID NO: 69) IgG6.sup.b
GCCCCCAAGACGGCCCCATCG APKTAPSVYPLAPCGRDT EU372653
GTCTACCCTCTGGCCCCCTGC SGPNVALGCLASSYFPEPV GGCAGGGACACGTCTGGCCC
TVTWNSGALTSGVHTFPS TAACGTGGCCTTGGGCTGCCT VLQPSGLYSLSSTVTVPAR
GGCCTCAAGCTACTTCCCCGA SSSRKCFTCNVNHPATTTK GCCAGTGACCGTGACCTGGA
VDLCVGRPCPICPACEGN ACTCGGGCGCCCTGACCAGT GPSVFIFPPKPKDTLMISRT
GGCGTGCACACCTTCCCATCC PEVTCVVVDVSQENPEVQ GTCCTGCAGCCGTCAGGGCTC
FSWYVDGEEVHTAETRPK TACTCCCTCAGCAGCACGGTG EEQFNSTYRVVSVLPIQHQ
ACCGTGCCGGCCAGGAGCTC DWLKGKEFECKVNNKDL GTCCAGAAAGTGCTTCACGTG
PAPITRIISKAKGPSREPQV CAATGTCAACCACCCGGCCAC YTLSPSAEELSRSKVSITCL
CACCACCAAGGTGGACCTGT VTGFYPPDIDVEWKSNGQ GTGTTGGACGACCATGTCCCA
PEPEGNYRSTPPQEDEDG TATGCCCAGCCTGTGAAGGGA TYFLYSKLAVDKARLQSG
ACGGGCCCTCGGTCTTCATCT GIHCAVMHEALHNHYTQ TCCCTCCAAAACCCAAGGAC KSISKT
(SEQ ID NO: 70) ACCCTCATGATCTCCCGGACC CCCGAGGTCACGTGCGTGGT
GGTAGATGTGAGCCAGGAAA ACCCGGAGGTCCAGTTCTCCT GGTACGTGGACGGCGAAGAG
GTGCACACGGCCGAGACGAG GCCAAAGGAGGAGCAGTTCA ACAGCACCTACCGTGTGGTCA
GCGTCCTGCCCATCCAGCACC AGGACTGGCTGAAGGGAAAG GAGTTCGAGTGCAAGGTCAA
CAACAAAGACCTCCCAGCCC CCATCACAAGGATCATCTCCA AGGCCAAAGGGCCGAGCCGG
GAGCCGCAGGTGTACACCCT GTCCCCATCCGCCGAGGAGCT GTCCAGGAGCAAAGTCAGCA
TAACCTGCCTGGTCACTGGCT TCTACCCACCTGACATCGATG TCGAGTGGAAGAGCAACGGA
CAGCCGGAGCCAGAGGGCAA TTACCGCTCCACCCCGCCCCA GGAGGACGAGGACGGGGACCT
ACTTCCTGTACAGCAAACTCG CGGTGGACAAGGCGAGGTTG CAGAGTGGAGGCATCCACTGT
GCGGTGATGCACGAGGCTCTG CACAACCACTACACCCAGAA GTCCATCTCCAAGACT (SEQ ID
NO: 71) Porcine Ig FP312898 http://ww Schwartz J.C. et Ig kappa
w.imgt.or al., Immunogeneti light (CK) g/IMGTre cs, 64, 303-311
chain variant pertoire/i (2012). PMID: constant 1 ndex.php 22109540
region ?section = LocusGe nes & repe rtoire = ge netable &
s pecies = Pi g & group = IGLC Ig CU694848 kappa (CK) variant 2
Ig CU467669 http://ww lambda w.imgt.or (CL) g/IMGTre variant
pertoire/i 1 ndex.php ?section = LocusGe nes & repe rtoire = ge
netable & s pecies = Pi g & group = IGKC Ig CU467599 lambda
(CK) variant 2 Water Water IgG1? GAGCGGCGTGCACACCTTCCC
SGVHTFPAVLQSSGLYSLS NW_005690903 Not None buffalo buffalo
GGCCGTCCTTCAGTCCTCCGG STVTAPASATKSQTFTCNV registered (Scienti- Ig
GCTCTACTCTCTCAGCAGCAC AHPASSTKVDKAVVPPCR fic heavy
GGTGACCGCGCCCGCCAGCG PKPCDCCPPPELPGGPSVF Name: chain
CCACAAAAAGCCAGACCTTC IFPPKPKDTLTISGTPEVTC Bubalus constant
ACCTGCAACGTAGCCCACCCG VVVDVGHDDPEVKFSWF bubalis) region
GCCAGCAGCACCAAGGTGGA VDDVEVNTARTKPREEQF (CH1- CAAGGCTGTTGTTCCCCCATG
NSTYRVVSALPIQHNDWT CH3) CAGACCCGAAACCCTGTGATTG GGKEFKCKVYNEGLPAPI
CTGCCCACCCCCTGAGCTCCC VRTISRTKGQAREPQVYV CGGAGGACCCTCTGTCTTCAT
LAPPQDELSKSTVSITCMV CTTCCCACCAAAACCCAAGG TGFYPDYIAVEWQKDGQP
ACACCCTCACAATCTCTGGAA ESEDKYGTTPPQLDSDGS CTCCTGAGGTCACGTGTGTGG
YFLYSRLRVNKNSWQEGG TGGTGGACGTGGGCCACGAT AYTCVVMHE (SEQ ID NO:
GACCCCGAGGTGAAGTTCTCC 72) TGGTTCGTGGACGATGTGGAG
GTAAACACAGCCAGGACGAA GCCAAGAGAGGAGCAGTTCA ACAGCACCTACCGCGTGGTCA
GCGCCCTGCCCATCCAGCACA ACGACTGGACTGGAGGAAAG GAGTTCAAGTGCAAGGTCTAC
AATGAAGGCCTCCCAGCCCCC ATCGTGAGGACCATCTCCAGG ACCAAAGGGCAGGCCCGGGA
GCCGCAGGTGTACGTCCTGGC CCCACCCCAGGACGAGCTCA GCAAAAGCACGGTCAGCATC
ACTTGCATGGTCACTGGCTTC TACCCAGACTACATCGCCGTA GAGTGGCAGAAAGATGGGCA
GCCTGAGTCAGAGGACAAATA TGGCACGACCCCGCCCCAGCT GGACAGCGATGGCTCCTACTT
CCTGTACAGCAGGCTCAGGGT GAACAAGAACAGCTGGCAAG AAGGAGGCGCCTACACGTGT
GTAGTGATGCATGAGGC (SEQ ID NO: 73) IgG2? GCCTCCATCACAGCCCCGAAA
ASITAPKVYPLTSCRGETS NW_005766143 GTCTACCCTCTGACTTCTTGC
SSTVTLGCLVSSYMPEPVT CGCGGGGAAACGTCCAGCTC VTWNSGALKSGVHTFPAV
CACCGTGACCCTGGGCTGCCT LQSSGLYSLSSTVTAPASA GGTCTCCAGCTACATGCCCGA
TKSQTFTCNVAHPASSTK GCCGGTGACCGTGACCTGGA VDTAVGFSSDCCKFPKPC
ACTCGGGTGCCCTGAAGAGC VRGPSVFIFPPKPKDTLMI GGCGTGCACACCTTCCCGGCC
TGNPEVTCVVVDVGRDN GTCCTTCAGTCCTCTGGGCTC PEVQFSWFVGDVEVHTG
TACTCTCTCAGCAGCACGGTG RSKPREEQFNSTYRVVSTL ACCGCGCCCGCCAGCGCCAC
PIQHNDWTGGKEFKCKV AAAAAGCCAGACCTTCACCT NNKGLPAPIVRTISRTKGQ
GCAACGTAGCCCACCCGGCC AREPQVYVLAPPQEELSK AGCAGCACCAAGGTGGACAC
STVSVTCMVTGFYPDYIA GGCTGTTGGGTTCTCCAGTGA VEWHRDRQAESEDKYRT
CTGCTGCAAGTTTCCTAAGCC TPPQLDSDGSYFLYSRLKV TTGTGTGAGGGGACCATCTGT
NKNSWQEGGAYTCVVMH CTTCATCTTCCCGCCGAAACC E (SEQ ID NO: 74)
CAAAGACACCCTGATGATCAC AGGAAATCCCGAGGTCACATG TGTGGTGGTGGACGTGGGCC
GGGATAACCCCGAGGTGCAG TTCTCCTGGTTCGTGGGTGAT GTGGAGGTGCACACGGGCAG
GTCGAAGCCGAGAGAGGAGC AGTTCAACAGCACCFACCGCG TGGTCAGCACCCTGCCCATCC
AGCACAATGACTGGACTGGA GGAAAGGAGITCAAGTGCAA GGTCAACAACAAAGGCCTCC
CAGCCCCCATCGTGAGGACCA TCTCCAGGACCAAAGGGCAG GCCCGGGAGCCGCAGGTGTA
CGTCCTGGCCCCACCCCAGGA AGAGCTCAGCAAAAGCACGG TCAGCGTCACTTGCATGGTCA
CTGGCTTCTACCCAGACTACA TCGCCGTAGAGTGGCATAGAG ACCGGCAGGCTGAGTCGGAG
GACAAGTACCGCACGACCCC GCCCCAGCTGGACAGCGATG GGTCCTACTTCCTGTACAGCA
GGCTCAAGGTGAACAAGAAC AGCTGGCAAGAAGGAGGCGC CTACACGTGTGTAGTGATGCA
TGAGGC (SEQ ID NO: 75) IgG3? GCCTCCACCACAGCCCCGAA
ASTTAPKVYPLASSCGDTS NW_005784206 AGTCTACCCTCTGGCATCCAG
SSTVTLGCLVSSYMPEPVT CTGCGGGGACACGTCCAGCT VTWNSGALKNGVHTFPA
CCACCGTGACCCTGGGCTGCC VRQSSGLYSLSSMVTMPT TGGTCTCCAGCTACATGCCCG
STAGTQTFTCNVAHPASST AGCCGGTGACCGTGACCTGG KVDTAVTARHPVPKTPET
AACTCGGGTGCCCTGAAGAA PIHPVKPPTQEPRDEKTPC CGGCGTGCACACCTTCCCGGC
QCPKCPEPLGGLSVFIFPP CGTCCGGCAGTCCTCCGGGCT KPKDTLTISGTPEVTCVVV
CTACTCTCTCAGCAGCATGGT DVGQDDPEVQFSWFVDD GACCATGCCCACCAGCACCGC
VEVHTARMKPREEQFNST AGGAACCCAGACCTTCACCT YRVVSALPIQHQDWLREK
GCAACGTAGCCCACCCGGCC EFKCKVNNKGLPAPIVRTI AGCAGCACCAAGGTGGACAC
SRTKGQAREPQVYVLAPP GGCTGTCACTGCAAGGCATCC REELSKSTLSLTCLITGFYP
GGTCCCGAAGACACCAGAGA EEVDVEWQRNGQPESED CACCTATCCATCCTGTAAAAC
KYHTTPPQLDADGSYFLY CCCCAACCCAGGAGCCCAGA SRLRVNRSSWQEGDHYTC
GATGAAAAGACACCCTGCCA AVMHEALRNHYKEKPISR GTGTCCCAAATGCCCAGAACC SPGK*
(SEQ ID NO: 76) TCTGGGAGGACTGTCTGTCTT CATCTTCCCACCGAAACCCAA
GGACACCCTCACAATCTCTGG AACGCCCGAGGTCACGTGTG TGGTGGTGGACGTGGGCCAG
GATGACCCCGAAGTGCAGTTC TCCTGGTTCGTGGATGACGTG GAGGTGCACACAGCCAGGAT
GAAGCCAAGAGAGGAGCAGT TCAACAGCACCTACCGCGTGG TCAGCGCCCTGCCCATCCAGC
ACCAGGACTGGCTGCGGGAA AAGGAGTTCAAGTGCAAGGT CAACAACAAAGGCCTCCCGG
CCCCCATCGTGAGGACCATCT CCAGGACCAAAGGGCAGGCC CGGGAGCCACAGGTGTATGTC
CTGGCCCCACCCCGGGAAGA GCTCAGCAAAAGCACGCTCA GCCTCACCTGCCTAATCACCG
GCTTCTACCCAGAAGAGGTAG ACGTGGAGTGGCAGAGAAAT GGGCAGCCTGAGTCAGAGGA
CAAGTACCACACGACCCCAC CCCAGCTGGACGCTGACGGC TCCTACTTCCTGTACAGCAGG
CTCAGGGTGAACAGGAGCAG CTGGCAGGAAGGAGACCACT ACACGTGTGCAGTGATGCATG
AAGCTTTACGGAATCACTACA AAGAGAAGCCCATCTCGAGG TCTCCGGGTAAATGA (SEQ ID
NO: 77) Water Ig CAGCCCAAGTCCGCACCCTCA QPKSAPSVTLFPPSTEELS
NW_005690786 Not None buffalo lambda? GTCACCCTGTTCCCACCCTCC
ANKATLVCLISDFYPGSMT registered Ig ACGGAGGAGCTCAGCGCCAA
VARKADGSTITRNVETTR light CAAGGCCACCCTGGTGTGTCT ASKQSNSKYAASSYLSLT
chain CATCAGCGACTTCTACCCGGG GSEWKSKGSYSCEVTHEG constant
TAGCATGACCGTGGCCAGGA STVTKTVKPSECS* (SEQ region
AGGCAGACGGCAGCACCATC ID NO: 78) (CL) ACCCGGAACGTGGAGACCAC
CCGGGCCTCCAAACAGAGCA ACAGCAAGTACGCGGCCAGC AGCTACCTGAGCCTGACGGG
CAGCGAGTGGAAATCGAAAG GCAGTTACAGCTGCGAGGTC ACGCACGAGGGGAGCACCGT
GACAAAGACAGTGAAGCCCT CAGAGTGTTCTTAG (SEQ ID NO: 79) Human Human
IgG4 GAGTCCAAATATGGTCCCCCA ESKYGPPCPSCPAPEFLGG K01316 http://ww
Ellison J. et al., (Scienti- Ig variant TGCCCATCATGCCCAGCACCT
PSVFLFPPKPKDTLMISRT w.imgt.or DNA, 1, 11-18 fic heavy 1
GAGTTCCTGGGGGGACCATCA PEVTCVVVDVSQEDPEVQ g/IMGTre (1981). PMID:
Name: chain GTCTTCCTGTTCCCCCCAAAA FNWYNDGVEVHNAKTKP pertoire/i
6299662 Homo constant CCCAAGGACACTCTCATGATC REEQFNSTYRVVSVLTVL
ndex.php sapiens) region TCCCGGACCCCTGAGGTCACG HQDWLNGKEYKCKVSNK
?section = (CH1- TGCGTGGTGGTGGACGTGAG GLPSSIEKTISKAKGQPRE LocusGe
CH3) CCAGGAAGACCCCGAGGTCC PQVYTLPPSQEEMTKNQV nes & repe
AGTTCAACTGGTACGTGGATG SLTCLVKGFYPSDIAVEWE rtoire = ge
GCGTGGAGGTGCATAATGCCA SNGQPENNYKTTPPVLDS netable & s
AGACAAAGCCGCGGGAGGAG DGSFFLYSRLTVDKSRWQ pecies = hu
CAGTTCAACAGCACGTACCGT EGNVFSCSVMHEALHNH man & gro
GTGGTCAGCGTCCTCACCGTC YTQKSLSLSLGK* (SEQ ID up = IGHC
CTGCACCAGGACTGGCTGAA NO: 12) CGGCAAGGAGTACAAGTGCA
AGGTCTCCAACAAAGGCCTC CCGTCCTCCATCGAGAAAACC ATCTCCAAAGCCAAAGGGCA
GCCCCGAGAGCCACAGGTGT ACACCCTGCCCCCATCCCAGG AGGAGATGACCAAGAACCAG
GTCAGCCTGACCTGCCTGGTC AAAGGCTTCTACCCCAGCGAC ATCGCCGTGGAGTGGGAGAG
CAATGGGCAGCCGGAGAACA ACTACAAGACCACGCCTCCCG TGCTGGACTCCGACGGCTCCT
TCTTCCTCTACAGCAGGCTAA CCGTGGACAAGAGCAGGTGG CAGGAGGGGAATGTCTTCTCA
TGCTCCGTGATGCATGAGGCT CTGCACAACCACTACACACA
GAAGAGCCTCTCCCTGTCTCT
GGGTAAATGA (SEQ ID NO: 14) 1gG4 GAGTCCAAATATGGTCCCCCG
ESKYGPPCPSCPAPEFLGG AJ001563 Brusco A. et al., variant
TGCCCATCATGCCCAGCACCT PSVFLFPPKPKDTLMISRT Eur. J. 2
GAGTTCCTGGGGGGACCATCA PEVTCVVVDVSQEDPEVQ Immunogenet.,
GTCTTCCTGTTCCCCCCAAAA FNWYVDGVEVHNAKTKP 25, 349-355
CCCAAGGACACTCTCATGATC REEQFNSTYRVVSVLTVV (1998). PMID:
TCCCGGACCCCTGAGGTCACG HQPWLNGKEYKCKVSNK 9805657
TGCGTGGTGGTGGACGTGAG GLPSSIEKTISKAKGQPRE CCAGGAAGACCCCGAGGTCC
PQVYTLPPSQEEMTKNQV AGTTCAACTGGTACGTGGATG SLTCLVKGFYPSDIAVEWE
GCGTGGAGGTGCATAATGCCA SNGQPENNYKTTPPVLDS AGACAAAGCCGCGGGAGGAG
DGSFFLYSRLTVDKSRWQ CAGTTCAACAGCACGTACCGT EGNVFSCSVMHEALHNH
GTGGTCAGCGTCCTCACCGTC YTQKSLSLSEGK (SEQ ID GTGCACCAGGACTGGCTGAA NO:
80) CGGCAAGGAGTACAAGTGCA AGGTCTCCAACAAAGGCCTC CCGTCCTCCATCGAGAAAACC
ATCTCCAAAGCCAAAGGGCA GCCCCGAGAGCCACAGGTGT ACACCCTGCCCCCATCCCAGG
AGGAGATGACCAAGAACCAG GTCAGCCTGACCTGCCTGGTC AAAGGCTTCTACCCCAGCGAC
ATCGCCGTGGAGTGGGAGAG CAATGGGCAGCCGGAGAACA ACTACAAGACCACGCCTCCCG
TGCTGGACTCCGACGGCTCCT TCTTCCTCTACAGCAGGCTAA CCGTGGACAAGAGCAGGTGG
CAGGAGGGGAATGTCTTCTCA TGCTCCGTGATGCATGAGGCT CTGCACAACCACTACACCXA
GAAGAGCCTCTCCCTGTCTCT GGGTAAATGA (SEQ ID NO: 81) IgG4
GCACCTGAGTTCCTGGGGGG APEFLGGSVFLFPPKPKD AJ001564 variant
ACCATCAGTCTTCCTGTTCCC TLMISKTPEVTCVVVDVS 3 CCCAAAACCCAAGGACACTC
QEDPEVQFNWYVDGVEV TCATGATCTCCCGGACCCCTG HNAKTKPREEQFNSTYRV
AGGTCACGTGCGTGGTGGTG VSVLTVLHQDWLNGKEY GACGTGAGCCAGGAAGACCC
KCKVSNKGLPSSIEKTISK CGAGGTCCAGTTCAACTGGTA AKGQPREPQVYTLPPSQE
CGTGGATGGCGTGGAGGTGCA EMTKNQVSLTCLVKGFYP TAATGCCAAGACAAAGCCGC
SDIAVEWESNGQPENNYK GGGAGGAGCAGTTCAACAGC TTPPVLDSDGSFFLYSKLT
ACGTACCGTGTGGTCAGCGTC VDKSRWQEGNVFSCSVM CTCACCGTCCTGCACCAGGAC
HEALHNHYTQKSLSLSLG TGGCTGAACGGCAAGGAGTA K* (SEQ ID NO: 82)
CAAGTGCAAGGTCTCCAACA AAGGCCTCCCGTCCTCCATCG AGAAAACCATCTCCAAAGCC
AAAGGGCAGCCCCGAGAGCC ACAGGTGTACACCCTGCCCCC ATCCCAGGAGGAGATGACCA
AGAACCAGGTCAGCCTGACC TGCCTGGTCAAAGGCTTCTAC CCCAGCGACATCGCCGTGGA
GTGGGAGAGCAATGGGCAGC CGGAGAACAACTACAAGACC ACGCCTCCCGTGCTGGACTCC
GACGGCTCCTTCTTCCTCTAC AGCAAGCTCACCGTGGACAA GAGCAGGTGGCAGGAGGGGA
ACGTCTTCTCATGCTCCGTGA TGCATGAGGCTCTGCACAACC ACTACACGCAGAAGAGCCTC
TCCCTGTCTCTGGGTAAATGA (SEQ ID NO: 83) Human Ig
ACTGTGGCTGCACCATCTGTC TVAAPSVFIFPPSDEQLKS X96754 http://ww None Ig
kappa TTCATCTTCCCGCCATCTGATG GTASVVCLLNNFYPREAK w.imgt.or light
(CK) AGCAGTTGAAATCTGGAACTG VQWKVDNALQSGNSQES g/IMGTre chain
CCTCTGTTGTGTGCCTGCTGA VTEQDSKDSTYSLSSTLTL perioire/i constant
ATAACTTCTATCCCAGAGAGG SKADYEKHKVYACEVTH ndex.php region
CCAAAGTACAGTGGAAGGTG QGLSSPVTKSFNRGEC* ?section =
GATAACGCCCTCCAATCGGGT (SEQ ID NO: 11) LocusGe AACTCCCAGGAGAGTGYCAC
nes & repe AGAGCAGGACAGCAAGGACA rtoire = ge
GCACCTACAGCCTCAGCAGC netable & s ACCCTGACGCTGAGCAAAGC pecies =
hu AGACTACGAGAAACACAAAG man & gro TCTACGCCTGCGAAGTCACCC up =
IGKC ATCAGGGCCTGAGCTCGCCCG TCACAAAGAGCTTCAACAGG GGAGAGTGTTAG (SEQ
ID NO: 13) Bovine Bovine IgG1 GCCTCCACCACAGCCCCGAA
ASTTAPKVYPLSSCCGDK X62916 hap://ww Symons D.B. et (Scienti- Ig
variant AGTCTACCCTCTGAGTTCTTG SSSTVTLGCLVSSYMPEPV w.imgt.or al. J.
fic heavy 1 CTGCGGGGACAAGTCCAGCT TVTWNSGALKSGVHTFPA g/IMGTre
Immunogenet., Name: chain CCACCGTGACCCTGGGCTGCC VLQSSGLYSLSSMVTVPG
pertoire/i 14, 273-283 Bos constant TGGTCTCCAGCTACATGCCCG
STSGQTFTCNVAHPASSTK ndex.php (1987). PMID: taurus) region
AGCCGGTGACCGTGACCTGG VDKAVDPTCKPSPCDCCP ?section = 3141517 (CH1-
AACTCGGGTGCCCTGAAGAG PPELPGGPSVFIFPPKPKDT LocusGe Symons D.B. et
CH3) CGGCGTGCACACCTTCCCGGC LTISGTPEVTCVVVDVGH nes & repe al.,
Mol. TGTCCTTCAGTCCTCCGGGCT DDPEVKFSWFVDDVEVNT rtoire = ge Immunol.,
26, GTACTCTCTCAGCAGCATGGT ATTKPREEQFNSTYRVVS netable & s
841-850 (1989). GACCGTGCCCGGCAGCACCT ALRIQHQDWTGGKEFKC pecies = bo
PMID: 2513487 CAGGACAGACCTTCACCTGC KVHNEGLPARIVRTISRTK vine &
gro Kacskovics I. and AACGTAGCCCACCCGGCCAG GPAREPQVYVLAPPQEEL up =
IGHC Butler J.E., Mot CAGCACCAAGGTGGACAAGG SKSTVSLTCMVTSFYPDYI
immunol., 33, CTGTTGATCCCACATGCAAAC AVEWQRNGQPESEDKYG 189-195
(1996). CATCACCCTGTGACTGTTGCC TTPPQLDADSSYFLYSKLR PMID: 8649440
CACCCCCTGAGCTCCCCGGAG VDRNSWQEGDTYTCVVM Rabbani H. et al.,
GACCCTCTGTCTTCATCTTCCC HEALHNHYTQKSTSKSAG Immunogenetics,
ACCGAAACCCAAGGACACCC K (SEQ ID NO: 84) 46, 326-331
TCACAATCTCGGGAACGCCCG (1997). PMID: AGGTCACGTGTGTGGTGGTG 9218535
GACGTGGGCCACGATGACCC Saini S.S. et al., CGAGGTGAAGTTCTCCTGGTT
Scand. J. CGTGGACGACGTGGAGGTAA Immunol. 65, 32-
ACACAGCCACGACGAAGCCG 8 (2007). PMID: AGAGAGGAGCAGTTCAACAG 17212764
CACCTACCGCGTGGTCAGCGC CCTGCGCATCCAGCACCAGGA CTGGACTGGAGGAAAGGAGT
TCAAGTGCAAGGTCCACAAC GAAGGCCTCCCGGCCCCCATC GTGAGGACCATCTCCAGGACC
AAAGGGCCGGCCCGGGAGCC GCAGGTGTATGTCCTGGCCCC ACCCCAGGAAGAGCTCAGCA
AAAGCACGGTCAGCCTCACC TGCATGGTCACCAGCTTCTAC CCAGACTACATCGCCGTGGAG
TGGCAGAGAAACGGGCAGCC TGAGTCGGAGGACAAGTACG GCACGACCCCGCCCCAGCTG
GACGCCGACAGCTCCTACTTC CTGTACAGCAAGCTCAGGGT GGACAGGAACAGCTGGCAGG
AAGGAGACACCTACACGTGT GTGGTGATGCACGAGGCCCTG CACAATCACTACACGCAGAA
GTCCACCTCTAAGTCTGCGGG TAAATGA (SEQ ID NO: 92) IgG1
GCCTCCACCACAGCCCCGAA ASTTAPKVYPLSSCCGDK X16701 variant
AGTCTACCCTCTGAGTTCTTG SSSTVTLGCLVSSYMPEPV (M25278) 2
CTGCGGGGACAAGTCCAGCT TVTWNSGALKSGVHTFPA CCACCGTGACCCTGGGCTGCC
VLQSSGLYSLSSMVTVPG TGGTCTCCAGCTACATGCCCG STSGQTFTCNVAHPASSTK
AGCCGGTGACCGTGACCTGG VDKAVDPTCKPSPCDCCP AACTCGGGTGCCCTGAAGAG
PPELPGGPSVFIFPPKPKDT CGGCGTGCACACCTTCCCGGC LTISGTPEVTCVVVDVGH
CGTCCTTCAGTCCTCCGGGCT DDPEVKFSWFVDDVEVNT GTACTCTCTCAGCAGCATGGT
ATTKPREEQFNSTYRVVS GACCGTGCCCGGCAGCACCT ALRIQHQDWTGGKEFKC
CAGGACAGACCTTCACCTGC KVHNEGLPAPIVRTISRTK AACGTAGCCCACCCGGCCAG
GPAREPQVYVLAPPQEEL CAGCACCAAGGTGGACAAGG SKSTVSLTCMVTSFYPDYI
CTGTTGATCCCACATGCAAAC AVEWQRNGQPESEDKYG CATCACCCTGTGACTGTTGCC
TTPPQLDADSSYFLYSKLR CACCCCCTGAGCTCCCCGGAG VDRNSWQEGDTYTCVVM
GACCCTCTGTCTTCATCTTCCC HEALHNHYTQKSTSKSAG ACCGAAACCCAAGGACACCC K*
(SEQ ID NO: 85) TCACAATCTCGGGAACGCCCG AGGTCACGTGTGTGGTGGTG
GACGTGGGCCACGATGACCC CGAGGTGAAGTTCTCCTGGTT CGTGGACGACGTGGAGGTAA
ACACAGCCACGACGAAGCCG AGAGAGGAGCAGTTCAACAG CACCTACCGCGTGGTCAGCGC
CCTGCGCATCCAGCACCAGGA CTGGACTGGAGGAAAGGAGT TCAAGTGCAAGGTCCACAAC
GAAGGCCTCCCGGCCCCCATC GTGAGGACCATCTCCAGGACC AAAGGGCCGGCCCGGGAGCC
GCAGGTGTATGTCCTGGCCCC ACCCCAGGAAGAGCTCAGCA AAAGCACGGTCAGCCATCACC
TGCATGGTCACCAGCTTCTAC CCAGACTACATCGCCGTGGAG TGGCAGAGAAACGGGCAGCC
TGAGTCGGAGGACAAGTACG GCACGACCCCGCCCCAGCTG GACGCCGACAGCTCCTACTTC
CTGTACAGCAAGCTCAGGGT GGACAGGAACAGCTGGCAGG AAGGAGACACCTACACGTGT
GTGGTGATGCACGAGGCCCTG CACAATCACTACACGCAGAA GTCCACCTCTAAGTCTGCGGG
TAAATGA (SEQ ID NO: 93) IgG1 GCCTCCACCACAGCCCCGAA
ASTTAPKVYPLSSCCGDK S82409 variant AGTCTACCCTCTGAGTTCTTG
SSSTVTLGCLVSSYMPEPV 3 CTGCGGGGACAAGTCCAGCT TVTWNSGALKSGVHTFPA
CCACCGTGACCCTGGGCTGCC VLQSSGLYSLSSMVTVPG TGGTCTCCAGCTACATGCCCG
STSGTQTFTCNVAHPASST AGCCGGTGACCGTGACCTGG KVDKAVDPRCKTTCDCCP
AACTCGGGTGCCCTGAAGAG PPELPGGPSVFIFPRKPKDT CGGCGTGCACACCTTCCCGGC
LTISGTPEVTCVVVDVGH CGTCCTTCAGTCCTCCGGGCT DDPEVKFSWFVDDVEVNT
CTACTCTCTCAGCAGCATGGT ATTKPREEQFNSTYRVVS GACCGTGCCCGGCAGCACCT
ALRIQHQDWTGGKEFKC CAGGAACCCAGACCTTCACCT KVHNEGLPARIVRTISRTK
GCAACGTAGCCCACCCGGCC GPAREPQVYVLAPPQEEL AGCAGCACCAAGGTGGACAA
SKSTVSLTCMVTSFYPDYI GGCTGTTGATCCCAGATGCAA AVEWQRNGQPESEDKYG
AACAACCTGTGACTGTTGCCC TIPPQLDADGSYFLYSRLR ACCGCCTGAGCTCCCTGGAG
VDRNSWQEGDTYTCVVM GACCCTGTCTTCATCTTCCC HEALHNHYTQKSTSKSAG
ACCGAAACCCAAGGACACCC K* (SEQ ID NO: 86) TCACAATCTCGGGAACGCCCG
AGGTCACGTGTGTGGTGGTG GACGTGGGCCACGATGACCC CGAGGTGAAGTTCTCCTGGTT
CGTGGACGACGTGGAGGTAA ACACAGCCACGACGAAGCCG AGAGAGGAGCAGTTCAACAG
CACCTACCGCGTGGTCAGCGC CCTGCGCATCCAGCACCAGGA CTGGACTGGAGGAAAGGAGT
TCAAGTGCAAGGTCCACAAC GAAGGCCTCCCAGCCCCCATC GTGAGGACCATCTCCAGGACC
AAAGGGCCGGCCCGGGAGCC GCAGGTGTATGTCCTGGCCCC ACCCCAGGAAGAGCTCAGCA
AAAGCACGGTCAGCCTCACC TGCATGGTCACCAGCTTCTAC CCAGACTACATCGCCGTGGAG
TGGCAGAGAAATGGGCAGCC TGAGTCAGAGGACAAGTACG GCACGACCCCTCCCCAGCTGG
ACGCCGACGGCTCCTACTTCC TGTACAGCAGGCTCAGGGTG GACAGGAACAGCTGGCAGGA
AGGAGACACCTACACGTGTG TGGTGATGCACGAGGCCCTGC ACAATCACTACACGCAGAAGT
CCACCTCTAAGTCTGCGGGTA AATGA (SEQ ID NO: 94) IgG2
GCCTCCACCACAGCCCCGAA ASTTAPKVYPLASSCGDTS S82407 variant
AGTCTACCCTCTGGCATCCAG SSTVTLGCLVSSYMPEPVT 1 CTGCGGAGACACATCCAGCTC
VTWNSGALKSGVHTFPAV CACCGTGACCCTGGGCTGCCT LQSSGLYSLSSMVTVPASS
GGTGTCCAGCTACATGCCCGA SGQTFTCNVAHPASSTKV
GCCGGTGACCGTGACCTGGA DKAVGVSIDCSKCHNQPC ACTCGGGTGCCCTGAAGAGC
VREPSVFIFPPKPKDTLMI GGCGTGCACACCTTCCCGGCT TGTPEVTCVVVNVGHDN
GTCCTTCAGTCCTCCGGGCTC PEVQFSWFVDDVEVHTAR TACTCTCTCAGCAGCATGGTG
SKPREEQFNSTYRVVSALP ACCGTGCCCGCCAGCAGCTC IQHQDWTGGKEFKCKVN
AGGACAGACCTTCACCTGCA NKGLSAPIVRIISRSKGPAR ACGTAGCCCACCCGGCCAGC
EPQVYVLDPPKEELSKSTL AGCACCAAGGTGGACAAGGC SVTCMVTGFYPEDVAVEW
TGTTGGGGTCTCCATTGACTG QRNRQTESEDKYRTTPPQ CTCCAAGTGTCATAACCAGCC
LDTDRSYFLYSKLRVDRN TTGCGTGAGGGAACCATCTGT SWQEGDAYTCVVMHEAL
CTTCATCTTCCCACCGAAACC HNHYMQKSTSKSAGK* CAAAGACACCCTGATGATCAC (SEQ
ID NO: 87) AGGAACGCCCGAGGTCACGT GTGTGGTGGTGAACGTGGGC
CACGATAACCCCGAGGTGCA GTTCTCCTGGTTCGTGGATGA CGTGGAGGTGCACACGGCCA
GGTCGAAGCCAAGAGAGGAG CAGTTCAACAGCACGTACCGC GTGGTCAGCGCCCTGCCCATC
CAGCACCAGGACTGGACTGG AGGAAAGGAGTTCAAGTGCA AGGTCAACAACAAAGGCCTC
TCGGCCCCCATCGTGAGGATC ATCTCCAGGAGCAAAGGGCC GGCCCGGGAGCCGCAGGTGT
ATGTCCTGGACCCACCCAAGG AAGAGCTCAGCAAAAGCACG CTCAGCGTCACCTGCATGGTC
ACCGGCTTCTACCCAGAAGAT GTAGCCGTGGAGTGGCAGAG AAACCGGCAGACTGAGTCGG
AGGACAAGTACCGCACGACC CCGCCCCAGCTGGACACCGA CCGCTCCTACTTCCTGTACAG
CAAGCTCAGGGTGGACAGGA ACAGCTGGCAGGAAGGAGAC GCCTACACGTGTGTGGTGATG
CACGAGGCCCTGCACAATCAC TACATGCAGAAGTCCACCTCT AAGTCTGCGGGTAAATGA (SEQ
ID NO: 95) IgG2 GCCTCCACCACAGCCCCGAA ASTTAPKVYPLSSCCGDK M36946
variant AGTCTACCCTCTGAGTTCTTG SSSTVTLGCLVSSYMPEPV (X06703) 2
CTGCGGGGACAAGTCCAGCT TVTWNSGALKSGVHTFPA CCACCGTGACCCTGGGCTGCC
VLQSSGLYSLSSMVTVPG TGGTGTCCAGCTACATGCCCG STSGQTFTCNVAHPASSTK
AGCCGGTGACCGTGACCTGG VDKAVGVSSDCSKPNNQ AACTCGGGTGCCCTGAAGAG
HCVREPSVFIFPPKPKDTL CGGCGTGCACACCTTCCCGGC MITGTPEVTCVVVNVGHD
CGTCCTTCAGTCCTCCGGGCT NPEVQFSWFVDDVEVHTA CTACTCTCTCAGCAGCATGGT
RTKPREEQFNSTYRVVSA GACCGTGCCCGGCAGCACCT LPIQHQDWTGGKEFKCKV
CAGGACAGACCTTCACCTGC NIKGLSASINRIIRSKGPA AACGTAGCCCACCCGGCCAG
REPQVYVLDPPKEELSKS CAGCACCAAGGTGGACAAGG TVSVTCMVIGFYPEDVDV
CTGTTGGGGTCTCCAGTGACT EWQRDRQTESEDKYRTTP GCTCCAAGCCTAATAACCAGC
PQLDADRSYFLYSKLRVD ATTGCGTGAGGGAACCATCTG RNSWQRGDTYTCVVMHE
TCTTCATCTTCCCACCGAAAC ALHNHYMQKSTSKSAGK CCAAAGACACCCTGATGATCA *
(SEQ ID NO: 88) CAGGAACGCCCGAGGTCACG TGTGTGGTGGTGAACGTGGG
CCACGATAACCCCGAGGTGCA GTTCTCCTGGTTCGTGGACGA CGTGGAGGTGCACACGGCCA
GGACGAAGCCGAGAGAGGAG CAGTTCAACAGCACGTACCGC GTGGTCAGCGCCCTGCCCATC
CAGCACCAGGACTGGACTGG AGGAAAGGAGTTCAAGTGCA AGGTCAACATCAAAGGCCTCT
CGGCCTCCATCGTGAGGATCA TCTCCAGGAGCAAAGGGCCG GCCCGGGAGCCGCAGGTGTAT
GTCCTGGACCCACCCAAGGA AGAGCTCAGCAAAAGCACGG TCAGCGTCACCTGCATGGTCA
TCGGCTTCTACCCAGAAGATG TAGACGTGGAGTGGCAGAGA GACCGGCAGACTGAGTCGGA
GGACAAGTACCGCACGACCC CGCCCCAGCTGGACGCCGAC CGCTCCTACTTCCTGTACAGC
AAGCTCAGGGTGGACAGGAA CAGCTGGCAGAGAGGAGACA CCTACACGTGTGTGGTGATGC
ACGAGGCCCTGCACAATCACT ACATGCAGAAGTCCACCTCTA AGTCTGCGGGTAAATGA (SEQ
ID NO: 96) IgG2 GCCTCCACCACAGCCCCGAA ASTTAPKVYPLSSCCGDK X16702
variant AGTCTACCCTCTGAGTTCTTG SSSGVTLGCLVSSYMPEPV (M25279) 3
CTGCGGGGACAAGTCCAGCT TVTWNSGALKSGVHTFPA CGGGGGTGACCCTGGGCTGC
VLQSSGLYSLSSMVTVPAS CTGGTCTCCAGCTACATGCCC SSGTQTFTCNVAHPASSTK
GAGCCGGTGACCGTGACCTG VDKAVGVSSDCSKPNNQ GAACTCGGGTGCCCTGAAGA
HCVREPSVFIFPPKPKDTL GCGGCGTGCACACCTTCCCGG MITGTPEVTCVVVNVGHD
CCGTCCTTCAGTCCTCCGGGC NPEVQFSWFVDDVEVHTA TCTACTCTCTCAGCAGCATGG
RTKPREEQFNSTYRVVSA TGACCGTGCCCGCCAGCAGCT LPIQHQDWTGGKEEKCKV
CAGGAACCCAGACCTTCACCT NIKGLSASIVRIISRSKGPA GCAACGTAGCCCACCCGGCC
REPQVYVLDPPKEELSKS AGCAGCACCAAGGTGGACAA TVSLTCMVIGFYPEDVDV
GGCTGTTGGGGTCTCCAGTGA EWQRDRQTESEDKYRTTP CTGCTCCAAGCCTAATAACCA
PQLDADRSYFLYSKLRVD GCATTGCGTGAGGGAACCATC RNSWQRGDTYTCVVMHE
TGTCTTCATCTTCCCACCGAA ALHNHYMQKSTSKSAGK ACCCAAAGACACCCTGATGAT *
(SEQ ID NO: 89) CACAGGAACGCCCGAGGTCA CGTGTGTGGTGGTGAACGTG
GGCCACGATAACCCCGAGGT GCAGTTCTCCTGGTTCGTGGA CGACGTGGAGGTGCACACGG
CCAGGACGAAGCCGAGAGAG GAGCAGTTCAACAGCACGTA CCGCGTGGTCAGCGCCCTGCC
CATCCAGCACCAGGACTGGA CTGGAGGAAAGGAGTTCAAG TGCAAGGTCAACATCAAAGG
CCTCTCGGCCTCCATCGTGAG GATCATCTCCAGGAGCAAAGG GCCGGCCCGGGAGCCGCAGG
TGTATGTCCTGGACCCACCCA AGGAAGAGCTCAGCAAAAGC ACGGTCAGCCTCACCTGCATG
GTCATCGGCTTCTACCCAGAA GATGTAGACGTGGAGTGGCA GAGAGACCGGCAGACTGAGT
CGGAGGACAAGTACCGCACG ACCCCGCCCCAGCTGGACGC CGACCGCTCCTACTTCCTGTA
CAGCAAGCTCAGGGTGGACA GGAACAGCTGGCAGAGAGGA GACACCTACACGTGTGTGGTG
ATGCACGAGGCCCTGCACAAT CACTACATGCAGAAGTCCACC TCTAAGTCTGCGGGTAAATGA
(SEQ ID NO: 97) IgG3 GCCTCCACCACAGCCCCGAA ASTTAPKVYPLASSCGDTS
U63638 variant AGTCTACCCTCTGGCATCCAG SSTVTLGCLVSSYMPEPVT 1
CTGCGGAGACACATCCAGCTC VTWNSGALKSGVHTFPAV CACCGTGACCCTGGGCTGCCT
RQSSGLYSLSSMVTVPASS GGTCTCCAGCTACATGCCCGA SETQTFTCNVAHPASSTKV
GCCGGTGACCCGTGACCTGGA DKAVTARRPVPTTPKTTIP ACTCGGGTGCCCTGAAGAGC
PGKPTTPKSEVEKTPCQCS GGCGTGCACACCTTCCCGGCC KCPEPLGGLSVFIFPPKPK
GTCCGGCAGTCCTCTGGGCTG DILTISGTPEVTCVVVDV TACTCTCTCAGCAGCATGGTG
GQDDPEVQFSWFVDDVE ACTGTGCCCGCCAGCAGCTCA VHTARTKPREEQFNSTYR
GAAACCCAGACCTTCACCTGC VVSALRIQHQDWLQGKEF AACGTAGCCCACCCGGCCAG
KCKVNNKGLPAPIVRTISR CAGCACCAAGGTGGACAAGG TKGQAREPQVYVLAPPRE
CTGTCACTGCAAGGCGTCCAG ELSKSTLSLTCLITGFYPEE TCCCGACGACGCCAAAGACA
IDVEWQRNGQPESEDKYH ACTATCCCTCCTGGAAAACCC TTAPQLDADGSYFLYSKL
ACAACCCCAAAGTCTGAAGT RVNKSSWQEGDHYTCAV TGAAAAGACACCCTGCCAGT
MHEALRNHYKEKSISRSP GTTCCAAATGCCCAGAACCTC GK* (SEQ ID NO: 90)
TGGGAGGACTGTCTGTCTTCA TCTTCCCACCGAAACCCAAGG ACACCCTCACAATCTCGGGAA
CGCCCGAGGTCACGTGTGTG GTGGTGGACGTGGGCCAGGA TGACCCCGAGGTGCAGTTCTC
CTGGTTCGTGGACGACGTGG AGGTGCACACGGCCAGGACG AAGCCGAGAGAGGAGCAGTT
CAACAGCACCTACCGCGTGGT CAGCGCCCTGCGCATCCAGCA CCAGGACTGGCTGCAGGGAA
AGGAGTTCAAGTGCAAGGTC AACAACAAAGGCCTCCCGGC CCCCATTGTGAGGACCATCTC
CAGGACCAAAGGGCAGGCCC GGGAGCCGCAGGTGTATGTCC TGGCCCCACCCCGGGAAGAG
CTCAGCAAAAGCACGCTCAG CCTCACCTGCCTGATCACCGG TTTCTACCCAGAAGAGATAGA
CGTGGAGTGGCAGAGAAATG GGCAGCCTGAGTCGGAGGAC AAGTACCACACGACCGCACC
CCAGCTGGAIGCTGACGGCTC CTACTTCCTGTACAGCAAGCT CAGGGTGAACAAGAGCAGCT
GGCAGGAAGGAGACCACTAC ACGTGTGCAGTGATGCACGA AGCTTTACGGAATCACTACAA
AGAGAAGTCCATCTCGAGGTC TCCGGGTAAATGA (SEQ ID NO: 98) IgG3
GCCTCCACCACAGCCCCGAA ASTTAPKVYPLASRCGDT 1163639 variant
AGTCTACCCTCTGGCATCCCG SSSTVTLGCLVSSYMPEPV 2 CTGCGGAGACACATCCAGCTC
TVTWNSGALKSGVHTFPA CACCGTGACCCTGGGCTGCCT VLQSSGLYSLSSMVTVPAS
GGTCTCCAGCTACATGCCCGA TSETQTFTCNVAHPASSTK GCCGGTGACCGTGACCTGGA
VDKAVTARRPVPTTPKTTI ACTCGGGTGCCCTGAAGAGT PPGKPTTQESEVEKTPCQC
GGCGTGCACACCTTCCCGGCC SKCPEPLGGLSVFIFPPKP GTCCTTCAGTCCTCCGGGCTG
KDTLTISGTPEVTCVVVD TACTCTCTCAGCAGCATGGTG VGQDDPEVQFSWFVDDV
ACCGTGCCCGCCAGCACCTCA EVHTARTKPREEQFNSTY GAAACCCAGACCTTCACCTGC
RVVSALRIQHQDWLQGKE AACGTAGCCCACCCGGCCAG FKCKVNNKGLPAPIVRTIS
CAGCACCAAGGTGGACAAGG RTKGQAREPQVYVLAPPR CTGTCCTGCAAGGCGTCCAG
EELSKSTLSLTCLITGFYPE TCCCGACGACGCCAAAGACA EIDVEWQRNGQPESEDKY
ACCATCCCTCCTGGAAAACCC HTFAPQLDADGSYFLYSR ACAACCCAGGAGTCTGAAGT
LRVNKSSWQEGDHYTCA TGAAAAGACACCCTGCCAGT VMHEALRNHYKEKSISRS
GTTCCAAATGCCCAGAACCTC PGK* (SEQ ID NO: 91) TGGGAGGACTGTCTGTCTTCA
TCTTCCCACCGAAACCCAAGG ACACCCTCACAATCTCGGGAA CGCCCGAGGTCACGTGTGTG
GTGGTGGACGTGGGCCAGGA TGACCCCGAGGTGCAGTTCTC CTGGTTCGTGGACGACGTGG
AGGTGCACACGGCCAGGACG AAGCCGAGAGAGGAGCAGTT CAACAGCACCTACCGCGTGGT
CAGCGCCCTGCGCATCCAGCA CCAGGACTGGCTGCAGGGAA AGGAGTTCAAGTGCAAGGTC
AACAACAAAGGCCTCCCGGC CCCCATTGTGAGGACCATCTC CAGGACCAAAGGGCAGGCCC
GGGAGCCCGCAGGTGTATGTCC TGGCCCCACCCCGGGAAGAG CTCAGCAAAAGCACGCTCAG
CCTCACCTGCCTGATCACCGG TTTCTACCCAGAAGAGATAGA CGTGGAGTGGCAGAGAAATG
GGCAGCCTGAGTCGGAGGAC AAGTACCACACGACCGCACC CCAGCTGGATGCTGACGGCTC
CTACTTCCTGTACAGCAGGCT CAGGGTGAACAAGAGCAGCT GGCAGGAAGGAGACCACTAC
ACGTGTGCAGTGATGCATGAA GCTTTACGGAATCACTACAAA
GAGAAGTCCATCTCGAGGTCT
CCGGGTAAATGA (SEQ ID NO: 99) Bovine, Ig CAGCCCAAGTCCCCACCCTCG
QPKSPPSVTLFPPSTEELN X62917 Not Chen L. et al., Ig light lambda
GTCACCCTGTTCCCGCCCTCC GNKATLVCLISDFYPGSVT registered Vet. Immunol.
chain ACGGAGGAGCTCAACGGCAA VVWKADGSTITRNVETTR Immunopathol,
constant CAAGGCCACCCTGGTGTGTCT ASKQSNSKYAASSYLSLTS 124, 284-294
region CATCAGCGACTTCTACCCGGG SDWKSKGSYSCEVTHEGS (2008). PMID: (CL)
TAGCGTGACCGTGGTCTGGAA TVTKTVKPSECS* (SEQ ID 18538861
GGCAGACGGCAGCACCATCA NO: 100) CCCGCAACGTGGAGACCACC
CGGGCCTCCAAACAGAGCAA CAGCAAGTACGCGGCCAGCA GCTACCTGAGCCTGACGAGC
AGCGACTGGAAATCGAAAGG CAGTTACAGCTGCGAGGTCAC GCACGAGGGGAGCACCGTGA
CGAAGACAGTGAAGCCCTCA GAGTGTTCTTAG (SEQ ID NO: 101)
[0105] The amino acid sequences as shown in SEQ ID NOS: 4, 3, 42,
44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76,
78, 12, 80, 82, 84-91, 100, 102 and 11 may have deletion(s),
substitution(s) or addition(s) of one or several (e.g., up to five,
about 10 at the most) amino acids. Even when such mutations have
been introduced, the resulting amino acid sequences are capable of
having the function as the constant region of Ig heavy chain or
light chain.
[0106] Although the constant region of wild-type human IgG1 has
ADCC activity and CDC activity, it is known that these activities
can be reduced by introducing amino acid substitutions and
deletions into specific sites. In the case of animals other than
human where the constant region of an immunoglobulin equivalent to
human IgG4 has not been identified, mutations may be introduced
into the relevant region of an immunoglobulin equivalent to human
IgG1 so that the resultant constant region with reduced ADCC
activity and CDC activity can be used.
[0107] The present invention provides an artificial genetic DNA
comprising (a') a DNA encoding a light chain comprising a light
chain variable region (VL) containing CDR1 having the amino acid
sequence of QSLLYSENQKDY (SEQ ID NO: 37), CDR2 having the amino
acid sequence of WAT and CDR3 having the amino acid sequence of
GQYLVYPFT (SEQ ID NO: 38) and the light chain constant region (CL)
of an antibody of an animal other than rat and (b') a DNA encoding
a heavy chain comprising a heavy chain variable region (VH)
containing CDR1 having the amino acid sequence of GYTFTSNF (SEQ ID
NO: 39), CDR2 having the amino acid sequence of IYPEYGNT (SEQ ID
NO: 40) and CDR3 having the amino acid sequence of ASEEAVISLVY (SEQ
ID NO: 41) and the heavy chain constant region (CH) of an antibody
of an animal other than rat. The present invention also provides a
DNA encoding a light chain comprising a VL containing CDR1 having
the amino acid sequence of QSLLYSENQKDY (SEQ ID NO: 37), CDR2
having the amino acid sequence of WAT and CDR3 having the amino
acid sequence of GQYLVYPFT (SEQ ID NO: 38) and the CL of an
antibody of an animal other than rat (i.e., the DNA of (a') above).
Further, the present invention also provides a DNA encoding a heavy
chain comprising a VH containing CDR1 having GYTFTSNF (SEQ ID NO:
39), CDR2 having the amino acid sequence of IYPEYGNT (SEQ ID NO:
40) and CDR3 having the amino acid sequence of ASEEAVISLVY (SEQ ID
NO: 41) and the CH of an antibody of an animal other than rat
(i.e., the DNA of (b') above).
[0108] For (a) a light chain comprising a light chain variable
region containing CDR1 having the amino acid sequence of
QSLLYSENQKDY (SEQ ID NO: 37), CDR2 having the amino acid sequence
of WAT and CDR3 having the amino acid sequence of GQYLVYPFT (SEQ ID
NO: 38) and the light chain constant region of an antibody of an
animal other than rat; and (b) a heavy chain comprising a heavy
chain variable region containing CDR1 having the amino acid
sequence of GYTFTSNF (SEQ ID NO: 39), CDR2 having the amino acid
sequence of IYPEYGNT (SEQ ID NO: 40) and CDR3 having the amino acid
sequence of ASEEAVISLVY (SEQ ID NO: 41) and the heavy chain
constant region of an antibody of an animal other than rat,
reference should be had to the foregoing description. The DNA of
(a') is a DNA (gene) encoding the light chain of (a); and the DNA
of (b') is a DNA (gene) encoding the heavy chain of (b). An
artificial genetic DNA comprising the DNA of (a') and the DNA of
('b) may be synthesized on commercial synthesizer. Restriction
enzyme recognition sites, KOZAK sequences, poly-A addition signal
sequences, promoter sequences, intron sequences or the like may be
added to the artificial genetic DNA.
[0109] The present invention also provides a vector comprising the
above-mentioned artificial genetic DNA.
[0110] As the vector, Escherichia coli-derived plasmids (e.g.,
pBR322, pBR325, pUC12 or pUC13): Bacillus subtilis-derived plasmids
(e.g., pUB110, pTP5 or pC194), yeast-derived plasmids (e.g., pSH19
or pSH15); bacteriophages such as .lamda. phage; animal viruses
such as retrovirus or vaccinia virus, or insect pathogen viruses
such as baculovirus may be used. In the Examples described later,
pDC6 (Japanese Patent No. 5704753, U.S. Pat. No. 9,096,878, EU
Patent 2385115, Hong Kong (China) patent HK1163739 and Australia
Patent 2009331326) was used.
[0111] The vector may also comprise promoters, enhancers, splicing
signals, poly-A addition signals, intron sequences, selection
markers, SV40 replication origins, and so forth.
[0112] The present invention also provides a host cell transformed
by the above vector. It is possible to prepare the anti-PD-L1
antibody of the invention by culturing the host cell and collecting
the antibody of interest from the resultant culture. Therefore, the
present invention also provides a method of preparing an antibody,
comprising culturing the above-described host cell and collecting
the anti-PD-L1 antibody of the invention from the culture. In the
method of the present invention for preparing an antibody, a vector
incorporating an artificial genetic DNA comprising a DNA encoding
the light chain and a DNA encoding the heavy chain may be
transfected into a host cell. Alternatively, a vector incorporating
a DNA encoding the light chain and a vector incorporating a DNA
encoding the heavy chain may be co-transfected into a host
cell.
[0113] Examples of the host cell include, but are not limited to,
bacterial cells (such as Escherichia bacteria, Bacillus bacteria or
Bacillus subtilis), fungal cells (such as yeast or Aspergillus),
insect cells (such as S2 cells or Sf cells), animal cells (such as
CHO cells, COS cells, HeLa cells, C127 cells, 3T3 cells, BHK cells
or HEK 293 cells) and plant cells. Among these, CHO-DG44 cell
(CHO-DG44(dfhr.sup.-/.sup.-)) which is a dihydrofolate reductase
deficient cell is preferable.
[0114] Introduction of a recombinant vector into a host cell may be
performed by the methods disclosed in Molecular Cloning 2nd
Edition, J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989
(e.g., the calcium phosphate method, the DEAE-dextran method,
transfection, microinjection, lipofection, electroporation,
transduction, scrape loading, the shotgun method, etc.) or by
infection.
[0115] The resultant transformant may be cultured in a medium,
followed by collection of the anti-PD-L1 antibody of the present
invention from the culture. When the antibody is secreted into the
medium, the medium may be recovered, followed by isolation and
purification of the antibody from the medium. When the antibody is
produced within the transformed cells, the cells may be lysed,
followed by isolation and purification of the antibody from the
cell lysate.
[0116] Examples of the medium include, but are not limited to,
OptiCHO medium, Dynamis medium, CD CHO medium, ActiCHO medium,
FortiCHO medium, Ex-Cell CD CHO medium, BalanCD CHO medium. ProCHO
5 medium and Cellvento CHO-100 medium.
[0117] The pH of the medium varies depending on the cell to be
cultured. Generally, a pH range from 6.8 to 7.6 is used; mostly, a
pH range from 7.0 to 7.4 is appropriate.
[0118] When the cell to be cultured is CHO cells, culture may be
performed by methods known to those skilled in the art. For
example, it is usually possible to perform culturing in a gas-phase
atmosphere having a CO.sub.2 concentration of 0-40%, preferably
2-10%, at 30-39.degree. C., preferably around 37.degree. C.
[0119] The appropriate period of culture is usually from one day to
three months, preferably from one day to three weeks.
[0120] Isolation and purification of the antibody may be performed
by known methods. Known isolation/purification methods which may be
used in the present invention include, but are not limited to,
methods using difference in solubility (such as salting-out or
solvent precipitation); methods using difference in molecular
weight (such as dialysis, ultrafiltration, gel filtration or
SDS-polyacrylamide gel electrophoresis); methods using difference
in electric charge (such as ion exchange chromatography); methods
using specific affinity (such as affinity chromatography); methods
using difference in hydrophobicity (such as reversed phase high
performance liquid chromatography); and methods using difference in
isoelectric point (such as isoelectric focusing).
[0121] The anti-PD-L1 antibody of the present invention may be used
as an antibody drug for animals or human. Therefore, the present
invention provides a pharmaceutical composition comprising the
above-described anti-PD-L1 antibody as an active ingredient.
[0122] The pharmaceutical composition of the present invention may
be used for prevention and/or treatment of cancers and/or
infections. Examples of cancers and/or infections include, but are
not limited to, neoplastic diseases (e.g., malignant melanoma, lung
cancer, gastric cancer, renal cancer, breast cancer, bladder
cancer, esophageal cancer, ovarian cancer and the like), leukemia,
Johne's disease, anaplasmosis, bacterial mastitis, mycotic
mastitis, mycoplasma infections (such as mycoplasma mastitis,
mycoplasma pneumonia or the like), tuberculosis, Theileria
orientalis infection, cryptosporidiosis, coccidiosis,
trypanosomiasis and leishmaniasis.
[0123] The anti-PD-L1 antibody of the present invention may be
dissolved in buffers such as PBS, physiological saline or sterile
water, optionally filter-sterilized with a filter or the like, and
then administered to animal subjects (including human) by
injection. To the solution of this antibody, additives (such as
coloring agents, emulsifiers, suspending agents, surfactants,
solubilizers, stabilizers, preservatives, antioxidants, buffers,
isotonizing agents, pH adjusters and the like) may be added. As
routes of administration, intravenous, intramuscular,
intraperitoneal, subcutaneous or intradermal administration and the
like may be selected. Transnasal or oral administration may also be
used.
[0124] The dose and the number of times and frequency of
administration of the anti-PD-L1 antibody of the present invention
may vary depending on the symptoms, age and body weight of the
animal subject, the method of administration, the dosage form and
so on. For example, 0.1-100 mg/kg body weight, preferably 1-10
mg/kg body weight, per adult animal may usually be administered at
least once, at such a frequency that enables confirmation of the
desired effect.
[0125] While the pharmaceutical composition of the present
invention may be used alone, it may be used in combination with
surgical operations, radiation therapies, other immunotherapies
such as cancer vaccine, or molecular target drugs. Synergistic
effect can be expected from such combinations.
EXAMPLES
[0126] Hereinbelow, the present invention will be described in more
detail with reference to the following Examples. However, the
present invention is not limited to these Examples.
[Example 1] Rat-Canine Chimeric Anti-PD-L1 Antibody
1. Introduction
[0127] Programmed cell death 1 (PD-1), an immunoinhibitory
receptor, and its ligand programmed cell death ligand 1 (PD-L1) are
molecules identified by Prof. Tasuku Honjo et al., Kyoto
University, as factors which inhibit excessive immune response and
are deeply involved in immunotolerance. Recently, it has been
elucidated that these molecules are also involved in
immunosuppression in tumors. In the subject Example, for the
purpose of establishing a novel therapy for canine neoplastic
diseases, a chimeric antibody gene was prepared in which a variable
region gene of a rat anti-bovine PD-L1 monoclonal antibody (4G12)
capable of inhibiting the binding of canine PD-1 to PD-L1 was
linked to a constant region gene of a canine immunoglobulin (IgG4).
The resultant chimeric antibody gene was introduced into Chinese
hamster ovary cells (CHO cells), which were cultured to produce a
rat-canine chimeric anti-PD-L1 antibody c4G12. The effect of this
chimeric antibody was confirmed in vitro and in vivo.
2. Materials and Methods
2.1 Bovine PD-L1 Monoclonal Antibody Producing Cells
[0128] The nucleotide sequence of bovine PD-L1 was identified
(Ikebuchi R, Konnai S. Shirai T, Sunden Y, Murata S, Onuma M,
Ohashi K. Vet Res. 2011 Sep. 26; 42:103). Based on the sequence
information, a recombinant bovine PD-L1 was prepared. Rat was
immunized with this recombinant protein to prepare a rat
anti-bovine PD-L1 antibody (Ikebuchi R, Konnai S, Okagawa T,
Yokoyama K, Nakajima C, Suzuki Y, Murata S, Ohashi K. Immunology.
2014 August; 142(4):551-61; Clone 4G12 which would later serve as
the variable region of the canine chimeric antibody of interest is
described in this article.)
2.2 Identification of Full-Length Canine PD-1 and PD-L1 Genes
[0129] To determine the full lengths of canine PD-1 and PD-L1
cDNAs, PCR primers were first designed based on the putative
nucleotide sequences of canine PD-1 and PD-L1 already registered at
The National Center for Biotechnology Information (NCBI) (GenBank
accession number: XM_543338 and XM_541302). Briefly, primers to
amplify the inner sequence of the open reading frame (ORF) of each
gene were designed (cPD-1 inner F and R, cPD-L1 inner F and R), and
PCR was performed. For the amplified products, nucleotide sequences
were determined with a capillary sequencer according to
conventional methods. Further, to determine the nucleotide
sequences of full-length PD-1 and PD-L1 cDNA, primers (cPD-1 5' GSP
and 3' GSP; cPD-L1 5' GSP and 3'GSP) were designed based on the
canine PD-1 and PD-L1 cDNA sequences determined above. 5'-RACE and
3'-RACE were then performed using the 5'-RACE system for rapid
amplification of cDNA ends and 3'-RACE system for rapid
amplification of cDNA ends (Invitrogen), respectively. The
resultant gene fragments of interest were sequenced as described
(Maekawa N, Konnai S, Ikebuchi R, Okagawa T, Adachi M, Takagi S,
Kagawa Y, Nakajima C, Suzuki Y, Murata S, Ohashi K. PLoS One. 2014
Jun. 10; 9(6):e98415).
TABLE-US-00002 Primer (cPD-1 inner F): (SEQ ID NO: 21)
AGGATGGCTCCTAGACTCCC Primer (cPD-1 inner R): (SEQ ID NO: 22)
AGACGATGGTGGCATACTCG Primer (cPD-L1 inner F): (SEQ ID NO: 23)
ATGAGAATGTTTAGTGTCTT Primer (cPD-L1 inner R): (SEQ ID NO: 24)
TTATGTCTCTTCAAATTGTATATC Primer (cPD-1 5'GSP): (SEQ ID NO: 25)
GTFGATCTGTGTGTTG Primer (cPD-1 3'GSP): (SEQ ID NO: 26)
CGGGACTTCCACATGAGCAT Primer (cPD-L3 5'GSP): (SEQ ID NO: 27)
TTTTAGACAGAAAGTGA Primer (cPD-L1 3'GSP): (SEQ ID NO: 28)
GACCAGCTCTTCTTGGGGAA
2.3 Construction of Canine PD-1 and PD-L1 Expressing COS-7
Cells
[0130] For preparing canine PD-1-EGFP and PD-L1-EGFP expression
plasmids, PCR was performed using a synthesized beagle PBMC-derived
cDNA as a template and primers designed by adding XhoI and BamHI
recognition sites (PD-1) and BglII and EcoRI recognition sites
(PD-L1) on the 5' side (cPD-1-EGFP F and R; cPD-L1-EGFP F and R).
The resultant PCR products were digested with XhoI (Takara) and
BamHI (Takara) (PD-1) and with BglII (New England Biolabs) and
EcoRI (Takara) (PD-L1), and then purified with FastGene Gel/PCR
Extraction Kit (NIPPON Genetics), followed by cloning into pEGFP-N2
vector (Clontech) treated with restriction enzymes in the same
manner. The resultant expression plasmids of interest were
extracted with QIAGEN Plasmid Midi kit (Qiagen) and stored at
-30.degree. C. until use in experiments. Hereinafter, the thus
prepared expression plasmids are designated as pEGFP-N2-cPD-1 and
pEGFP-N2-cPD-L1.
TABLE-US-00003 Primer (cPD-1-EGFP F): (SEQ ID NO: 29)
CCGCTCGAGATGGGGAGCCGGCGGGGGCC Primer (cPD-1-EGFP R): (SEQ ID NO:
30) CGCGGATCCTGAGGGGCCACAGGCCGGGTC Primer (cPD-L1-EGFP F): (SEQ ID
NO: 31) GAAGATCTATGAGAATGTTTAGTGTC Primer (cPD-L1-EGFP R): (SEQ ID
NO: 32) GGAATTCTGTCTCTTCAAATTGTATATC
[0131] COS-7 cells were subcultured at a density of
5.times.10.sup.4 cells/cm.sup.2 in 6-well plates, and then cultured
overnight in RPMI 1640 medium containing 10% inactivated fetal
bovine serum and 0.01% L-glutamine at 37.degree. C. in the presence
of 5% CO.sub.2. The pEGFP-N2-cPD-1, pEGFP-N2-cPD-L1 or pEGFP-N2
(negative control) was introduced into COS-7 cells at 0.4
.mu.g/cm.sup.2 using Lipofectamine 2000 (Invitrogen). The cells
were cultured for 48 hours (cPD-1-EGFP expressing cell and
cPD-L1-EGFP expressing cell). In order to confirm the expression of
canine PD-1 and PD-L1 in the thus prepared expressing cells,
intracellular localization of enhanced green fluorescent protein
(EGFP) was visualized with an inverted confocal laser microscope
LSM700 (ZEISS) (Maekawa N, Konnai S, Ikebuchi R, Okagawa T, Adachi
M, Takagi S, Kagawa Y, Nakajima C, Suzuki Y, Murata S. Ohashi K.
PLoS One. 2014 Jun. 10; 9(6):e98415).
2.4 Construction of Recombinant Canine PD-1, PD-L1 and CD80
[0132] In order to amplify the extracellular regions of canine
PD-1, PD-L1 and CD80 estimated from their putative amino acid
sequences, primers were designed. Briefly, primers having an NheI
or EcoRV recognition sequence (PD-1 and PD-L1) added on the 5' side
(cPD-1-Ig F and R; cPD-L1-Ig F and R) or having an EcoRV or KpnI
(CD80) recognition sequence added on the 5' side (cCD80-Ig F and R)
were designed. PCR was performed using a synthesized beagle
PBMC-derived cDNA as a template. The PCR products were digested
with NheI (Takara) and EcoRV (Takara) or with EcoRV (Takara) and
KpnI (New England Biolabs) and purified with FastGene Gel/PCR
Extraction Kit (NIPPON Genetics). The thus purified DNAs were
individually cloned into pCXN2.1-Rabbit IgG Fc vector (Niwa et al.,
1991; Zettimeissl et al., 1990; kindly provided by Dr. T. Yokomizo,
Juntendo University Graduate School of Medicine, and modified in
the inventors' laboratory) treated with restriction enzymes in the
same manner. The expression plasmids were purified with QIAGEN
Plasmid Midi kit (Qiagen) and stored at -30.degree. C. until use in
experiments. Hereinafter, the thus prepared expression plasmids are
designated as pCXN2.1-cPD-1-Ig, pCXN2.1-cPD-L1-Ig and
pCXN2.1-cCD80-Ig, respectively.
TABLE-US-00004 Primer (cPD-1-Ig F): (SEQ ID NO: 33)
CCGCTCGAGATGGGGAGCCGGCGGGGGCC Primer (cPD-1-Ig R): (SEQ ID NO: 34)
CGCGGATATCCAGCCCCTGCAACTGGCCGC Primer (cPD-L1-Ig F): (SEQ ID NO:
35) CGCGGCTAGCATGAGAATGTTTAGTGTCTT Primer (c-PD-L1-Ig R): (SEQ ID
NO: 36) CGCGGATATCAGTCCTCTCACTTGCTGGAA Primer (cCD80-Ig F): (SEQ ID
NO: 129) CGCGGATATCATGGATTACACAGCGAAGTG Primer (cCD80-Ig R): (SEQ
ID NO: 130) CGGGGTACCCCAGAGCTGTTGCTGGTTAT
[0133] These expression vectors were individually transfected into
Expi293F cells (Life Technologies) to obtain a culture supernatant
containing a recombinant Ig fusion protein. The recombinant protein
produced was purified from the supernatant with Ab Capcher Extra
(Protein A mutant; ProteNova). After buffer exchange with
phosphate-buffered physiological saline (PBS; pH 7.4) using
PD-MidiTrap G-25 (GE Healthcare), each recombinant protein was
stored at -30.degree. C. until use in experiments (cPD-1-Ig,
cPD-L1-Ig and cCD80-Ig). The concentration of each protein was
measured with Pierce BCA Protein Assay Kit (Thermo Fisher
Scientific) before use in subsequent experiments.
2.5 Identification of Rat Anti-Bovine PD-L1 Monoclonal Antibody
Showing Cross-Reactivity with Canine PD-L1
[0134] In order to identify rat anti-bovine PD-L1 monoclonal
antibody showing cross-reactivity with canine PD-L1, flow cytometry
was performed using the anti-bovine PD-L1 antibody prepared in 2.1
above. The anti-bovine PD-L1 antibody (10 .mu.g/ml) was reacted
with 2.times.10.sup.5-1.times.10.sup.6 cells at room temperature
for 30 min. After washing, the anti-bovine PD-L1 antibody was
detected with allophycocyanine-labeled anti-rat Ig goat antibody
(Beckman Coulter). FACS Verse (Becton, Dickinson and Company) was
used for analysis. As negative controls, rat IgG2a (.kappa.)
isotype control (BD Biosciences), rat IgG1 (.kappa.) isotype
control (BD Biosciences) and rat IgM (.kappa.) isotype control (BD
Biosciences) were used. For every washing operation and dilution of
antibodies, 10% inactivated goat serum-supplemented PBS was used
(Maekawa N, Konnai S, Ikebuchi R, Okagawa T, Adachi M, Takagi S,
Kagawa Y, Nakajima C, Suzuki Y, Murata S, Ohashi K. PLoS One. 2014
Jun. 10; 9(6):e98415 which is an article describing the use of
three bovine PD-L1 monoclonal antibodies: 4G12 (Rat IgG2a
(.kappa.)), 5A2 (Rat IgG1 (.kappa.)) and 6G7 (Rat IgM
(.kappa.)).
2.6 Selection Test of Variable Region for Establishment of
Rat-Canine Chimeric Anti-PD-L1 Antibody
[0135] Out of 10 clones of rat anti-bovine PD-L1 monoclonal
antibody which showed cross-reactivity with canine PD-L1, 4G12 (Rat
IgG2a (.kappa.)), 5A2 (Rat IgG1 (.kappa.)) and 6G7 (Rat IgM
(.kappa.)) were selected and check was made to see whether these
antibodies would inhibit canine PD-1/PD-L1 binding. Briefly, canine
PD-1-Ig (prepared in 2.4 above) was immobilized on flat bottomed
96-well plates and blocked with 1% BSA and 0.05% Tween
20-containing PBS. Canine PD-L1-Ig (prepared in 2.4 above) was
biotinylated using Lightning-Link Biotin Conjugation Kit (Innova
Bioscience) and reacted with various concentrations (0, 2.5, 5 and
10 .mu.g/ml) of rat anti-bovine PD-L1 antibodies 4G12, 5A2 and 6G7
at 37.degree. C. for 30 min, followed by addition to the 96-well
plates. The binding of cPD-L1-Ig to cPD-1-Ig was measured by color
reaction using Neutravidin-HRP (Thermo Fisher Scientific) and TMB
one component substrate (Bethyl Laboratories). As a result, rat
anti-bovine PD-L1 monoclonal antibodies 4G12 and 607 showed a good
inhibitory activity against canine PD-1/PD-L1 binding, whereas 5A2
showed no binding inhibitory activity (FIG. 1).
2.7 Preparation of Rat-Canine Chimeric Anti-PD-L1 Antibody
Expressing Vector (FIG. 2)
[0136] Using rat anti-bovine PD-L1 monoclonal antibodies 4G12 and
6G7 which showed a good inhibitory activity against canine
PD-1/PD-L1 binding (FIG. 1) as the variable region, two types of
rat-canine chimeric anti-PD-L1 antibodies were established.
[0137] Briefly, heavy chain and light chain variable region genes
were identified from hybridomas producing rat anti-bovine PD-L1
monoclonal antibodies 4G12 and 6G7. Further, the heavy chain and
light chain variable region genes of the above rat antibodies were
linked to the constant region of heavy chain IgG4 and the constant
region of light chain Lambda of a known canine antibody,
respectively, to prepare nucleotide sequences, followed by codon
optimization (SEQ ID NOS: 9 and 10 (amino acid sequences), SEQ ID
NOS: 19 and 20 (nucleotide sequences after codon optimization).
Then, synthesis of genes was performed so that NotI restriction
enzyme recognition site, KOZAK sequence, chimeric antibody's light
chain sequence, poly-A addition signal sequence (PABGH), promoter
sequence (PCMV). SacI restriction enzyme recognition site, intron
sequence (INRBG), KOZAK sequence, chimeric antibody's heavy chain
sequence and XbaI restriction enzyme recognition site would be
located in this order. The synthesized gene strands were
individually incorporated into the cloning site (NotI and XbaI
restriction enzyme recognition sequences downstream of PCMV and
between INRBG and PABGH) of expression vector pDC6 (kindly provided
by Prof. S. Suzuki. Hokkaido University Research Center for
Zoonosis Control) using restriction enzyme recognition sequences so
that the above-listed sequences would be located in the
above-mentioned order (FIG. 2). Thus, rat-canine chimeric
anti-PD-L1 antibody expressing vectors were constructed. Each of
the expression vectors was transfected into Expi293F cells (Life
Technologies) to obtain a culture supernatant containing a chimeric
antibody. The chimeric antibody was purified from the supernatant
with Ab Capcher Extra (Protein A mutant; ProteNova) and further
purified by gel filtration chromatography. SDS-PAGE was performed
under non-reducing conditions using 10% acrylamide gel. Bands were
stained with Quick-CBB kit (Wako Pure Chemical) and decolorized in
distilled water. Although contaminant proteins were observed after
protein A purification alone, a highly purified antibody could be
obtained by performing gel filtration chromatography (FIG. 3). It
was confirmed by flow cytometry that the resultant purified
antibodies specifically bound to canine PD-L1 expressing cells
(data not shown). When the inhibitory activity of the two chimeric
antibodies against canine PD-1/PD-L1 binding was examined by the
method described in 2.6 above, rat-canine chimeric anti-PD-L1
antibody c4G12 showed a binding inhibitory activity similar to that
of its original rat anti-bovine PD-L1 monoclonal antibody 4G12,
whereas binding inhibition capacity was clearly attenuated in
rat-canine chimeric anti-PD-L1 antibody c6G7 (FIG. 4) Therefore,
rat-canine chimeric anti-PD-L1 antibody c4G12 was selected as a
therapeutic antibody, which incorporated the variable region
sequences of rat anti-bovine PD-L1 monoclonal antibody 4G12 (SEQ ID
NOS: 2 and 1 (amino acid sequences) and SEQ ID NOS: 16 and 15
(nucleotide sequences after codon optimization)). The amino acid
sequence and the nucleotide sequence (after codon optimization) of
the light chain of c4G12 are shown in SEQ ID NOS: 9 and 19, and the
amino acid sequence and the nucleotide sequence (after codon
optimization) of the heavy chain of c4G12 are shown in SEQ ID NOS:
10 and 20.
2.8 Expression of Rat-Canine Chimeric Anti-PD-L1 Antibody c4G12
[0138] Rat-canine chimeric anti-PD-L1 antibody c4G12 expressing
vector pDC6 as used in 2.7 above was transfected into CHO-DG44
cells (CHO-DG44(dfhr.sup.-/.sup.-)) which were dihydrofolate
reductase deficient cells and high expression clones were selected
by dot blotting. Further, gene amplification treatment was
performed by adding load on cells in a medium containing 60 nM
methotrexate (Mtx). Cells stably expressing rat-canine chimeric
anti-PD-L1 antibody c4G12 (clone name: 4.3F1) after gene
amplification were transferred to Mtx-free Opti-CHO medium and
cultured under shaking for 14 days (125 rpm, 37.degree. C., 5%
CO.sub.2). Cell survival rate was calculated by trypan blue
staining (FIG. 5). Chimeric antibody production in the culture
supernatant was measured by ELISA (FIG. 5). The culture supernatant
at day 14 was centrifuged at 10,000 g for 10 min to remove cells,
then passed through a 0.22 .mu.m filter before the process
proceeded to purification steps for the antibody.
[0139] It should be noted that by exchanging the medium with
Dynamis medium and doing appropriate feeding, antibody production
was improved about two-fold compared to the conventional production
(data not shown).
2.9 Purification of Rat-Canine Chimeric Anti-PD-L1 Antibody
c4G12
[0140] The culture supernatant provided as described above was
purified with Ab Capcher Extra (ProteNova). An open column method
was used for binding to resin; PBS pH 7.4 was used as equilibration
buffer and wash buffer. As elution buffer, IgG Elution Buffer
(Thermo Scientific) was used. As neutralization buffer, 1 M Tris
was used. The purified antibody was concentrated and
buffer-exchanged with PBS by ultrafiltration using Amicon Ultra-15
(50 kDa, Millipore). The resultant antibody was passed through a
0.22 .mu.m filter for use in respective experiments.
2.10 Confirmation of Purification of Rat-Canine Chimeric Anti-PD-L1
Antibody c4G12 (FIG. 6)
[0141] In order to confirm the purity of the purified antibody,
antibody proteins were detected by SDS-PAGE and CBB staining. Using
SuperSep Ace 5-20% (Wako) gradient gel, rat anti-bovine PD-L1
monoclonal antibody 4G12 and rat-canine chimeric anti-PD-L1
antibody c4G12 were electrophoresed under reducing conditions and
non-reducing conditions. Bands were stained with Quick-CBB kit
(Wako) and decolored in distilled water. Bands were observed at
positions of molecular weights corresponding to antibodies. No
bands of contaminant proteins were recognized visually.
2.11 Measurement of Binding Avidities to cPD-L1-His of Rat
Anti-Bovine PD-L1
[0142] Monoclonal Antibody 4G12 and Rat-Canine Chimeric Anti-PD-L1
Antibody c4G12 In order to amplify the extracellular region of
canine PD-L1 estimated from its putative amino acid sequence,
primers were designed. Briefly, a primer having an NheI recognition
sequence added on the 5' side (cPD-L1-His F) and a primer having an
EcoRV recognition sequence and 6.times.His tag sequence added on
the 5' side (cPD-L1-His R) were designed. PCR was performed using a
synthesized beagle PBMC-derived cDNA as a template. The PCR
products were digested with NheI (Takara) and EcoRV (Takara) and
purified with FastGene Gel/PCR Extraction Kit (NIPPON Genetics).
The thus purified DNA was cloned into pCXN2.1 vector (Niwa et al.,
1991; kindly provided by Dr. T. Yokomizo, Juntendo University
Graduate School of Medicine) treated with restriction enzymes in
the same manner. The expression plasmids were purified with QIAGEN
Plasmid Midi kit (Qiagen) and stored at -30.degree. C. until use in
experiments. Hereinafter, the thus prepared expression plasmid is
designated as pCXN2.1-cPD-L1-His.
TABLE-US-00005 Primer (cPD-L1-His F): (SEQ ID NO: 131)
CGCGGCTAGCATGAGAATGTTTAGTGTCTT Primer (cPD-L1-His R): (SEQ ID NO:
132) CGCGGATATCTTAATGGTGATGGTGATGGT GAGTCCTCTCACTTGCTGG
[0143] The expression vector was transfected into Expi293F cells
(Life Technologies) to obtain a culture supernatant containing a
recombinant protein. The recombinant protein produced was purified
from the supernatant using TALON Metal Affinity Resin (Clontech),
and the buffer was exchanged with PBS using Amicon Ultra-4
Ultracel-3 (Merck Millipore). The thus obtained recombinant protein
was stored at 4.degree. C. until use in experiments (cPD-L1-His).
The protein concentration was measured with Pierce BCA Protein
Assay Kit (Thermo Fisher Scientific) for use in subsequent
experiments.
[0144] Using a biomolecular interaction analyzer (Biacore X100),
the binding avidities to cPD-L1-His of rat anti-bovine PD-L1
monoclonal antibody 4G12 and rat-canine chimeric anti-PD-L1
antibody c4G12 were assessed. Briefly, anti-histidine antibody was
fixed on CM5 censor chip, followed by capturing of cPD-L1-His.
Subsequently, monoclonal antibodies were added as analyte to
observe specific binding. Both antibodies exhibited specific
binding and their avidities were almost comparable (Table 1).
Further, the binding avidities of canine PD-1-Ig and CD80-Ig to
cPD-L1-His were measured in the same manner and found to be clearly
lower than that of rat-canine chimeric anti-PD-L1 antibody c4G12
(Table 1).
TABLE-US-00006 TABLE 1 Binding Avidity of Each Antibody and
Recombinant Protein to Canine PD-L1-His ka (.times.10.sup.6/Ms) kd
(.times.10.sup.-3/s) KD (nM) 4G12 2.42 .+-. 0.10 4.54 .+-. 0.19
1.88 .+-. 0.06 c4G12 3.14 .+-. 0.19 7.19 .+-. 0.26 2.30 .+-. 0.07
cPD-1 25.4 .+-. 4.89 cCD80 24.3 .+-. 0.89
2.12 Inhibitory Activity of Rat-Canine Chimeric Anti-PD-L1 Antibody
c4G12 Against Canine PD-1/PD-L1 Binding and CD80/PD-L1 Binding
(FIG. 7)
[0145] Using the canine PD-1-Ig, PD-L1-Ig and CD80-Ig (described
above), anti-PD-L1 antibody was tested for its ability to inhibit
canine PD-1/PD-L1 binding and CD80/PD-L1 binding. Briefly, canine
PD-1-Ig or CD80-Ig was immobilized on flat-bottom 96-well plates.
Canine PD-L1-Ig was reacted with various concentrations (0, 2.5, 5
and 10 .mu.g/ml) of rat anti-bovine PD-L1 antibody 4G12 or
rat-canine chimeric anti-PD-L1 antibody c4G12 according to the same
procedures as described in 2.6 above, and the binding of canine
PD-L1-Ig was assessed. No change in binding inhibition activity was
observed due to the chimerization of antibody.
2.13. Canine Immune Cell Activating Effect of Rat-Canine Chimeric
Anti-PD-L1 Antibody c4G12 (FIG. 8)
[0146] Canine PBMCs were cultured under stimulation with a
super-antigen Staphylococcal Enterotoxin B (SEB) for three days,
and changes in cytokine production by addition of rat-canine
chimeric anti-PD-L1 antibody c4G12 were measured by ELISA using
Duoset ELISA canine IL-2 or IFN-.gamma. (R&D systems).
Rat-canine chimeric anti-PD-L1 antibody c4G12 increased the
production of IL-2 and IFN-.gamma. from canine PBMCs. Further,
nucleic acid analogue EdU was added to the culture medium at day 2
of the culture under SEB stimulation. Two hours later, uptake of
EdU was measured by flow cytometry using Click-iT Plus EdU flow
cytometry assay kit (Life Technologies). As a result, EdU uptake in
canine CD4.sup.+ and CD8.sup.+ lymphocytes was enhanced by addition
of rat-canine chimeric anti-PD-L1 antibody c4G12, indicating an
elevated cell proliferation capacity.
2.14 Selection of Tumor-Affected Dogs to be Used in Canine
Inoculation Test
[0147] Since the subject treatment is expected to manifest a higher
efficacy when PD-L1 is being expressed in tumors, PD-L1 expression
analysis at the tumor site of dogs was performed by
immunohistochemical staining. Briefly, tumor tissue samples fixed
with formaldehyde and embedded in paraffin were sliced into 4 .mu.m
thick sections with a microtome, attached to and dried on
silane-coated slide glass (Matsunami Glass) and deparaffinized with
xylene/alcohol. While the resultant sections were soaked in citrate
buffer [citric acid (Wako Pure Chemical) 0.37 g, trisodium citrate
dihydrate (Kishida Chemical) 2.4 g, distilled water 1000 ml],
antigen retrieval treatment was performed for 10 min with
microwave, followed by staining using a Nichirei automatic
immuno-staining device. As pretreatment, sample sections were
soaked in 0.3% hydrogen peroxide-containing methanol solution at
room temperature for 15 min and washed with PBS. Then, anti-bovine
PD-L1 monoclonal antibody was added and reaction was conducted at
room temperature for 30 min. After washing with PBS, histofine
simple stain MAX-PO (Rat) (Nichirei Bioscience) was added and
reaction was carried at room temperature for 30 min, followed by
coloring with 3,3'-diaminobenzidine tetrahydrochloride and
observation with a light microscope. Dogs with oral melanoma or
undifferentiated sarcoma in which tumor cells were PD-L1 positive
were used in the following inoculation test (clinical trial).
Anti-bovine PD-L1 monoclonal antibody was established from a rat
anti-bovine PD-L1 monoclonal antibody producing hybridoma (Ikebuchi
R. Konnai S, Okagawa T, Yokoyama K, Nakajima C, Suzuki Y. Murata S,
Ohashi K. Immunology. 2014 August; 142(4):551-61).
2.15 Inoculation Test on Dogs
[0148] With respect to the rat-canine chimeric anti-PD-L1 antibody
c4G12 to be inoculated into dogs in the clinical trial, the culture
supernatant obtained by the procedures described in 2.8 above was
purified by affinity chromatography using MabSelect SuRe LX (GE
Healthcare) and then by hydroxyapatite chromatography using
BioScale CHT20-1 prepacked column (Bio-Rad) in order to remove
contaminants and polymeric proteins. Aggregate-containing fractions
were further purified by anion exchange chromatography using
HiScreen Q-Sepharose HP prepacked column (GE Healthcare).
(1) Safety Test: The established rat-canine chimeric anti-PD-L1
antibody c4G12 was administered intravenously into a dog (beagle,
spayed female, 13-year-old, about 10 kg in body weight) at 2 mg/kg,
every 2 weeks, 3 times in total. There was observed no anaphylaxis
or other adverse effects that would cause any trouble in clinical
trials. (2) Clinical Trial 1: The established rat-canine chimeric
anti-PD-L1 antibody c4G12 was administered intravenously into a
PD-L1 positive dog with relapsed oral melanoma (FIG. 9A) (miniature
dachshund, male, 11-year-old, about 7.5 kg in body weight) at 2
mg/kg or 5 mg/kg, every 2 weeks. 22 times in total. At week 10
after the start of treatment, a remarkable reduction in tumor size
was recognized. At week 34 after the start of treatment, a still
further reduction was confirmed (FIG. 10). During the observation
period of 44 weeks, no metastases to lymph nodes or the lung were
observed. When 30% or more reduction in the longest diameter of
tumor compared to that at the baseline is defined as PR (partial
response), the criterion of PR was satisfied at weeks 16-20 and at
week 34 and thereafter (FIG. 11). (3) Clinical Trial 2: Rat-canine
chimeric anti-PD-L1 antibody c4G12 was administered intravenously
into a dog with undifferentiated sarcoma whose primary lesion was
PD-L1 positive (FIG. 9B) and who had a plurality of metastatic
lesions in muscles throughout the body (west highland white
terrier, castrated male, 12-year-old, about 8 kg in body weight) at
5 mg/kg, every 2 weeks, 2 times in total. At week 3 from the start
of treatment, a clear regression of tumor was recognized (FIG. 12).
(4) Clinical Trial 3: Rat-canine chimeric anti-PD-L1 antibody c4G12
was administered intravenously into a dog with oral melanoma whose
primary lesion had been removed by surgery (beagle, spayed female,
I1-year-old, about 10 kg in body weight) at 2 mg/kg or 5 mg/kg,
every 2 weeks, 9 times in total. At week 18 after the start of
treatment, a plurality of pulmonary metastatic lesions disappeared
(FIG. 13), (5) Clinical Trial 4: Rat-canine chimeric anti-PD-L1
antibody c4G12 was administered intravenously into 4 dogs with oral
melanoma with pulmonary metastasis at 2 mg/kg or 5 mg/kg, every 2
weeks. Although no clear reduction in tumor size was observed
during the observation period, the duration of the treated dogs'
survival after confirmation of pulmonary metastasis tended to be
longer than that of a control group (antibody not administered,
historical control group: n=15) (FIG. 14). Therefore, the survival
duration may have been extended by antibody administration.
2.16 CDR Analysis of Anti-PD-L1 Antibody
[0149] The complementarity-determining regions (CDRs) of rat
anti-bovine PD-L1 antibody 4G12 were determined using NCBI IGBLAST
(http://www.ncbi.nlm.nih.gov/igblast/). The results are shown in
FIG. 15.
[Example 2] Application of Anti-PD-L1 Antibody to Other Animal
Species
1.1 Identification of Ovine, Porcine and Water Buffalo PD-L1
Genes
[0150] In order to determine the full-lengths of the coding
sequences (CDSs) of ovine, porcine and water buffalo PD-L1 cDNAs,
primers for amplifying the full lengths of CDSs from the nucleotide
sequences of ovine, porcine and water buffalo PD-L1 genes (GenBank
accession number; XM_004004362, NM_001025221 and XM_613366) were
first designed (ovPD-L1 CDS F and R; poPD-L1 CDS F and R; buPD-L1
CDS F1, R1, F2 and R2), and then PCR was performed. For the
resultant amplified products, nucleotide sequences were determined
with a capillary sequencer according to conventional methods
(Mingala C N, Konnai S, Ikebuchi R, Ohashi K. Comp. Immunol.
Microbiol. Infect. Dis. 2011 January; 34(1):55-63; Water buffalo
PD-L1 gene was identified in this article).
TABLE-US-00007 Primer (ovPD-L1 CDS F): (SEQ ID NO: 109)
ATGAGGATATATAGTGTCTTAACAT Primer (ovPD-L1 CDS R): (SEQ ID NO: 110)
TTACGICTCCTCAAAATGTG Primer (poPD-L1 CDS F): (SEQ ID NO: 111)
ATGAGGAFAIGTAGTATCTTTACAT Primer (poPD-L1 CDS R): (SEQ ID NO: 112)
TTACGTCTCCTCAAATTGTGT Primer (buPD-L1 CDS F1): (SEQ ID NO: 113)
ATGAGGATATATAGTGTCTT Primer (buPD-L1 CDS R1): (SEQ ID NO: 114)
GCCACTCAGGACTTGGTGAT Primer (buPD-L1 CDS F2): (SEQ ID NO: 115)
GGGGGITTACTGTTGCTTGA Primer (buPD-L1 CDS R2): (SEQ ID NO: 116)
TTACGTCTCCTCAAATTGT
1.2 Construction of Ovine PD-1, Ovine PD-L1, Porcine PD-1 and
Porcine PD-L1 Expressing COS-7 Cells
[0151] In order to prepare ovine PD-1, ovine PD-L1, porcine PD-1
and porcine PD-L1 expressing plasmids, PCR was performed using a
synthesized ovine or porcine PBMC-derived cDNA as a template and
primers designed by adding BglII and SmaI (ovine PD-1), HindIII and
SmaI (porcine PD-1), or XhoI and SmaI (ovine and porcine PD-L1)
recognition sites on the 5' side (ovPD-1-EGFP F and R; ovPD-L1-EGFP
F and R; poPD-1-EGFP F and R; or poPD-L1-EGFP F and R). The
resultant PCR products were digested with BglII (Takara) and SmaI
(Takara) (ovine PD-1), HindIII (Takara) and SmaI (Takara) (porcine
PD-1), and XhoI (Takara) and SmaI (Takara) (ovine and porcine
PD-L1), then purified with FastGene Gel/PCR Extraction Kit (NIPPON
Genetics) and cloned into pEGFP-N2 vector (Clontech) treated with
restriction enzymes in the same manner. Expression plasmids were
extracted using FastGene Xpress Plasmid PLUS Kit (NIPPON Genetics)
and stored at -30.degree. C. until use in experiments. Hereinafter,
the thus prepared plasmid is designated as pEGFP-N2-ovPD-1,
pEGFP-N2-ovPD-L1, pEGFP-N2-poPD-1 or pEGFP-N2-poPD-L1.
TABLE-US-00008 Primer (ovPD-1-EGFP F): (SEQ ID NO: 117)
GAAGATCTATGGGGACCCCGCGGGCGCCG Primer (ovPD-1-EGFP R): (SEQ ID NO:
118) GACCCGGGGAGGGGCCAGGAGCAGTGTCC Primer (ovPD-L1-EGFP F): (SEQ ID
NO: 119) CCGCTCGAGATGAGGATATATAGTGTCT Primer (ovPD-L1-EGFP R): (SEQ
ID NO: 120) ATCCCGGGCGTCTCCTCAAAATGTGTAG Primer (poPD-1-EGFP F):
(SEQ ID NO: 121) ACTAAGCTrATGGGGACCCCGCGGG Primer (poPD-1-EGFPR):
(SEQ ID NO: 122) ACTCCCGGGGAGGGGCCAAGAGCAGT Primer (poPD-L1-EGFP
F): (SEQ ID NO: 123) CCGCTCGAGATGAGGATATGTAGTATCTT Primer
(poPD-L1-EGFP R): (SEQ ID NO: 124)
ATCCCGGGCGTCTCCTCAAATTGTGTATC
[0152] COS-7 cells were subcultured at a density of
5.times.10.sup.4 cells/cm.sup.2 in 6-well plates, and then cultured
overnight in RPMI 1640 medium containing 10% inactivated fetal
bovine serum and 0.01% L-glutamine at 37.degree. C. in the presence
of 5% CO.sub.2. The pEGFP-N2-ovPD-1, pEGFP-N2-ovPD-L1,
pEGFP-N2-poPD-1, pEGFP-N2-poPD-L1 or pEGFP-N2 (negative control)
was introduced into COS-7 cells at 0.4 .mu.g/cm.sup.2 using
Lipofectamine 2000 (Invitrogen). The cells were cultured for 48
hours (ovPD-1-EGFP expressing cell, ovPD-L1-EGFP expressing cell,
poPD-1-EGFP expressing cell, and poPD-L1-EGFP expressing cell). In
order to confirm the expression of ovine PD-1, ovine PD-L1, porcine
PD-1 and porcine PD-L1 in the thus prepared expressing cells,
intracellular localization of EGFP was visualized with an inverted
confocal laser microscope LSM700 (ZEISS) or an all-in-one
fluorescence microscope BZ-9000 (KEYENCE).
1.3 Construction of Recombinant Ovine PD-L1 and Porcine PD-L1
[0153] In order to amplify the extracellular regions of ovine PD-L1
or porcine PD-L1 estimated from their putative amino acid
sequences, primers were designed. Briefly, primers having an NheI
or EcoRV recognition sequence added on the 5' side (ovPD-L1-Ig F
and R, or poPD-L1-Ig F and R) were designed. PCR was performed
using a synthesized ovine or porcine PBMC-derived cDNA as a
template. The PCR products were digested with NheI (Takara) and
EcoRV (Takara) and purified with FastGene Gel/PCR Extraction Kit
(NIPPON Genetics). The thus purified DNAs were individually cloned
into pCXN2.1-Rabbit IgG Fc vector (Niwa et al., 1991. Zettimeissl
et al., 1990, kindly provided by Dr. T. Yokomizo, Juntendo
University Graduate School of Medicine, and modified in the
inventors' laboratory) treated with restriction enzymes in the same
manner. The expression plasmids were purified with FastGene Xpress
Plasmid PLUS Kit (NIPPON Genetics) and stored at -30.degree. C.
until use in experiments. Hereinafter, the thus prepared expression
plasmids are designated as pCXN2.1-ovPD-L1-Ig and
pCXN2.1-poPD-L1-Ig, respectively.
TABLE-US-00009 Primer (ovPD-L1-Ig F): (SEQ ID NO: 125)
GACGCTAGCATGAGGATATATAGTGTCT Primer (ovPD-L1-Ig R): (SEQ 1D NO:
126) GCTCTGATATCCCTCGTTTTTGCTGGAT Primer (poPD-L1-Ig F): (SEQ ID
NO: 127) GACGCTAGCATGAGGATATGTAGTATCTT Primer (poPD-L1-Ig R): (SEQ
ID NO: 128) AGCTTGATATCCCTCTTTCTTGCTGGATC
[0154] Thirty micrograms of pCXN2.1-ovPD-L1-Ig or
pCXN2.1-poPD-L1-Ig was introduced into 7.5.times.10.sup.7 Expi293F
cells (Life Technologies) using Expifectamin (Life Technologies).
After 6-day shaking culture, a culture supernatant was collected.
The culture supernatant contained an Fc fusion recombinant protein.
The produced Fc recombinant protein was purified from the
supernatant using Ab-Capcher Extra (ProteNova). After purification,
the buffer was exchanged with PBS (pH 7.4) using PD-10 Desalting
Column (GE Healthcare). The resultant recombinant protein was
stored at -30.degree. C. until use in Experiment (ovine PD-L1-Ig).
Concentrations of purified ovine PD-L1-Ig and porcine PD-L1-Ig were
measured with Rabbit IgG ELISA Quantitation Set (BETHYL). For each
washing operation in ELISA, Auto Palte Washer BIO WASHER 50 (DS
Pharma Biomedical) was used. Absorbance was measured with
Microplate Reader MTP-650FA (Corona Electric).
1.4 Reactivity of Rat Anti-Bovine PD-L1 Antibody 4G12 with Ovine
and Porcine PD-L1
[0155] It was confirmed by flow cytometry that rat anti-bovine
PD-L1 monoclonal antibody cross-reacts with ovine and porcine
PD-L1. Ovine or Porcine PD-L1-EGFP expressing COS-7 cells were
blocked with 10% inactivated goat serum supplemented PBS at room
temperature for 15 min and reacted with 10 .mu.g/ml of rat
anti-bovine PD-L1 antibody 4G12 at room temperature for 30 min.
After washing, the cells were reacted with allophycocyanine-labeled
anti-rat Ig goat antibody (Beckman Coulter) at room temperature for
30 min. For analysis, FACS Verse (BD Bioscience) was used. As a
negative control antibody, rat IgG2a (.kappa.) isotype control (BD
Bioscience) was used. For every washing operation and dilution of
antibodies, 1% bovine serum albumin supplemented PBS was used.
[0156] Experimental results are shown in FIG. 16. It was confirmed
that rat anti-bovine PD-L1 antibody 4G12 binds to ovine and porcine
PD-L1.
1.5 Reactivity of Rat Anti-Bovine PD-L1 Antibody 4G12 with Water
Buffalo Leukocytes
[0157] Peripheral blood of water buffalo (Bubalus bubalis; Asian
water buffalo) was hemolyzed with ACK buffer to isolate leukocytes.
After blocking with 10% inactivated goat serum supplemented PBS at
room temperature for 15 min, reaction with rat anti-bovine PD-L1
antibody 4G12, peridinin-chlorophyll-protein complex/cyanin
5.5-labeled anti-bovine CD14 antibody (mouse IgG1, CAM36A, VMRD)
and anti-bovine CD11b antibody (mouse IgG2b, CC126, AbD Serotec)
was conducted at room temperature for 30 min. After washing,
reaction with allophycocyanine-labelled anti-rat Ig goat antibody
(Beckman Coulter) and fluorescein isothiocyanate-labeled anti-mouse
IgG2 goat antibody (Beckman Coulter) was conducted at room
temperature for 30 min. For analysis, FACS Calibur (BD Biosciences)
was used. As a negative control antibody, rat IgG2a (.kappa.)
isotype control (BD Biosciences) was used. For every washing
operation and dilution of antibodies, 10% inactivated goat serum
supplemented PBS was used.
[0158] Experimental results are shown in FIG. 17. Rat anti-bovine
PD-L1 antibody 4G12 strongly bound to blood macrophages (CD14.sup.+
CD11b.sup.+ cells) of water buffalo. On the other hand, rat
anti-bovine PD-L1 antibody 4G12 weakly bound to lymphocytes
(CD14.sup.- CD11b.sup.- cells) of water buffalo. This difference in
binding property is believed to reflect the expression levels of
PD-L1 in macrophages and lymphocytes.
1.6 Inhibition Test on Ovine or Porcine PD-1/PD-L1 Binding with Rat
Anti-Bovine PD-L1 Antibody 4G12
[0159] Using ovine PD-1-EGFP expressing COS-7 cells and ovine
PD-L1-Ig recombinant protein, or porcine PD-1-EGFP expressing COS-7
cells and porcine PD-L1-Ig recombinant protein, inhibition of ovine
or porcine PD-1/PD-L1 binding by rat anti-bovine PD-L1 antibody
(4G12) was tested. Briefly, rat anti-bovine PD-L1 antibody 4G12 of
various concentrations (0, 1, 5, 10, 20, 50 .mu.g/ml) was reacted
in advance with ovine PD-L1-Ig (final concentration 1 .mu.g/ml) or
porcine PD-L1-Ig (final concentration 5 .mu.g/ml) at 37.degree. C.
for 30 min. Subsequently, the antibody 4G12 was reacted with
2.times.10.sup.5 ovine PD-1-EGFP expressing COS-7 cells or porcine
PD-1-EGFP expressing COS-7 cells at 37.degree. C. for 30 min. After
washing, ovine PD-L1-Ig or porcine PD-L1-Ig bound to cell surfaces
was detected with Alexa Fluor 647-labeled anti-rabbit IgG (H+L)
goat F(ab')2 (Life Technologies). For analysis, FACS Verse (BD
Biosciences) was used. As a negative control antibody, rat IgG2a
(.kappa.) isotype control (BD Biosciences) was used. Taking the
proportion of PD-L1-Ig bound cells without antibody addition as
100%, the proportion of PD-L1-Ig bound cells at each antibody
concentration was shown as relative value.
[0160] The results revealed that rat anti-bovine PD-L1 antibody
4G12 is capable of inhibiting ovine PD-1/PD-L1 and porcine
PD-1/PD-L1 binding in a concentration dependent manner (FIG.
18).
Example 31
1. Introduction
[0161] Programmed cell death 1 (PD-1), an immunoinhibitory
receptor, and its ligand programmed cell death ligand 1 (PD-L1) are
molecules identified by Prof. Tasuku Honjo et al., Kyoto
University, as factors which inhibit excessive immune response and
are deeply involved in immunotolerance. Recently, it has been
elucidated that these molecules are also involved in
immunosuppression in tumors. In the subject Example, for the
purpose of establishing a novel therapy for bovine infections, the
present inventors have prepared a chimeric antibody gene by linking
the variable region gene of rat anti-bovine PD-L1 monoclonal
antibody (4G12) capable of inhibiting the binding of bovine PD-1
and PD-L1 to the constant region gene of a bovine immunoglobulin
(IgG1 with mutations having been introduced into the putative
binding sites for Fc.gamma. receptors in CH2 domain to inhibit ADCC
activity; see FIG. 19 for amino acid numbers and mutations: 250
E.fwdarw.P, 251 L.fwdarw.V, 252 P.fwdarw.A, 253 G.fwdarw.deletion.
347 A.fwdarw.S, 348 P.fwdarw.S; Ikebuchi R. Konnai S, Okagawa T.
Yokoyama K, Nakajima C. Suzuki Y, Murata S. Ohashi K. Immunology
2014 August; 142(4):551-561). This chimeric antibody gene was
introduced into Chinese hamster ovary cells (CHO cells). By
culturing/proliferating the resultant cells, the present inventors
have obtained a rat-bovine chimeric anti-bovine PD-L1 antibody
(ch4G12) and confirmed its effect in vitro and in vivo.
2. Materials and Methods
Construction of Bovine PD-1 and PD-L1 Expressing Cells
[0162] The nucleotide sequences of the full length cDNAs of bovine
PD-1 gene (GenBank accession number AB510901; Ikebuchi R, Konnai S.
Sunden Y, Onuma M, Ohashi K. Microbiol. Immunol. 2010 May;
54(5):291-298) and bovine PD-L1 gene (GenBank accession number
AB510902: Ikebuchi R, Konnai S. Shirai T. Sunden Y. Murata S, Onuma
M, Ohashi K. Vet. Res. 2011 Sep. 26; 42:103) were determined. Based
on the resultant genetic information, bovine PD-1 and bovine PD-L1
membrane expressing cells were prepared. First, for preparing
bovine PD-1 or PD-L1 expressing plasmid, PCR was performed using a
synthesized bovine PBMC-derived cDNA as a template and designed
primers having NotI and HindIII (bovine PD-1) recognition sites and
NheI and XhoI (bovine PD-L1) recognition sites on the 5' side
(boPD-1-myc F and R; boPD-L1-EGFP F and R). The PCR products were
digested with NotI (Takara) and HindIII (Takara; bovine PD-1), NheI
(Takara) and XhoI (Takara; bovine PD-L1), purified with FastGene
Gel/PCR Extraction Kit (NIPPON Genetics) and cloned into pCMV-Tag1
vector (Agilent Technologies; bovine PD-1) or pEGFP-N2 vector
(Clontech; bovine PD-L1) treated with restriction enzymes in the
same manner. The resultant expression plasmid of interest was
extracted with QIAGEN Plasmid Midi kit (Qiagen) and stored at
-30.degree. C. until use in experiments. Hereinafter, the thus
prepared expression plasmid is designated as pCMV-Tag1-boPD-1.
TABLE-US-00010 Primer (boPD-1-mvc F): (SEQ ID NO: 133)
ATATGCGGCCGCATGGGGACCCCGCGGGCGCT Primer (boPD-1-mvc R): (SEQ ID NO:
134) GCGCAAGCTTTCAGAGGGGCCAGGAGCAGT Primer (boPD-L1-EGFP F): (SEQ
ID NO: 135) CTAGCTAGCACCATGAGGATATATAGTGTCTTAAC Primer
(boPD-L1-EGFP R): (SEQ ID NO: 136)
CAATCTCGAGTTACAGACAGAAGATGACTGC
[0163] Bovine PD-1 membrane expressing cells were prepared by the
procedures described below. First, 2.5 .mu.g of pCMV-Tag1-boPD-1
was introduced into 4.times.10.sup.6 CHO-DG44 cells using
Lipofectamine LTX (Invitrogen). Forty-eight hours later, the medium
was exchanged with CD DG44 medium (Life Technologies) containing
800 .mu.g/ml G418 (Enzo Life Science). 20 ml/L GlutaMAX supplement
(Life Technologies), and 18 ml/L 10% Pluronic F-68 (Life
Technologies), followed by selection. The resultant expression
cells were reacted with rat anti-bovine PD-1 antibody 5D2 at room
temperature. After washing, the cells were further reacted with
anti-rat IgG microbeads-labeled antibody (Miltenyi Biotec) at room
temperature. Cells expressing bovine PD-1 at high levels were
isolated with Auto MACS (Miltenyi Biotec). Subsequently,
re-isolation was performed in the same manner to obtain still
higher purity. The resultant expression cells were subjected to
cloning by limiting dilution to thereby obtain a CHO DG44 cell
clone expressing bovine PD-1 at high level (bovine PD-1 expressing
cells).
[0164] Bovine PD-L1 membrane expressing cells were prepared by the
procedures described below. First, 2.5 .mu.g of pEGFP-N2-boPD-L1 or
pEGFP-N2 (negative control) was introduced into 4.times.10.sup.6
CHO-DG44 cells using Lipofectamine LTX (Invitrogen). Forty-eight
hours later, the medium was exchanged with CD DG44 medium (Life
Technologies) containing G418 (Enzo Life Science) 800 .mu.g/ml.
GlutaMAX supplement (Life Technologies) 20 ml/L, and 10% Pluronic
F-68 (Life Technologies) 18 ml/L, followed by selection and cloning
by limiting dilution (bovine PD-L1 expressing cell clone). In order
to confirm the expression of bovine PD-L1 in the thus prepared
expressing cell clone, intracellular localization of EGFP was
visualized with an inverted confocal laser microscope LSM700
(ZEISS).
[0165] Construction of Soluble Bovine PD-1 and PD-L1 Bovine PD-1-Ig
expressing plasmid was constructed by the procedures described
below. Briefly, the signal peptide and the extracellular region of
bovine PD-1 (GenBank accession number AB510901) were linked to the
Fe domain of the constant region of a known bovine IgG1 (GenBank
accession number X62916) to prepare a gene sequence. After codons
were optimized for CHO cells, gene synthesis was performed in such
a manner that NotI recognition sequence, KOZAK sequence, bovine
PD-1 signal peptide sequence, bovine PD-1 gene extracellular region
sequence, bovine IgG1 Fc region sequence, and XbaI recognition
sequence would be located in the gene in this order. It should be
noted here that bovine IgG1 was mutated to inhibit ADCC activity;
more specifically, mutations were introduced into the putative
binding sites for Fc.gamma. receptors of CH2 domain (sites of
mutation: 185 E.fwdarw.P, 186 L.fwdarw.V, 187 P.fwdarw.A, 189
G.fwdarw.deletion, 281 A.fwdarw.S, 282 P.fwdarw.S; Ikebuchi R,
Konnai S, Okagawa T, Yokoyama K, Nakajima C. Suzuki Y, Murata S,
Ohashi K. Immunology 2014 August; 142(4):551-561; the amino acid
sequence of PD-1-Ig and the sites of mutation are disclosed in FIG.
2 of this article). The synthesized gene strand was digested with
NotI (Takara) and XbaI (Takara), purified with FastGene Gel/PCR
Extraction Kit (NIPPON Genetics), and incorporated into the cloning
site (NotI and XbaI restriction enzyme recognition sequences
downstream of PCMV and between INRBG and PABGH) of expression
vector pDN11 (kindly provided by Prof. S. Suzuki, Hokkaido
University Research Center for Zoonosis Control) treated with
restriction enzymes in the same manner, whereby bovine PD-1-Ig
expressing vector was constructed. The expression plasmid was
purified with QIAGEN Plasmid Midi kit (Qiagen) and stored at
-30.degree. C. until use in experiments. Hereinafter, the thus
prepared expression plasmid is designated as pDN11-boPD-1-Ig.
[0166] Bovine PD-L1-Ig expressing plasmid was constructed by the
procedures described below. In order to amplify the signal peptide
and the extracellular region of bovine PD-L1 (GenBank accession
number AB510902), primers were designed that had NheI and EcoRV
recognition sites added on the 5' side (boPD-L1-Ig F and R). PCR
was performed using a synthesized bovine PBMC-derived cDNA as a
template. The PCR products were digested with NheI (Takara) and
EcoRV (Takara), purified with FastGene Gel/PCR Extraction Kit
(NIPPON Genetics) and cloned into pCXN2.1-Rabbit IgG1 Fc vector
(Niwa et al.. 1991: Zettlmeissl et al., 1990; kindly provided by
Dr. T. Yokomizo, Juntendo University Graduate School of Medicine,
and modified in the inventors' laboratory) treated with restriction
enzymes in the same manner. The expression plasmid was purified
with QIAGEN Plasmid Midi kit (Qiagen) or FastGene Xpress Plasmid
PLUS Kit (NIPPON Genetics) and stored at -30.degree. C. until use
in experiments. Hereinafter, the thus prepared expression plasmid
is designated as pCXN2.1-boPD-L1-Ig.
TABLE-US-00011 Primer (boPD-L1-Ig F): (SEQ ID NO: 137)
GCTAGCATGAGGATATATAGTGTCTTAAC Primer (boPD-L1-Ig R): (SEQ ID NO:
138) GATATCATTCCTCTTTTTTGCTGGAT
[0167] Soluble bovine PD-1-Ig expressing cells were prepared by the
procedures described below. Briefly, 2.5 .mu.g of pDN11-boPD-1-Ig
was introduced into 4.times.10.sup.6 CHO-DG44 cells using
Lipofectamine LTX (Invitrogen). Forty-eight hours later, the medium
was exchanged with OptiCHO AGT medium (Life Technologies)
containing 800 .mu.g/ml G418 (Enzo Life Science) and 20 ml/L
GlutaMAX supplement (Life Technologies). After cultured for 3
weeks, the cells were subjected to selection. Briefly, the
concentrations of the Fc fusion recombinant protein in the culture
supernatants of the resultant cell clones were measured by ELISA
using anti-bovine IgG F(c) rabbit polyclonal antibody (Rockland) to
thereby select those cell clones that express the Fc fusion
recombinant protein at high levels. The resultant highly expressing
cell clone was transferred to a G418-free medium and cultured under
shaking for 14 days, followed by collection of a culture
supernatant. The culture supernatant containing the Fc fusion
recombinants protein was ultrafiltered with Centricon Plus-70
(Millipore). Then, the Fc fusion recombinant protein was purified
with Ab-Capcher Extra (ProteNova). After purification, the buffer
was exchanged with phosphate-buffered physiological saline (PBS; pH
7.4) using PD-10 Desalting Column (GE Healthcare). The resultant
protein was stored at -30.degree. C. until use in experiments
(bovine PD-1-Ig). The concentration of the purified bovine PD-1-Ig
was measured by ELISA using IgG F(c) rabbit polyclonal antibody
(Rockland). For each washing operation in ELISA, Auto Plate Washer
BIO WASHER 50 (DS Pharma Biomedical) was used. Absorbance was
measured with Microplate Reader MTP-650FA (Corona Electric).
[0168] Soluble bovine PD-L1-Ig expressing cells were prepared by
the procedures described below. Briefly, 30 .mu.g of
pCXN2.1-boPD-L1-Ig was introduced into 7.5.times.10.sup.7 Expi293F
cells (Life Technologies) using Expifectamine (Life Technologies).
After 7-day culture under shaking, the culture supernatant was
collected. The recombinant protein was purified from the
supernatant using Ab-Capcher Extra (ProteNova; bovine PD-L1-Ig).
After purification, the buffer was exchanged with PBS (pH 7.4)
using PD MiniTrap G-25 (GE Healthcare). The resultant protein was
stored at -30.degree. C. until use in experiments (bovine
PD-L1-Ig). The concentration of the purified bovine PD-L1-Ig was
measured using Rabbit IgG ELISA Quantitation Set (Bethyl). For each
washing operation in ELISA, Auto Plate Washer BIO WASHER 50 (DS
Pharma Biomedical) was used. Absorbance was measured with
Microplate Reader MTP-650FA (Corona Electric).
Preparation of Rat Anti-Bovine PD-L1 Monoclonal Antibody Producing
Cells
[0169] Rat was immunized in the footpad with bovine PD-L1-Ig
(Ikebuchi R, Konnai S, Okagawa T, Yokoyama K, Nakajima C. Suzuki Y,
Murata S, Ohashi K. Immunology 2014 August; 142(4):551-561: bovine
PD-L1-Ig was prepared by the method disclosed in this article and
used for immunization). Hybridomas were established by the iliac
lymph node method to thereby obtain rat anti-bovine PD-L1
monoclonal antibody producing hybridoma 4G12. With respect to the
method of establishment of rat anti-bovine PD-L1 monoclonal
antibody, details are disclosed in the following non-patent
document (Ikebuchi R. Konnai S, Okagawa T. Yokoyama K, Nakajima C,
Suzuki Y, Murata S. Ohashi K. Vet. Res. 2013 Jul. 22; 44:59;
Ikebuchi R, Konnai S, Okagawa T, Yokoyama K, Nakajima C, Suzuki Y,
Murata S. Ohashi K. Immunology 2014 August; 142(4):551-561).
Preparation of Rat-Bovine Chimeric Anti-Bovine PD-L1 Antibody
Expressing Vector
[0170] Rat-bovine chimeric anti-bovine PD-L1 antibody ch4G12 was
established by fusing the antibody constant regions of bovine IgG1
and Ig.lamda. with rat anti-bovine PD-L1 antibody 4G12 being used
as an antibody variable region.
[0171] First, the genes of heavy chain and light chain variable
regions were identified from a hybridoma that would produce rat
anti-bovine PD-L1 antibody 4G12. Subsequently, a gene sequence was
prepared in which the heavy chain and the light chain variable
regions of the antibody 4G12 were linked to known constant regions
of bovine IgG1 (heavy chain; modified from GenBank Accession number
X62916) and bovine Ig.lamda. (light chain; GenBank Accession number
X62917), respectively, and codon optimization was carried out
[rat-bovine chimeric anti-bovine PD-L1 antibody ch4G12: SEQ ID NOS:
105 and 106 (amino acid sequences), SEQ ID NOS: 107 and 108
(nucleotide sequences after codon optimization)]. It should be
noted that in order to suppress the ADCC activity of bovine IgG1,
mutations were added to the putative binding sites of Fc.gamma.
receptors in CH2 domain (See FIG. 19 for amino acid numbers and
mutations: 250 E.fwdarw.P. 251 L.fwdarw.V, 252 P.fwdarw.A, 253
G.fwdarw.deletion, 347 A.fwdarw.S, 348 P.fwdarw.S; Ikebuchi R,
Konnai S, Okagawa T, Yokoyama K, Nakajima C, Suzuki Y. Murata S.
Ohashi K. Immunology 2014 August; 142(4):551-561). Then, the gene
was artificially synthesized in such a manner that NotI recognition
sequence, KOZAK sequence, chimeric antibody light chain sequence,
poly-A addition signal sequence (PABGH), promoter sequence (PCMV),
SacI recognition sequence, intron sequence (INRBG), KOZAK sequence,
chimeric antibody heavy chain sequence and XbaI recognition
sequence would be located in this order. The synthesized gene
strand was digested with NotI (Takara) and XbaI (Takara), purified
with FastGene Gel/PCR Extraction Kit (NIPPON Genetics) and cloned
into the cloning site (NotI and XbaI restriction enzyme recognition
sequences downstream of PCMV and between INRBG and PABGH) of
expression plasmid pDC6 (kindly provided by Prof. S. Suzuki,
Hokkaido University Research Center for Zoonosis Control) treated
with restriction enzymes in the same manner (FIG. 20). The
resultant plasmid was extracted with QIAGEN Plasmid Midi kit
(Qiagen) and stored at -30.degree. C. until use in experiments.
Hereinafter, the thus prepared expression plasmid is designated as
pDC6-boPD-L1ch4G12.
Expression of Rat-Bovine Chimeric Anti-Bovine PD-L1 Antibody
[0172] The pDC6-boPD-L1ch4G12 was transfected into CHO-DG44 cells
(CHO-DG44 (dfhr.sup.-/.sup.-)) which were a dihydrofolate reductase
deficient cell. Forty-eight hours later, the medium was exchanged
with OptiCHO AGT medium (Life Technologies) containing 20 ml/L
GlutaMAX supplement (Life Technologies). After cultured for 3
weeks, the cells were subjected to selection and cloning by
limiting dilution. Subsequently, the concentrations of the chimeric
antibody in the culture supernatants were measured by dot blotting
and ELISA using anti-bovine IgG F(c) rabbit polyclonal antibody
(Rockland) to thereby select high expression clones. Further, the
selected clones expressing rat-bovine chimeric anti-bovine PD-L1
antibody at high levels were subjected to gene amplification
treatment by adding a load with 60 nM methotrexate (Mtx)-containing
medium. The thus established cell clone stably expressing
rat-bovine chimeric anti-bovine PD-L1 antibody was transferred into
Mtx-free Opti-CHO AGT medium and cultured under shaking for 14 days
(125 rpm, 37.degree. C., 5% CO.sub.2). Chimeric antibody production
in the culture supernatant was measured by ELISA using anti-bovine
IgG F(c) rabbit polyclonal antibody (Rockland). For each washing
operation in ELISA, Auto Plate Washer BIO WASHER 50 (DS Pharma
Biomedical) was used. Absorbance was measured with Microplate
Reader MTP-650FA (Corona Electric). The culture supernatant at day
14 was centrifuged at 10,000 g for 10 min to remove cells, and the
centrifugal supernatant was passed through a Steritop-GP 0.22 .mu.m
filter (Millipore) for sterilization and then stored at 4.degree.
C. until it was subjected to purification.
Purification of Rat-Bovine Chimeric Anti-Bovine PD-L1 Antibody
[0173] From the culture supernatant prepared as described above,
each chimeric antibody was purified using Ab Capcher Extra
(ProteNova). An open column method was used for binding to resin;
PBS pH 7.4 was used as an equilibration buffer and a wash buffer.
As an elution buffer, IgG Elution Buffer (Thermo Fisher Scientific)
was used. As a neutralization buffer, 1M Tris (pH 9.0) was used.
The purified antibody was subjected to buffer exchange with PBS (pH
7.4) using PD-10 Desalting Column (GE Healthcare) and concentrated
using Amicon Ultra-15 (50 kDa, Millipore). The thus purified
chimeric antibody was passed through a 0.22 .mu.m syringe filter
(Millipore) for sterilization and stored at 4.degree. C. until use
in experiments.
Confirmation of the Purity of Purified Rat-Bovine Chimeric
Anti-Bovine PD-L1 Antibody (FIG. 21)
[0174] In order to confirm the purity of purified rat-bovine
chimeric anti-bovine PD-L1 antibody, antibody proteins were
detected by SDS-PAGE and CBB staining. Using 10% acrylamide gel,
the purified rat-bovine chimeric antibody was electrophoresed under
reducing conditions (reduction with 2-mercaptoethanol from
Sigma-Aldrich) and non-reducing conditions. Bands were stained with
Quick-CBB kit (Wako) and decolored in distilled water. The results
are shown in FIG. 21. Bands were observed at predicted positions,
that is, at 25 kDa and 50 kDa under reducing conditions and at 150
kDa under non-reducing conditions.
Binding Specificity of Rat-Bovine Chimeric Anti-Bovine PD-L1
Antibody (FIG. 22)
[0175] It was confirmed by flow cytometry that the rat-bovine
chimeric anti-bovine PD-L1 antibody specifically binds to the
bovine PD-L1 expressing cells (described above). First, rat
anti-bovine PD-L1 antibody 4G12 or rat-bovine chimeric anti-bovine
PD-L1 antibody ch4G12 was reacted with bovine PD-L1 expressing
cells at room temperature for 30 min. After washing, APC-labeled
anti-rat Ig goat antibody (Southern Biotech) or Alexa Fluor
647-labeled anti-bovine IgG (H+L) goat F(ab')2 (Jackson
ImmunoResearch) was reacted at room temperature for 30 min. As
negative control antibody, rat IgG2a (.kappa.) isotype control (BD
Biosciences) or bovine IgG1 antibody (Bethyl) was used. After
washing, each rat antibody or rat-bovine chimeric antibody bound to
cell surfaces was detected by FACS Verse (BD Biosciences). For
every washing operation and dilution of antibody, PBS supplemented
with 1% bovine serum albumin (Sigma-Aldrich) was used.
[0176] The experimental results are shown in FIG. 22. It was
revealed that rat-bovine chimeric anti-bovine PD-L1 antibody ch4G12
binds to bovine PD-L1 expressing cells in the same manner as rat
anti-bovine PD-L1 antibody 4G12.
Inhibitory Activity of Rat-Bovine Chimeric Anti-PD-L1 Antibody
Against Bovine PD-1/PD-L1 Binding
(1) Binding Inhibition Test on Bovine PD-L1 Expressing Cells and
Soluble Bovine PD-1 (FIG. 23)
[0177] Using bovine PD-L1 expressing cells (described above) and
bovine PD-1-Ig (described above), bovine PD-1/PD-L1 binding
inhibition by anti-bovine PD-L1 antibody was tested. First,
2.times.10.sup.5 bovine PD-L1 expressing cells were reacted with
various concentrations (0, 0.32, 0.63, 1.25, 2.5, 5 or 10 .mu.g/ml)
of rat anti-bovine PD-L1 antibody 4G12 or rat-bovine chimeric
anti-bovine PD-L1 antibody ch4G12 at room temperature for 30 min.
As negative control antibody, rat IgG2a (.kappa.) isotype control
(BD Biosciences) or bovine IgG1 antibody (Bethyl) was used. After
washing, bovine PD-1-Ig labeled with biotin using Lightning-Link
Type A Biotin Labeling Kit (Innova Bioscience) was added to a final
concentration of 2 .mu.g/ml, followed by reaction for another 30
min at room temperature. Subsequently, after washing, bovine
PD-1-Ig bound to cell surfaces was detected with APC-labeled
streptavidin (BioLegend). For analysis, FACS Verse (BD Biosciences)
was used. For every washing operation and dilution of antibody, PBS
supplemented with 1% bovine serum albumin (Sigma-Aldrich) was used.
Taking the proportion of PD-1-Ig bound cells without antibody
addition as 100%, the proportion of PD-1-Ig bound cells at each
antibody concentration was shown as relative value.
[0178] The experimental results are shown in FIG. 23. It was
revealed that like rat anti-bovine PD-L1 antibody 4G12, rat-bovine
chimeric anti-bovine PD-L1 antibody ch4G12 is capable of inhibiting
bovine PD-1/PD-L1 binding in a concentration dependent manner.
(2) Binding Inhibition Test on Bovine PD-1 Expressing Cells and
Soluble Bovine PD-L1 (FIG. 24)
[0179] Using bovine PD-1 expressing cells (described above) and
bovine PD-L1-Ig (described above), bovine pD-1/PD-L1 binding
inhibition by anti-bovine PD-L1 antibody was tested. First, rat
anti-bovine PD-L1 antibody 4G12 or rat-bovine chimeric anti-bovine
PD-L1 antibody ch4G12 at a final concentration of 0, 0.32, 0.63,
1.25, 2.5, 5 or 10 .mu.g/ml and bovine PD-L1-Ig at a final
concentration of 1 .mu.g/ml were placed in 96-well plates, where
they were reacted at room temperature for 30 min. The resultant
mixture was reacted with 2.times.10.sup.5 bovine PD-1 expressing
cells at room temperature for 30 min. As negative control antibody,
rat IgG2a (.kappa.) isotype control (BD Biosciences) or bovine IgG1
antibody (Bethyl) was used. After washing, Alexa Fluor 647-labeled
anti-rabbit IgG (H+L) goat F(ab')2 (Life Technologies) was reacted
at room temperature for 30 min to thereby detect bovine PD-L1-Ig
bound to cell surfaces. For analysis, FACS Verse (BD Biosciences)
was used. For every washing operation and dilution of antibody, PBS
supplemented with 1% bovine serum albumin (Sigma-Aldrich) was used.
Taking the proportion of PD-L1-Ig bound cells without antibody
addition as 100%, the proportion of PD-L1-Ig bound cells at each
antibody concentration was shown as relative value.
[0180] The experimental results are shown in FIG. 24. It was
revealed that like rat anti-bovine PD-L1 antibody 4G12, rat-bovine
chimeric anti-bovine PD-L1 antibody ch4G12 is capable of inhibiting
bovine PD-1/PD-L1 binding in a concentration dependent manner.
Biological Activity Test Using Rat-Bovine Chimeric Anti-Bovine
PD-L1 Antibody
(1) Effect on Cell Proliferation (FIG. 25)
[0181] In order to confirm that bovine PD-1/PD-L1 binding
inhibition by rat-bovine chimeric anti-PD-L1 antibody activates
lymphocytes, a biological activity test was performed using cell
proliferation as an indicator. Briefly, bovine PBMCs isolated from
peripheral blood of healthy cattle were suspended in PBS to give a
concentration of 10.times.10.sup.6 cells/ml, and reacted with
carboxyfluorescein succinimidyl ester (CFSE) at room temperature
for 20 min. After washing twice with RPMI 1640 medium
(Sigma-Aldrich) containing 10.degree./o inactivated fetal bovine
serum (Cell Culture Technologies), antibiotics (streptomycin 200
.mu.g/ml, penicillin 200 U/ml) (Life Technologies) and 0.01%
L-glutamine (Life Technologies), the PBMCs were reacted with
anti-bovine CD3 mouse antibody (WSU Monoclonal Antibody Center) at
4.degree. C. for 30 min. After washing, the PBMCs were reacted with
anti-mouse IgG1 microbeads (Miltenyi Biotec) at 4.degree. C. for 15
min, followed by isolation of CD3-positive T cells using
autoMACS.TM. Pro(Miltenyi Biotec). To the isolated CD3-positive T
cells, anti-bovine CD3 mouse antibody (WSU Monoclonal Antibody
Center) and anti-bovine CD28 mouse antibody (Bio-Rad) were added.
Then, the cells were co-cultured with bovine PD-L1 expressing cells
(CD3-positive T cells: bovine PD-L1 expressing cells=10:1) in the
presence or absence of 10 .mu.g/ml of rat-bovine chimeric
anti-bovine PD-L1 antibody ch4G12. As a control for antibodies,
serum-derived bovine IgG (Sigma-Aldrich) was used; as a control for
PD-L1 expressing cells, EGFP expressing cells transfected with
pEGFP-N2 were used. After a 6-day coculture, cells were harvested
and reacted with anti-bovine CD4 mouse antibody and anti-bovine CD8
mouse antibody (Bio-Rad) at room temperature for 30 min. For the
labeling of antibodies, Zenon Mouse IgG1 Labeling Kits (Life
Technologies) or Lightning-Link Kit (Innova Biosciences) was used.
For analysis, FACS Verse (BD Biosciences) was used. For washing
operation after culturing and dilution of antibody, PBS
supplemented with 1% bovine serum albumin (Sigma-Aldrich) was
used.
[0182] The experimental results are shown in FIG. 25. Proliferation
of CD4-positive and CD8-positive T cells was significantly
suppressed by co-culture with bovine PD-L1 expressing cells. It was
revealed that rat-bovine chimeric anti-bovine PD-L1 antibody ch4G12
inhibits this suppression in CD4-positive T cells.
(2) Effect on IFN-.gamma. Production (FIG. 26)
[0183] In order to confirm that bovine PD-1/PD-L1 binding
inhibition by rat-bovine chimeric anti-PD-L1 antibody activates
lymphocytes, a biological activity test was performed using
IFN-.gamma. production as an indicator. Briefly, PBMCs isolated
from peripheral blood of BLV-infected cattle were suspended in RPMI
medium (Sigma-Aldrich) containing 10% inactivated fetal bovine
serum (Cell Culture Technologies), antibiotics (streptomycin 200
.mu.g/ml, penicillin 200 U/ml) (Life Technologies) and 0.01%
L-glutamine (Life Technologies) to give a concentration of
4.times.10.sup.6 cells/ml. To the PBMCs, 10 .mu.g/ml of rat
anti-bovine PD-L1 antibody 4G12 or rat-bovine chimeric anti-bovine
PD-L1 antibody ch4G12, and 2% BLV-infected fetal lamp kidney cell
(FLK-BLV) culture supernatant were added; culturing was then
performed at 37.degree. C. under 5% CO.sub.2 for 6 days. As control
antibodies, serum-derived rat IgG (Sigma-Aldrich) and serum-derived
bovine IgG (Sigma-Aldrich) were used. After a 6-day culture, a
culture supernatant was collected, and IFN-.gamma. production was
measured with Bovine IFN-.gamma. ELISA Kit (BETYL). For each
washing operation in ELISA, Auto Plate Washer BIO WASHER 50 (DS
Pharma Biomedical) was used. Absorbance was measured with
Microplate Reader MTP-650FA (Corona Electric).
[0184] The experimental results are shown in FIG. 26. It was
revealed that rat-bovine chimeric anti-bovine PD-L1 antibody ch4G12
increases bovine PBMCs' IFN-.gamma. response to BLV antigen in the
same manner as rat anti-bovine PD-L1 antibody 4G12 (n=10).
CDR Analysis of Rat Anti-Bovine PD-L1 Antibody
[0185] The complementarity-determining regions (CDRs) of rat
anti-bovine PD-L1 antibody 4G12 were determined using NCBI IGBLAST
(http://www.ncbi.nlm.nih.gov/igblast/). The results are shown in
FIG. 19.
Inoculation Test on Cattle
[0186] Established rat-bovine chimeric anti-bovine PD-L1 antibody
ch4G12 (about 26W mg; 1 mg/kg) was intravenously administered into
experimentally BLV-infected calf (Holstein, male, 7 months old, 267
kg). Blood samples were collected chronologically from the infected
calf, followed by isolation of PBMCs by density gradient
centrifugation.
(1) Cell Proliferation Response of T Cells to BLV Antigen (FIG.
27)
[0187] Bovine PBMCs were suspended in PBS and reacted with CFSE at
room temperature for 20 min. After washing twice with RPMI 1640
medium (Sigma-Aldrich) containing 10% inactivated fetal bovine
serum (Cell Culture Technologies), antibiotics (streptomycin 200
.mu.g/ml, penicillin 200 U/ml) (Life Technologies) and 0.01%
L-glutamine (Life Technologies), the cell concentration was
adjusted to 4.times.10.sup.6 cells/ml using the same medium.
Culture supernatant of 2% BLV-infected fetal lamp kidney cells
(FLK-BLV) was added to the PBMCs, which were then cultured at
37.degree. C. under 5% CO.sub.2 for 6 days. As a control, culture
supernatant of 2% BLV-not-infected fetal lamp kidney cells (FLK)
was used. After a 6-day culture, PBMCs were collected and reacted
with anti-bovine CD4 mouse antibody, anti-bovine CD8 mouse antibody
and anti-bovine IgM mouse antibody (Bio-Rad) at 4.degree. C. for 20
min. For the labeling of antibodies, Zenon Mouse IgG1 Labeling Kits
(Life Technologies) or Lightning-Link Kit (Innova Biosciences) was
used. For analysis, FACS Verse (BD Biosciences) was used. For every
washing operation and dilution of antibody. PBS supplemented with
1% bovine serum albumin (Sigma-Aldrich) was used.
[0188] The experimental results are shown in FIG. 27. As a result
of antibody administration, BLV-specific cell proliferation
response of CD4-positive T cells increased compared to the response
before administration.
(2) Changes in the BLV Provirus Load (FIG. 28)
[0189] DNA was extracted from isolated bovine PBMCs using Wizard
DNA Purification kit (Promega). The concentration of the extracted
DNA was quantitatively determined, taking the absorbance (260 nm)
measured with Nanodrop 8000 Spectrophotometer (Thermo Fisher
Scientific) as a reference. In order to measure the BLV provirus
load in PBMCs, real time PCR was performed using Cycleave PCR
Reaction Mix SP (TaKaRa) and Probe/Primer/Positive control for
bovine leukemia virus detection (TaKaRa). Light Cycler 480 System
II (Roche Diagnosis) was used for measurement.
[0190] The experimental results are shown in FIG. 28. The BLV
provirus load significantly decreased until the end of test period
compared to the load before administration.
[0191] All publications, patents and patent applications cited
herein are incorporated herein by reference in their entirety.
INDUSTRIAL APPLICABILITY
[0192] The anti-PD-L1 antibody of the present invention is
applicable to prevention and/or treatment of cancers and infections
in animals.
TABLE-US-00012 SEQUENCE LISTING FREE TEXT <SEQ ID NO: 1> SEQ
ID NO: 1 shows the amino acid sequence of the light chain variable
region (VL) of rat anti-bovine PD-L1antibody.
MESQTHVLISLLLSVSGTYGDIAITQSPSSVAVSVGETVTLSCKSSQSLLYSENQKDYL
GWYQQKPGQTPKPLIYWATNRHTGVPDRFTGSGSGTDFTLIISSVQAEDLADYYCGQ
YLVYPFTFGPGTKLELK <SEQ ID NO: 2> SEQ ID NO: 2 shows the amino
acid sequence of the heavy chain variable region (VH) of rat
anti-bovine PD-L1antibody.
MGWSQIILFLVAAATCVHSQVQLQQSGAELVKPGSSVKISCKASGYTFTSNFMHWVK
QQPGNGLEWIGWIYPEYGNTKYNQKFDGKATLTADKSSSTAYMQLSSLTSEDSAVYF
CASEEAVISLVYWGQGTLVTVSS <SEQ ID NO: 3> SEQ ID NO: 3 shows the
amino acid sequence of the light chain constant region (CL) of a
canine antibody.
QPKASPSVTLFPPSSEELGANKATLVCLISDFYPSGVTVAWKASGSPVTQGVETTKPSK
QSNNKYAASSYLSLTPDKWKSHSSFSCLVTHEGSTVEKKVAPAECS <SEQ ID NO: 4>
SEQ ID NO: 4 shows the amino acid sequence of the heavy chain
constant region (CH) of a canine antibody.
ASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVSWNSGSLTSGVHTFPSVLQS
SGLYSLSSTVTVPSSRWPSETFTCNVVHPASNTKVDKPVPKESTCKCISPCPVPESLGG
PSVFIFPPKPKDILRITRTPEITCVVLDLGREDPEVQISWFVDGKEVHTAKTQPREQQFN
STYRVVSVLPIEHQDWLTGKEFKCRVNHIGLPSPIERTISKARGQAHQPSVYVLPPSPK
ELSSSDTVTLTCLIKDFFPPEIDVEWQSNGQPEPESKYHTTAPQLDEDGSYFLYSKLSV
DKSRWQQGDTFTCAVMIHEALQNHYTDLSLSHSPGK <SEQ ID NO: 5> SEQ ID
NO: 5 shows the nucleotide sequence of the VL of rat anti-bovine
PD-L1 antibody.
ATGGAATCACAGACGCATGTCCTCATTTCCCTTCTGCTCTCGGTATCTGGTACCTAT
GGGGACATTGCGATAACCCAGTCTCCATCCTCTGTGGCTGTGTCAGTAGGAGAGA
CGGTCACTCTGAGCTGCAAGTCCAGTCAGAGTCTTTTATACAGTGAAAACCAAAA
GGACTATTTGGGCTCGGTACCAGCAGAAACCAGGGCAGACTCCTAAACCCCTTATCT
ACTGGGCAACCAACCGGCACACTGGGGTCCCTGATCGCTTCACAGGTAGTGGATC
CGGGACAGACTTCACTCTGATCATCAGCAGTGTGCAGGCTGAAGACCTGGCTGAT
TATTACTGTGGGCAGTACCTTGTCTATCCGTTCACGTTTGGACCTGGGACCAAGCT GGAACTGAAA
The nucleotide sequence of SEQ ID NO: 5 after codon optimization is
shown in <SEQ ID NO: 15>.
ATGGAATCTCAAACTCATGTTTTGATTTCATTACTTCTGAGTGTTTCCGGAACCTAC
GGTGATATCGCTATCACTCAATCTCCCTCCTCTGITGCTGTGTCTGTGGGCGAAAC
CGTTACCCTGTCCTGCAAGTCCAGTCAGTCTCTTCTCTACTCCGAGAATCAAAAGG
ACTACCTGGGCTGGTACCAACAGAAGCCCGGCCAGACCCCAAAGCCACTGATATA
CTGGGCAACCAACAGGCACACCGGAGTGCCCGACAGGTTCACAGGCAGTGGATC
TGGCACCGACTTTACCTTGATCATTTCAAGCGTGCAGGCTGAAGATCTGGCCGACT
ACTACTGTGGTCAGTATCTGGTGTATCCTTTCACTTTCGGGCCAGGGACAAAATTG GAATTGAAG
<SEQ ID NO: 6> SEQ ID NO: 6 shows the nucleotide sequence of
the VH of rat anti-bovine PD-L1 antibody.
ATGGGATGGAGCCAGATCATCCTCTTTCTGGTGGCAGCAGCTACATGTGTTCACTC
CCAGGTACAGCTGCAGCAATCTGGGGCTGAATTAGTGAAGCCTGGGTCCTCAGTG
AAAATTTCCTGCAAGGCTTCTGGCTACACCTTCACCAGTAACTTTATGCACTGGGT
AAAGCAGCAGCCTGGAAATGGCCTTGAGTGGATTGGGTGGATTTATCCTGAATATG
GTAATACTAAGTACAATCAAAAGTTCGATGGGAAGGCAACACTCACTGCAGACAA
ATCCTCCAGCACAGCCTATATGCAGCTCAGCAGCCTGACATCTGAGGACTCTGCAG
TCTATTTCTGTGCAAGTGAGGAGGCAGTTATATCCCTTGTTTACTGGGGCCAAGGC
ACTCTGGTCACTGTCTCTTCA The nucleotide sequence of SEQ ID NO: 6 after
codon optimization is shown in <SEQ ID NO: 16>.
ATGGGTTGGTCTCAAATTATCTTGTTTTTGGTTGCTGCAGCCACTTGTGTTCATTCT
CAGGTGCAGCTGCAACAAAGCGGCGCAGAACTGGTGAAACCTGGCAGCAGCGTG
AAAATATCTTGTAAGGCCAGCGGATATACTTTCACCTCCAATTTCATGCATTGGGTC
AAACAGCAGCCCGGCAACGGACTCGAGTGGATCGGCTGGATCTACCCCGAGTATG
GCAACACAAAATATAACCAAAAATTTGATGAAAGGCTACCCTGACTGCCGATAA
GTCCTCCAGCACCGCATACATGCAACTCTCCTCCCTGACCTCCGAGGATAGCGCTG
TCTACTTCTGTGCTTCCGAAGAGGCTGTCATATCCTTGGTCTATTGGGGCCAAGGA
ACTCTGGTGACCGTCTCATCT <SEQ ID NO: 7> SEQ ID NO: 7 shows the
nucleotide sequence of the CL of a canine antibody.
CAGCCCAAGGCCTCCCCCTCGGTCACACTCTTCCCGCCCTCCTCTGAGGAGCTCG
GCGCCAACAAGGCCACCCTGGTGTGCCTCATCAGCGACTTCTACCCCAGCGGCGT
GACGGTGGCCTGGAAGGCAAGCGGCAGCCCCGTCACCCAGGGCGTGGAGACCAC
CAAGCCCTCCAAGCAGAGCAACAACAAGTACGCGGCCAGCAGCTACCTGAGCCT
GACGCCTGACAAGTGGAAATCTCACAGCAGCTTCAGCTGCCTGGTCACGCACGA
GGGGAGCACCGTGGAGAAGAAGGTGGCCCCCGCAGAGTGCTCTTAG The nucleotide
sequence of SEQ ID NO: 7 after codon optimization is shown in
<SEQ ID NO: 17>.
CAGCCCAAAGCCTCTCCCAGCGTCACCCTCTTCCCACCTTCCAGTGAGGAGCTGG
GGGCAAACAAAGCCACTTTGGTGTGTCTCATCTCCGATTTTTACCCCTCCGGGGTC
ACAGTCGCATGGAAGGCCTCCGGATCCCCTGTGACACAGGGAGTGGAGACAACA
AAACCTAGCAAGCAGAGTAACAAATAAGTATGCCGCCTCAAGCTATCAGCCTTAC
TCCTGATAAGTGGAAGTCACATAGCAGTTTTAGTTGCCTCGTAACACATGAGGGTT
CAACTGTGGAGAAAAAAGTAGCTCCAGCTGAGTGCTCATGA <SEQ ID NO: 8> SEQ
ID NO: 8 is the nucleotide sequence of the CH of a canine antibody.
GCCTCCACCACGGCCCCCTCGGTTTTCCeCACTGGCCCCCAGCTGCGGGTCCACTT
CCGGCTCCACGGTGGCCCTGGCCTGCCTGGTGTCAGGCTACTTCCCCGAGCCTGT
AACTGTGTCCTGGAATTCCGGCTCCTTGACCAGCGGTGTGCACACCTTCCCGTCC
GTCCTGCAGTCCTCAGGGCTCTACTCCCTCAGCAGCACGGTGACAGTGCCCTCCA
GCAGGTGGCCCAGCGAGACCTTCACCTGCAACGTGGTCCACCCGGCCAGCAACA
CTAAAGTAGACAAGCCAGTGCCCAAAGAGTCCACCTGCAAGTGTATATCCCCATG
CCCAGTCCCTGAATCACTGGGAGGGCCTTCGGTCTTCATCTTTCCCCCGAAACCCA
AGGACATCCTCAGGATTACCCGAACACCCGAGATCACCTGTGTGGTGTTAGATCTG
GGCCGTGAGGACCCTGAGGTGCAGATCAGCTGGTTCGTGGATGGTAAGGAGGTG
CACACAGCCAAGACGCAGCCTCGTGAGCAGCAGTTCAACAGCACCTACCGTGTG
GTCAGCGTCCTCCCCATTGAGCACCAGGACTGGCTCACCGGAAAGGAGTTCAAGT
GCAGAGTCAACCACATAGGCCTCCCGTCCCCCATCGAGAGGACTATCTCCAAAGC
CAGAGGGCAAGCCCATCAGCCCAGTGTGTATGTCCTGCCACCATCCCCAAAGGAG
TTGTCATCCAGTGACACGGTCACCCTGACCTGCCTGATCAAAGACTTCTTCCCACC
TGAGATTGATGTGGAGTGGCAGAGCAATGGACAGCCGGAGCCCGAGAGCAAGTA
CCACACGACTGCGCCCCAGCTGGACGAGGACGGGTCCTACTTCCTGTACAGCAAG
CTCTCTGTGGACAAGAGCCGCTGGCAGCAGGGAGACACCTTCACATGTGCGGTGA
TGCATGAAGCTCTACAGAACCACTACACAGATCTATCCTCTCCCATTCTCCGGGT AAATGA The
nucleotide sequence of SEQ ID NO: 8 after codon optimization is
shown in <SEQ ID NO: 18>.
GCTAGCACAACCGCTCCCTCCGTTTTTCCCCTCGCCCCATCCTGCGGGTCAACCAG
CGGATCCACCGTCGCTCTGGCTTGTCTGGTGTCAGGATACTTCCCCGAGCCTGTCA
CCGTTTCTTGGAATAGCGGCAGCCTTACTTCCGGCGTGCATACCTTCCCTAGCGTG
CTTCAGTCCTCCGGTCTGTATTCCCTCAGCTCCACCGTAACTGTCCCAAGCTCAAG
GTGGCCCTCTGAGACATTTACCTGCAATGTGGTCCATCCTGCTTCAAATACCAAAG
TGGACAAGCCCGTCCCAAAAGAGTCTACCTGCAAATGTATCAGTCCTTGTCCCGT
GCCCGAGTCTCTGGGCGGACCCTCAGTCTTTATCTTCCCACCCAAGCCAAAGGAC
ATATTGCGCATTACACGGACACCCGAAATCACCTGTGTTGTGTTGGATCTCGGCCG
GGAAGATCCTGAGGTGCAGATTAGTTGGTTTGTTGATGGCAAGGAGGTGCACACA
GCAAAAACACAGCCCAGAGAACAGCAGTTCAACAGTACTTATAGAGTAGTGAGT
GTGTTGCCTATAGAGCATCAGGACTGGCTGACAGGCAAAGAATTCAAATGTAGGG
TTAACCACATTGGCCTCCCTAGTCCAATCGAGAGGACAATCTCTAAAGCCCGAGG
CCAGGCTCATCAGCCTTCTGTGTACGTTCTGCCTCCTAGTCCTAAGGAACTGTCTT
CTTCAGACACAGTAACACTCACTTGCCTGATTAAGGACTTTTTTCCTCCAGAGATT
GATGTGGAATGGCAGTCTAACGGGCAGCCAGAGCCAGAATCTAAGTACCACACTA
CTGCACCACAGCTGGATGAGGATGGGTCTTACTTCCTGTACAGTAAGCTGAGTGT
GGACAAGTCTCGATGGCAGCAGGGGGATACTTTTACTTGCGCAGTAATGCACGAA
GCATTGCAGAACCACTACACTGACCTGTCACTTAGTCACTCACCAGGGAAGTAA <SEQ ID
NO: 9> SEQ ID NO: 9 shows the amino acid sequence of a chimeric
light chain consisting of the VL of rat anti-bovine Pd-L1 antibody
and the CL of a canine antibody.
MESQTHVLISLLLSVSGTYGDIAITQSPSSVAVSVGETVTLSCKSSQSLLYSENQKDYL
GWYQQKPGQTPKPLIYWATNRHTGVPDRFTGSGSGTDFTLIISSVQAEDLADYYCGQ
YLVYPFTFGPGTKLELKQPKASPSVTLFPPSSEELGANKATLVCLISDFYPSGVTVAWK
ASGSPVTQGVETTKPSKQSNNKYAASSYLSLTPDKWKSHSSFSCLVTHEGSTVEKKV APAECS
<SEQ ID NO: 10> SEQ ID NO: 10 shows the amino acid sequence
of a chimeric heavy chain consisting of the VH of rat anti-bovine
PD-L1 antibody and the CH of a canine antibody.
MGWSQIILFLVAAAFCVHSQVQLQQSGAELVKPGSSVKISCKASGYTFTSNFMHWVK
QQPGNGLEWIGWIYPEYGNTKYNQKFDGKATLTADKSSSTAYMQLSSLTSEDSAVYF
CASEEAVISLVYWGQGTLVTVSSASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEP
VTVSWNSGSLTSGVHTFPSVLQSSGLYSLSSTVTVPSSRWPSETFTCNVVHPASNTKV
DKPVPKESTCKCISPCPVPESLGGPSVFIFPPKPKDILRITRTPEITCVVLDLGREDPEVQ
ISWFVDGKEVHTAKTQPREQQFNSTYRVVSVLPIEHQDWLTGKEFKCRVNHIGLPSPI
ERTISKARGQAHQPSVYVLPPSPKELSSSDTVTLTCLIKDFFPPEIDVEWQSNGQPEPES
KYHTTAPQLDEDGSYFLYSKLSVDKSRWQQGDTFTCAVMHEALQNHYTDLSLSHSP GK <SEQ
ID NO: 19> This sequence shows the nucleotide sequence (after
codon optimization) of a chimeric light chain consisting of the VL
of rat anti-bovine PD-L1 antibody and the CL of a canine antibody.
ATGGAATCTCAAACTCATGTTTTGATTTCATTACTTCTGAGTGTTTCCGGAACCTAC
GGTGATATCGCTATCACTCAATCTCCCTCCTCTGTTGCTGTGTCTGTGGGCGAAAC
CGTTACCCTGTCCTGCAAGTCCAGTCAGTCTCTTCTCTACTCCGAGAATCAAAAGG
ACTACCTGGGCTGGTACCAACAGAAGCCCGGCCAGACCCCAAAGCCACTGATATA
CTGGGCAACCAACAGGCACACCGGAGTGCCCGACAGGTTCACAGGCAGTGGATC
TGGCACCGACTTTACCTTGATCATTTCAAGCGTGCAGGCTGAAGATCTGGCCGACT
ACTACTGTGGTCAGTATCTGGTGTATCCTTTCACTTTCGGGCCAGGGACAAAATTG
GAATTGAAGCAGCCCAAAGCCTCTCCCAGCGTCACCCTCTTCCCACCTTCGAGTG
AGGAGCTGGGGGCAAACAAAGCCACTTTGGTGTGTCTCATCTCCGATTTTTACCC
CTCCGGGGTCACAGTCGCATGGAAGGCCTCCGGATCCCCTGTGACACAGGGAGTG
GAGACAACAAAACCTAGCAAGCAGAGTAACAATAAGTATGCCGCCTCAAGCTATC
TCAGCCTTACTCCTGATAAGTGGAAGTCACATAGCAGTTTTAGTTGCCTCGTAACA
CATGAGGGTTCAACTGTGGAGAAAAAAGTAGCTCCAGCTGAGTGCTCATGA <SEQ ID NO:
20> SEQ ID NO: 20 shows the nucleotide sequence (after codon
optimization) of a chimeric heavy chain consisting of the VH of rat
anti-bovine PD-L1 antibody and the CH of a canine antibody.
ATGGGTTGGTCTCAAATTATCTTGTTTTTGGTTGCTGCAGCCACTTGTGTTCATTCT
CAGGTGCAGCTGCAACAAAGCGGCGCAGAACTGGTGAAACCTGGCAGCAGCGTG
AAAATATCTTGTAAGGCCAGCGGATATACTTTCACCTCCAATTTCATGCATTGGGTC
AAACAGCAGCCCGGCAACGGACTCGAGTGGATCGGCTGGATCTACCCCGAGTATG
GCAACACAAAATATAACCAAAAATTTGATGGAAAGGCTACCCTGACTGCCGATAA
GTCCTCCAGCACCGCATACATGCAACTCTCCTCCCTGACCTCCGAGGATAGCGCTG
TCTACTTCTGTGCTTCCGAAGAGGCTGTCATATCCTTGGTCTATTGGGGCCAAGGA
ACTCTGGTGACCGTCTCATCTGCTAGCACAACCGCTCCCTCCGTTTTTCCCCTCGC
CCCATCCTGCGGGTCAACCAGCGGATCCACCGTCGCTCTGGCTTGTCTGGTGTCA
GGATACTTCCCCGAGCCTGTCACCGTTTCTTGGAATAGCGGCAGCCTTACTTCCGG
CGTGCATACCTTCCCTAGCGTGCTTCAGTCCTCCGGTCTGTATTCCCTCAGCTCCAC
CGTAACTGTCCCAAGCTCAAGGTGGCCCTCTGAGACATTTACCTGCAATGTGGTCC
ATCCTGCTTCAAATACCAAAGTGGACAAGCCCGTCCCAAAAGAGTCTACCTGCAA
ATGTATCAGTCCTTGTCCCGTGCCCGAGTCTCTGGGCGGACCCTCAGTCTTTATCTT
CCCACCCAAGCCAAAGGACATATTGCGCATTACACGGACACCCGAAATCACCTGT
GTTGTGTTGGATCTCGGCCGGGAAGATCCTGAGGTGCAGATTAGTTGGTTTGTTGA
TGGCAAGGAGGTGCACACAGCAAAAACACAGCCCAGAGAACAGCAGTTCAACA
GTACTTATAGAGTAGTGAGTGTGTTGCCTATAGAGCATCAGGACTGGCTGACAGGC
AAAGAATTCAAATGTAGGGTTAACCACATTGGCCTCCCTAGTCCAATCGAGAGGA
CAATCTCTAAAGCCCGAGGCCAGGCTCATCAGCCTTCTGTGTACGTTCTGCCTCCT
AGTCCTAAGGAACTGTCTTCTTCAGACACAGTAACACTCACTTGCCTGATTAAGG
ACTTTTTTCCTCCAGAGATTGATGTGGAATGGCAGTCTAACGGGCAGCCAGAGCC
AGAATCTAAGTACCACACTACTGCACCACAGCTGGATGAGGATGGGTCTTACTTCC
TGTACAGTAAGCTGAGTGTGGACAAGTCTCGATGGCAGCAGGGGGATACTTTTAC
TTGCGCAGTAATGCACGAAGCATTGCAGAACCACTACACTGACCTGTCACTTAGT
CACTCACCAGGGAAGTAA <SEQ ID NO: 11> SEQ ID NO: 11 shows the
amino acid sequence of the CL of a human antibody.
TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTE
QDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC <SEQ ID NO:
12> SEQ ID NO: 12 shows the amino acid sequence of the CH
(CH1--CH3) of a human antibody (IgG4 variant 1).
STKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS
GLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEELGGP
SVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREE
QFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTL
PPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSR
LTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK <SEQ ID NO: 13> SEQ ID
NO: 13 shows the nucleotide sequence of the CL of a human antibody.
ACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATC
TGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAA
GTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCA
CAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGA
GCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGG
GCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG <SEQ ID NO:
14> SEQ ID NO: 14 shows the nucleotide sequence of the CH
(CH1--CH3) of a human antibody (IgG4 variant 1).
TCCACCAAGGGCCCATCCGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCG
AGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGA
CGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGT
CCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGC
AGCTTGGGCACGAAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACC
AAGGTGGACAAGAGAGTTGAGTCCAAATATGGTCCCCCATGCCCATCATGCCCAG
CACCTGAGTTCCTGGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGA
CACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGC
CAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATA
ATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTACCGTGTGGTCA
GCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCA
AGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCTCCAAAGCCAA
AGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGAT
GACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGA
CATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCAC
GCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTAACCGTG
GACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGG
CTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCTGGGTAAATGA <SEQ ID
NOS: 21-36> SEQ ID NOS: 21-36 show the nucleotide sequences of
primers cPD-1 inner F, cPD-1 inner R, cPD-L1 inner F, cPD-L1 inner
R, cPD-1 5' GSP, cPD-1 3' GSP, cPD-L1 5' GSP, cPD-L1 3' GSP,
cPD-1-EGFP F, cPD-1-EGFP R, cPD-L1-EGFP F, cPD-L1-EGFP R, cPD-1-Ig,
cPD- 1-Ig R, cPD-L1-Ig F and cPD-L1-Ig R in this order. <SEQ ID
NO: 37> SEQ ID NO: 37 shows the amino acid sequence
(QSLLYSENQKDY) of CDR1 of the VL of rat anti-bovine PD-L1 antibody
4G12. <SEQ ID NO: 38> SEQ ID NO: 38 shows the amino acid
sequence (QSLLYSENQKDY) of CDR3 of the VL of rat anti-bovine PD-L1
antibody 4G12, <SEQ ID NO: 39> SEQ ID NO: 39 shows the amino
acid sequence (GYTFTSNF) of CDR1 of the VH of rat anti-bovine PD-L1
antibody 4G12. <SEQ ID NO: 40> SEQ ID NO: 40 shows the amino
acid sequence (IYPEYGNT) of CDR2 of the VH of rat anti-bovine PD-L1
antibody 4G12. <SEQ ID NO: 41> SEQ ID NO: 41 shows the amino
acid sequence (ASEEAVISLVY) of CDR3 of the VH of rat anti-bovine
PD-L1 antibody 4G12. <SEQ ID NO: 42> SEQ ID NO: 42 shows the
amino acid sequence of the CH (CH1--CH3) of ovine antibody (IgG1).
<SEQ ID NO: 43> SEQ ID NO: 43 shows the nucleotide sequence
of the CH (CH1--CH3) of ovine antibody (IgG1). <SEQ ID NO:
44> SEQ ID NO: 44 he amino acid sequence of the CH (CH1--CH3) of
ovine antibody (IgG2). <SEQ ID NO: 45> SEQ ID NO: 45 shows
the nucleotide sequence of the CH (CH1---CH3) of ovine antibody
(IgG2). <SEQ ID NO: 46> SEQ ID NO: 46 shows the amino acid
sequence of the light chain (Ig kappa(CK)) constant region of an
ovine antibody. <SEQ ID NO: 47> SEQ ID NO: 47 shows the
nucleotide sequence of the light chain (Ig kappa(CK)) constant
region of an ovine antibody. <SEQ ID NO: 48> SEQ ID NO: 48
shows the amino acid sequence of the light chain (Ig lambda(CL))
constant region of an ovine antibody.
<SEQ ID NO: 49> SEQ ID NO: 49 shows the nucleotide sequence
of the light chain (Ig lambda(CL)) constant region of an ovine
antibody. <SEQ ID NO: 50> SEQ ID NO: 50 shows the amino acid
sequence of the CH (CH1--CH3) of porcine antibody (IgG1.sup.a).
<SEQ ID NO: 51> SEQ ID NO: 51 shows the nucleotide acid
sequence of the CH (CH1--CH3) of porcine antibody (IgG1.sup.a).
<SEQ ID NO: 52> SEQ ID NO: 52 shows the amino acid sequence
of the CH (CH1--CH3) of porcine antibody (IgG1.sup.b). <SEQ ID
NO: 53> SEQ ID NO: 53 shows the nucleotide sequence of the CH
(CH1--CH3) of porcine antibody (IgG1.sup.b). <SEQ ID NO: 54>
SEQ ID NO: 54 shows the amino acid sequence of the CH (CH1--CH3) of
porcine antibody (IgG2.sup.a). <SEQ ID NO: 55> SEQ ID NO: 55
shows the nucleotide sequence of the CH (CH1--CH3) of porcine
antibody (IgG2.sup.a). <SEQ ID NO: 56> SEQ ID NO: 56 shows
the amino acid sequence of the CH (CH1--CH3) of porcine antibody
(IgG2.sup.b). <SEQ ID NO: 57> SEQ ID NO: 57 shows the
nucleotide sequence of the CH (CH1--CH3) of porcine antibody
(IgG2.sup.b). <SEQ ID NO: 58> SEQ ID NO: 58 shows the amino
acid sequence of the CH (CH1--CH3) of porcine antibody (IgG3).
<SEQ ID NO: 59> SEQ ID NO: 59 shows the nucleotide sequence
of the CH (CH1--CH3) of porcine antibody (IgG3). <SEQ ID NO:
60> SEQ ID NO: 60 shows the amino acid sequence of the CH
(CH1--CH3) of porcine antibody (IgG4.sup.a). <SEQ ID NO: 61>
SEQ ID NO: 61 shows the nucleotide sequence of the CH (CH1--CH3) of
porcine antibody (IgG4.sup.a). <SEQ ID NO: 62> SEQ ID NO: 62
shows the amino acid sequence of the CH (CH1--CH3) of porcine
antibody (IgG4.sup.b). <SEQ ID NO: 63> SEQ ID NO: 63 shows
the nucleotide sequence of the CH (CH1--CH3) of porcine antibody
(IgG4.sup.b). <SEQ ID NO: 64> SEQ ID NO: 64 shows the amino
acid sequence of the CH (CH1--CH3) of porcine antibody
(IgG5.sup.a). <SEQ ID NO: 65> SEQ ID NO: 65 shows the
nucleotide sequence of the CH (CH1--CH3) of porcine antibody
(IgG5.sup.a). <SEQ ID NO: 66> SEQ ID NO: 66 shows the amino
acid sequence of the CH (CH1--CH3) of porcine antibody
(IgG5.sup.b). <SEQ ID NO: 67> SEQ ID NO: 67 shows the
nucleotide sequence of the CH (CH1--CH3) of porcine antibody
(IgG5.sup.b). <SEQ ID NO: 68> SEQ ID NO: 68 shows the amino
acid sequence of the CH (CH1--CH3) of porcine antibody
(IgG6.sup.a). <SEQ ID NO: 69> SEQ ID NO: 69 shows the
nucleotide sequence of the CH (CH1--CH3) of porcine antibody
(IgG6.sup.a). <SEQ ID NO: 70> SEQ ID NO: 70 shows the amino
acid sequence of the CH (CH1--CH3) of porcine antibody
(IgG6.sup.b). <SEQ ID NO: 71> SEQ ID NO: 71 shows the
nucleotide sequence of the CH (CH1--CH3) of porcine antibody
(IgG6.sup.b). <SEQ ID NO: 72> SEQ ID NO: 72 shows the amino
acid sequence of the CH (CH1--CH3) of a water buffalo antibody
(estimated to be IgG1). <SEQ ID NO: 73> SEQ ID NO: 73 shows
the nucleotide sequence of the CH (CH1--CH3) of a water buffalo
antibody (estimated to be IgG1). <SEQ ID NO: 74> SEQ ID NO:
74 shows the amino acid sequence of the CH (CH1--CH3) of a water
buffalo antibody (estimated to be IgG2). <SEQ ID NO: 75> SEQ
ID NO: 75 shows the nucleotide sequence of the CH (CH1--CH3) of a
water buffalo antibody (estimated to be IgG2). <SEQ ID NO:
76> SEQ ID NO: 76 shows the amino acid sequence of the CH
(CH1--CH3) of a water buffalo antibody (estimated to be IgG3).
<SEQ ID NO: 77> SEQ ID NO: 77 shows the nucleotide sequence
of the CH (CH1--CH3) of a water buffalo antibody (estimated to be
IgG3). <SEQ ID NO: 78> SEQ ID NO: 78 shows the amino acid
sequence of the light chain (estimated to be Ig lambda) constant
region (CL) of a water buffalo antibody. <SEQ ID NO: 79> SEQ
ID NO: 79 shows the nucleotide sequence of the light chain
(estimated to be Ig lambda) constant region (CL) of a water buffalo
antibody. <SEQ ID NO: 80> SEQ ID NO: 80 shows the amino acid
sequence of the CH (CH1--CH3) of human antibody (IgG4 variant 2).
<SEQ ID NO: 81> SEQ ID NO: 81 shows the nucleotide sequence
of the CH (CH1--CH3) of human antibody (IgG4 variant 2). <SEQ ID
NO: 82> SEQ ID NO: 82 shows the amino acid sequence of the CH
(CH1--CH3) of human antibody (IgG4 variant 3). <SEQ ID NO:
83> SEQ ID NO: 83 shows the nucleotide sequence of the CH
(CH1-CH3) of human antibody (IgG4 variant 3). <SEQ ID NO: 84>
SEQ ID NO: 84 shows the amino acid sequence of the CH (CH1--CH3) of
bovine antibody (IgG1 variant 1). <SEQ ID NO: 85> SEQ ID NO:
85 shows the amino acid sequence of the CH (CH1--CH3) of bovine
antibody (IgG1 variant 2). <SEQ ID NO: 86> SEQ ID NO: 86
shows the amino acid sequence of the CH (CH1--CH3) of bovine
antibody (IgG1 variant 3). <SEQ ID NO: 87> SEQ ID NO: 87
shows the amino acid sequence of the CH (CH1--CH3) of bovine
antibody (IgG2 variant 1). <SEQ ID NO: 88> SEQ ID NO: 88
shows the amino acid sequence of the CH (CH1--CH3) of bovine
antibody (IgG2 variant 2). <SEQ ID NO: 89> SEQ ID NO: 89
shows the amino acid sequence of the CH (CH1--CH3) of bovine
antibody (IgG2 variant 3). <SEQ ID NO: 90> SEQ ID NO: 90
shows the amino acid sequence of the CH (CH1--CH3) of bovine
antibody (IgG3 variant 1). <SEQ ID NO: 91> SEQ ID NO: 91
shows the amino acid sequence of the CH (CH1--CH3) of bovine
antibody (IgG3 variant 2). <SEQ ID NO: 92> SEQ ID NO: 92
shows the nucleotide sequence of the CH (CH1--CH3) of bovine
antibody (IgG1 variant 1). <SEQ ID NO: 93> SEQ ID NO: 93
shows the nucleotide sequence of the CH (CH1--CH3) of bovine
antibody (IgG1 variant 2). <SEQ ID NO: 94> SEQ ID NO: 94
shows the nucleotide sequence of the CH (CH1--CH3) of bovine
antibody (IgG1 variant 3). <SEQ ID NO: 95> SEQ ID NO: 95
shows the nucleotide sequence of the CH (CH1--CH3) of bovine
antibody (IgG2 variant 1). <SEQ ID NO: 96> SEQ ID NO: 96
shows the nucleotide sequence of the CH (CH1--CH3) of bovine
antibody (IgG2 variant 2). <SEQ ID NO: 97> SEQ ID NO: 97
shows the nucleotide sequence of the CH (CH1--CH3) of bovine
antibody (IgG2 variant 3). <SEQ ID NO: 98> SEQ ID NO: 98
shows the nucleotide sequence of the CH (CH1--CH3) of bovine
antibody (IgG3 variant 1).
<SEQ ID NO: 99> SEQ ID NO: 99 shows the nucleotide sequence
of the CH (CH1--CH3) of bovine antibody (IgG3 variant 2). <SEQ
ID NO: 100> SEQ ID NO: 100 shows the amino acid sequence of the
CL of a bovine antibody (bovine Ig lambda, GenBank: X62917).
QPKSPPSVTLFPPSTEELNGNKATLVCLISDFYPGSVTVVWKADGSTITRNVETTRASK
QSNSKYAASSYLSLTSSDWKSKGSYSCEVTHEGSTVTKTVKPSECS <SEQ ID NO:
101> SEQ ID NO: 101 shows the nucleotide sequence of the CL of a
bovine antibody (bovine Ig lambda, GenBank: X62917).
CAGCCCAAGTCCCCACCCTCGGTCACCCTGTTCCCGCCCTCCACGGAGGAGCTCA
ACGGCAACAAGGCCACCCTGGTGTGTCTCATCAGCGACTTCTACCCGGGTAGCGT
GACCGTGGTCTGGAAGGCAGACGGCAGCACCATCACCCGCAACGTGGAGACCAC
CCGGGCCTCCAAACAGAGCAACAGCAAGTACGCGGCCAGCAGCTACCTGAGCCT
GACGAGCAGCGACTGGAAATCGAAAGGCAGTTACAGCTGCGAGGTCACGCACGA
GGGGAGCACCGTGACGAAGACAGTGAAGCCCTCAGAGTGTTCTTAG The nucleotide
sequence of SEQ ID NO: 101 after codon optimization is shown in
<SEQ ID NO: 104>.
CAGCCTAAGAGTCCTCCTTCTGTAACACTCTTTCCCCCCTCTACCGAGGAACTCAA
CGGCAATAAAGCTACCTTGGTTTGCCTTATTTCTGATTTCTACCCCGGGTCTGTGAC
CGTGGTGTGGAAAGCTGATGGGTCCACCATTACTCGGAATGTGGAAACCACCCGG
GCTTCTAAGCAGTCCAACTCTAAATACGCAGCATCCTCCTATTTGAGTCTTACTAGT
AGTGACTGGAAGTCAAAGGGTAGTTACAGTTGCGAAGTCACACATGAAGGTTCA
ACAGTGACAAAGACAGTCAAGCCCTCAGAGTGCTCATAG <SEQ ID NO: 102> SEQ
ID NO: 102 shows the amino acid sequence of the CH of a bovine
antibody (bovine IgG1, modified from GenBank: X62916). The sites of
mutation are underlined. Amino acid numbers and mutations:
113E.fwdarw.P, 114L.fwdarw.V, 115P.fwdarw.A, 116G.fwdarw.deletion,
209A.fwdarw.S, 210P.fwdarw.S
ASTTAPKVYPLSSCCGDKSSSTVTLGCLVSSYMPEPVTVTWNSGALKSGVHTFPAVL
QSSGLYSLSSMVTVPGSTSGQTFTCNVAHPASSTKVDKAVDPTCKPSPCDCCPPPPVA
GPSVFIFPPKPKDTLTISGTPEVTCVVVDVGHDDPEVKFSWFVDDVEVNTATTKPREE
QFNSTYRVVSALRIQHQDWTGGKEFKCKVHNEGLPSSIVRTISRTKGPAREPQVYVLA
PPQEELSKSTVSLTCMVTSFYPDYIAVEWQRNGQPESEDKYGTTPPQLDADSSYFLYS
KLRVDRNSWQEGDTYTCVVMHEALHNHYTQKSTSKSAGK <SEQ ID NO: 103> SEQ
ID NO: 103 shows the nucleotide sequence (after codon optimization)
of the CH of a bovine antibody (bovine IgG1, modified from GenBank:
X62916). GCTAGCACAACTGCTCCTAAGGTGTACCCCCTGAGCTCTTGCTGCGGCGACAAGT
CTAGCAGCACCGTGACCCTCGGATGCCTCGTCAGCAGCTATATGCCTGAGCCAGTT
ACAGTGACATGGAATTCTGGTGCCCTTAAGTCCGGCGTCCATACCTTCCCTGCTGT
GCTGCAGTCCTCTGGCCTGTACAGTTTGTCCTCTATGGTGACAGTACCCGGTTCCA
CCTCCGGACAGACCTTTACCTGTAATGTGGCTCATCCCGCCTCCTCCACAAAGGTG
GATAAGGCTGTTGACCCTACCTGTAAACCCAGTCCATGCGACTGCTGTCCCCCCCC
TCCAGTTGCCGGACCCTCAGTCTTTATTTTCCCACCCAAACCCAAAGACACCCTGA
CAATCTCTGGAACACCAGAAGTCACCTGCGTCGTCGTGGATGTGGGCCACGACGA
TCCTGAGGTAAAATTCTCATGGTTCGTCGACGATGTGGAAGTGAATACAGCTACTA
CAAAACCTCGCGAAGAGCAGTTTAACTCTACCTATCGAGTGGTTTCTGCTTTGCGG
ATTCAGCATCAGGATTGGACAGGCGGCAAAGAGTTTAAATGTAAAGTCCATAACG
AGGGACTTCCTTCTAGTATCGTGCGCACTATCAGTAGAACTAAAGGGCCTGCTCGG
GAACCTCAGGTGTACGTCCTGGCACCTCCACAGGAAGAGCTGAGTAAGTCTACAG
TTTCTCTGACTTGTATGGTAACATCTTTTTATCCAGATTACATCGCAGTTGAATGGC
AGAGGAACGGGCAGCCAGAGAGTGAGGATAAGTACGGGACTACTCCACCACAGC
TGGACGCAGACTCAAGTTACTTCCTGTACTCAAAGCTGAGGGTTGACAGAAACTC
ATGGCAGGAGGGGGACACTTACACTTGCGTAGTTATGCACGAGGCACTTCACAAC
CACTACACTCAGAAGAGTACTTCAAAGAGTGCAGGGAAGTAA <SEQ ID NO: 105>
SEQ ID NO: 105 shows the amino acid sequence of a chimeric light
chain consisting of the VL of rat anti-bovine PD-L1 antibody and
the CL of a bovine antibody.
MESQTHVLISLLLSVSGTYGDIAITQSPSSVAVSVGETVTLSCKSSQSLLYSENQKDYL
GWYQQKPGQTPKPLIYWATNRHTGVPDRFTGSGSGTDFTLIISSVQAEDLADYYCGQ
YLVYPFTFGPGTKLELKQPKSPPSVTLFPPSTEELNGNKATLVCLISDFYPGSVTVVWK
ADGSTITRNVETTRASKQSNSKYAASSYLSLTSSDWKSKGSYSCEVTHEGSTVTKTV KPSECS
<SEQ ID NO: 106> SEQ ID NO: 106 shows the amino acid sequence
of a chimeric heavy chain consisting of the VH of rat anti-bovine
PD-L1 antibody and the CH of a bovine antibody (bovine IgG1,
modified from GenBank: X62916).
MGWSQIILFLVAAATCVHSQVQLQQSGAELVKPGSSVKISCKASGYTFTSNFMHWVK
QQPGNGLEWIGWIYPEYGNTKYNQKFDGKATLTADKSSSTAYMQLSSLTSEDSAVYF
CASEEAVISLYWGQGTLVTVSSASTTAPKVYPLSSCCGDKSSSTVTLGCLVSSYMPE
PVTVTWNSGALKSGVHTFPAVLQSSGLYSLSSMVTVPGSTSGQTFTCNVAHPASSTKV
DKAVDPTCKPSPCDCCPPPPVAGPSVFIFFPKPKDTLTISGTPEVTCVVVDVGHDDPEV
KFSWFVDDVEVNTATTKPREEQFNSTYRVVSALRIQHQDWTGGKEFKCKVHNEGLP
SSIVRTISRTKGPAREPQVYVLAPPQEELSKSTVSLTCMVTSFYPDYIAVEWQRNGQPE
SEDKYGTTPPQLDADSSYFLYSKLRVDRNSWQEGDTYTCVVMHEALHNHYTQKSTS KSAGK
<SEQ ID NO: 107> This sequence shows the nucleotide sequence
(after codon optimization) of a chimeric light chain consisting of
the VL of rat anti-bovine PD-L1 antibody and the CL of a bovine
antibody. ATGGAATCTCAAACTCATGTTTTGATTTCATTACTTCTGAGTGTTTCCGGAACCTAC
GGTGATATCGCTATCACTCAATCTCCCTCCTCTGTTGCTGTGTCTGTGGGCGAAAC
CGTTACCCTGTCCTGCAAGTCCAGTCAGTCTCTTCTCTACTCCGAGAATCAAAAGG
ACTACCTGGGCTGGTACCAACAGAAGCCCGGCCAGACCCCAAAGCCACTGATATA
CTGGGCAACCAACCAACGGCACACCGGAGTGCCCGACAGGTTCACAGGCAGTGGATC
TGGCACCGACTTTACCTTGATCATTTCAAGCGTGCAGGCTGAAGATCTGGCCGACT
ACTACTGTGGTCAGTATCTGGTGTATCCTTTCACTTTCGGGCCAGGGACAAAACTC
GAGCTCAAACAGCCTAAGAGTCCTCCTTCTGTAACACTCTTTCCCCCCTCTACCGA
GGAACTCAACGGCAATAAAGCTACCTTGGTTTGCCTTATTTCTGATTTCTACCCCG
GGTCTGTGACCGTGGTGTGGAAAGCTGATGGGTCCACCATTACTCGGAATGTGGA
AACCACCCGGGCTTCTAAGCAGTCCAACTCTAAATACGCAGCATCCTCCTATTTGA
GTCTTACTAGTAGTGACTGGAAGTCAAAGGGTAGTTACAGTTGCGAAGTCACACA
TGAAGGTTCAACAGTGACAAAGACAGTCAAGCCCTCAGAGTGCTCATAG <SEQ ID NO:
108> This sequence shows the nucleotide sequence (after codon
optimization) of a chimeric heavy chain consisting of the VH of rat
anti-bovine PD-L1 antibody and the CH of a bovine antibody (bovine
IgG1, modified from GenBank: X62916).
ATGGGGTGGTCCCAGATTATATTGTTCCTCGTCGCCGCCGCCACTTGCGTACACAG
CCAAGTGCAACTTCAACAAAGCGGTGCAGAACTGGTAAAGCCCGGTAGCTCTGT
GAAAATATCCTGTAAAGCCAGTGGCTACACATTTACCAGCAACTTTAGCACTGGG
TGAAGCAACAGCCCGGAAATGGCTTGGAGTGGATTGGCTGGATCTATCCCGAATAT
GGTAACACCAAGTATAATCAGAAGTTCGACGGTAAGGCCACCCTCACCGCCGATA
AGTCATCCTCCACCGCCTATATGCAGCTCAGCAGCCTGACCAGCGAGGATTCCGCT
GTGTACTTCTGTGCCAGCGAAGAGGCTGTGTGATCTCATTGGTGTATTGGGGACAGG
GCACCCTCGTCACCGTGTCCAGCGCTAGCACAACTGCTCCTAAGGTGTACCCCCT
GAGCTCTTGCTGCGGCGACAAGTCTAGCAGCACCGTGACCCTCGGATGCCTCGTC
AGCAGCTATAGCCTGAGCCAGTTACAGTGACATGGAATTCTGGTGCCCTTAAGTC
CGGCGTCCATACCTTCCCTGCTGTGCTGCAGTCCTCTGGCCTGTACAGTTTGTCCT
CTATGGTGACAGTACCCGGTTCCACCTCCGGACAGACCTTTACCTGTAATGTGGCT
CATCCCGCCTCCTCCACAAAGGTGGATAAGGCTGTTGACCCTACCTGTAAACCCA
GTCCATGCGACTGCTGTCCCCCCCCTCCAGTTGCCGGACCCTCAGTCTTTATTTTC
CCACCCAAACCCAAAGACACCCTGACAATCTCTGGAACACCAGAAGTCACCTGC
GTCGTCGTGGATGTGGGCCACGACGATCCTGAGGTAAAATTCTCATGGTTCGTCGA
CGATGTGGAAGTGAATACAGCTACTACAAAACCTCGCGAAGAGCAGTTTAACTCT
ACCTATCGAGTGGTTTCTGCTTTGCGGATTCAGCATCAGGATTGGACAGGCGGCAA
AGAGTTTAAATGTAAAGTCCATAACGAGGGACTTCCTTCTAGTATCGTGCGCACTA
TCAGTAGAACTAAAGGGCCTGCTCGGGAACCTCAGGTGTACGTCCTGGCACCTCC
ACAGGAAGAGCTGAGTAAGTCTACAGTTTCTCTGACTTGTATGGTAACATCTTTTT
ATCCAGATTACATCGCAGTTGAATGGCAGAGGAACGGGCAGCCAGAGAGTGAGG
ATAAGTACGGGACTACTCCACCACAGCTGGACGCAGACTCAAGTTACTTCCTGTA
CTCAAAGCTGAGGGTTGACAGAAACTCATGGCAGGAGGGGGACACTTACACTTG
CGTAGTTATGCACGAGGCACTTCACAACCACTACACTCAGAAGAGTACTTCAAAG
AGTGCAGGGAAGTAA <SEQ ID NOS: 109-132> SEQ ID NOS: 109-132
show the nucleotide sequences of primers ovPD-L1 CDS F, ovPD-L1 CDS
R, poPD-L1 CDS F, poPD-L1 CDS R, buPD-L1 CDS F1, buPD-L1 CDS R1,
buPD-L1 CDS F2, buPD-L1 CDS R2, ovPD-1-EGFP F, ovPD-1-EGFP R,
ovPD-L1-EGFP F, ovPD- L1-EGFP R, poPD-1-EGFP F, poPD-1-EGFP R,
poPD-L1-EGFP F, poPD-L1-EGFP R, ovPD-L1-Ig F, ovPD-L1-Ig R,
poPD-L1-Ig F, poPD-L1-Ig R, cCD80-Ig F, cCD80-Ig R, cPD- L1-His F
and cPD-L1-His R in this order. <SEQ ID NOS: 133-138> SEQ ID
NOS: 133-138 show nucleotide sequences of primers boPD-1-myc F,
boPD-1-myc R, boPD-L1-EGFP F, boPD-L1-EGFP R, boPD-L1-Ig F and
boPD-L1-Ig R in this order.
Sequence CWU 1
1
1381133PRTRattus norvegicus 1Met Glu Ser Gln Thr His Val Leu Ile
Ser Leu Leu Leu Ser Val Ser1 5 10 15Gly Thr Tyr Gly Asp Ile Ala Ile
Thr Gln Ser Pro Ser Ser Val Ala 20 25 30Val Ser Val Gly Glu Thr Val
Thr Leu Ser Cys Lys Ser Ser Gln Ser 35 40 45Leu Leu Tyr Ser Glu Asn
Gln Lys Asp Tyr Leu Gly Trp Tyr Gln Gln 50 55 60Lys Pro Gly Gln Thr
Pro Lys Pro Leu Ile Tyr Trp Ala Thr Asn Arg65 70 75 80His Thr Gly
Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp 85 90 95Phe Thr
Leu Ile Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Asp Tyr 100 105
110Tyr Cys Gly Gln Tyr Leu Val Tyr Pro Phe Thr Phe Gly Pro Gly Thr
115 120 125Lys Leu Glu Leu Lys 1302137PRTRattus norvegicus 2Met Gly
Trp Ser Gln Ile Ile Leu Phe Leu Val Ala Ala Ala Thr Cys1 5 10 15Val
His Ser Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys 20 25
30Pro Gly Ser Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe
35 40 45Thr Ser Asn Phe Met His Trp Val Lys Gln Gln Pro Gly Asn Gly
Leu 50 55 60Glu Trp Ile Gly Trp Ile Tyr Pro Glu Tyr Gly Asn Thr Lys
Tyr Asn65 70 75 80Gln Lys Phe Asp Gly Lys Ala Thr Leu Thr Ala Asp
Lys Ser Ser Ser 85 90 95Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser
Glu Asp Ser Ala Val 100 105 110Tyr Phe Cys Ala Ser Glu Glu Ala Val
Ile Ser Leu Val Tyr Trp Gly 115 120 125Gln Gly Thr Leu Val Thr Val
Ser Ser 130 1353105PRTCanis lupus 3Gln Pro Lys Ala Ser Pro Ser Val
Thr Leu Phe Pro Pro Ser Ser Glu1 5 10 15Glu Leu Gly Ala Asn Lys Ala
Thr Leu Val Cys Leu Ile Ser Asp Phe 20 25 30Tyr Pro Ser Gly Val Thr
Val Ala Trp Lys Ala Ser Gly Ser Pro Val 35 40 45Thr Gln Gly Val Glu
Thr Thr Lys Pro Ser Lys Gln Ser Asn Asn Lys 50 55 60Tyr Ala Ala Ser
Ser Tyr Leu Ser Leu Thr Pro Asp Lys Trp Lys Ser65 70 75 80His Ser
Ser Phe Ser Cys Leu Val Thr His Glu Gly Ser Thr Val Glu 85 90 95Lys
Lys Val Ala Pro Ala Glu Cys Ser 100 1054331PRTCanis lupus 4Ala Ser
Thr Thr Ala Pro Ser Val Phe Pro Leu Ala Pro Ser Cys Gly1 5 10 15Ser
Thr Ser Gly Ser Thr Val Ala Leu Ala Cys Leu Val Ser Gly Tyr 20 25
30Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ser Leu Thr Ser
35 40 45Gly Val His Thr Phe Pro Ser Val Leu Gln Ser Ser Gly Leu Tyr
Ser 50 55 60Leu Ser Ser Thr Val Thr Val Pro Ser Ser Arg Trp Pro Ser
Glu Thr65 70 75 80Phe Thr Cys Asn Val Val His Pro Ala Ser Asn Thr
Lys Val Asp Lys 85 90 95Pro Val Pro Lys Glu Ser Thr Cys Lys Cys Ile
Ser Pro Cys Pro Val 100 105 110Pro Glu Ser Leu Gly Gly Pro Ser Val
Phe Ile Phe Pro Pro Lys Pro 115 120 125Lys Asp Ile Leu Arg Ile Thr
Arg Thr Pro Glu Ile Thr Cys Val Val 130 135 140Leu Asp Leu Gly Arg
Glu Asp Pro Glu Val Gln Ile Ser Trp Phe Val145 150 155 160Asp Gly
Lys Glu Val His Thr Ala Lys Thr Gln Pro Arg Glu Gln Gln 165 170
175Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Pro Ile Glu His Gln
180 185 190Asp Trp Leu Thr Gly Lys Glu Phe Lys Cys Arg Val Asn His
Ile Gly 195 200 205Leu Pro Ser Pro Ile Glu Arg Thr Ile Ser Lys Ala
Arg Gly Gln Ala 210 215 220His Gln Pro Ser Val Tyr Val Leu Pro Pro
Ser Pro Lys Glu Leu Ser225 230 235 240Ser Ser Asp Thr Val Thr Leu
Thr Cys Leu Ile Lys Asp Phe Phe Pro 245 250 255Pro Glu Ile Asp Val
Glu Trp Gln Ser Asn Gly Gln Pro Glu Pro Glu 260 265 270Ser Lys Tyr
His Thr Thr Ala Pro Gln Leu Asp Glu Asp Gly Ser Tyr 275 280 285Phe
Leu Tyr Ser Lys Leu Ser Val Asp Lys Ser Arg Trp Gln Gln Gly 290 295
300Asp Thr Phe Thr Cys Ala Val Met His Glu Ala Leu Gln Asn His
Tyr305 310 315 320Thr Asp Leu Ser Leu Ser His Ser Pro Gly Lys 325
3305399DNARattus norvegicus 5atggaatcac agacgcatgt cctcatttcc
cttctgctct cggtatctgg tacctatggg 60gacattgcga taacccagtc tccatcctct
gtggctgtgt cagtaggaga gacggtcact 120ctgagctgca agtccagtca
gagtctttta tacagtgaaa accaaaagga ctatttgggc 180tggtaccagc
agaaaccagg gcagactcct aaacccctta tctactgggc aaccaaccgg
240cacactgggg tccctgatcg cttcacaggt agtggatccg ggacagactt
cactctgatc 300atcagcagtg tgcaggctga agacctggct gattattact
gtgggcagta ccttgtctat 360ccgttcacgt ttggacctgg gaccaagctg gaactgaaa
3996411DNARattus norvegicus 6atgggatgga gccagatcat cctctttctg
gtggcagcag ctacatgtgt tcactcccag 60gtacagctgc agcaatctgg ggctgaatta
gtgaagcctg ggtcctcagt gaaaatttcc 120tgcaaggctt ctggctacac
cttcaccagt aactttatgc actgggtaaa gcagcagcct 180ggaaatggcc
ttgagtggat tgggtggatt tatcctgaat atggtaatac taagtacaat
240caaaagttcg atgggaaggc aacactcact gcagacaaat cctccagcac
agcctatatg 300cagctcagca gcctgacatc tgaggactct gcagtctatt
tctgtgcaag tgaggaggca 360gttatatccc ttgtttactg gggccaaggc
actctggtca ctgtctcttc a 4117318DNACanis lupus 7cagcccaagg
cctccccctc ggtcacactc ttcccgccct cctctgagga gctcggcgcc 60aacaaggcca
ccctggtgtg cctcatcagc gacttctacc ccagcggcgt gacggtggcc
120tggaaggcaa gcggcagccc cgtcacccag ggcgtggaga ccaccaagcc
ctccaagcag 180agcaacaaca agtacgcggc cagcagctac ctgagcctga
cgcctgacaa gtggaaatct 240cacagcagct tcagctgcct ggtcacgcac
gaggggagca ccgtggagaa gaaggtggcc 300cccgcagagt gctcttag
3188996DNACanis lupus 8gcctccacca cggccccctc ggttttccca ctggccccca
gctgcgggtc cacttccggc 60tccacggtgg ccctggcctg cctggtgtca ggctacttcc
ccgagcctgt aactgtgtcc 120tggaattccg gctccttgac cagcggtgtg
cacaccttcc cgtccgtcct gcagtcctca 180gggctctact ccctcagcag
cacggtgaca gtgccctcca gcaggtggcc cagcgagacc 240ttcacctgca
acgtggtcca cccggccagc aacactaaag tagacaagcc agtgcccaaa
300gagtccacct gcaagtgtat atccccatgc ccagtccctg aatcactggg
agggccttcg 360gtcttcatct ttcccccgaa acccaaggac atcctcagga
ttacccgaac acccgagatc 420acctgtgtgg tgttagatct gggccgtgag
gaccctgagg tgcagatcag ctggttcgtg 480gatggtaagg aggtgcacac
agccaagacg cagcctcgtg agcagcagtt caacagcacc 540taccgtgtgg
tcagcgtcct ccccattgag caccaggact ggctcaccgg aaaggagttc
600aagtgcagag tcaaccacat aggcctcccg tcccccatcg agaggactat
ctccaaagcc 660agagggcaag cccatcagcc cagtgtgtat gtcctgccac
catccccaaa ggagttgtca 720tccagtgaca cggtcaccct gacctgcctg
atcaaagact tcttcccacc tgagattgat 780gtggagtggc agagcaatgg
acagccggag cccgagagca agtaccacac gactgcgccc 840cagctggacg
aggacgggtc ctacttcctg tacagcaagc tctctgtgga caagagccgc
900tggcagcagg gagacacctt cacatgtgcg gtgatgcatg aagctctaca
gaaccactac 960acagatctat ccctctccca ttctccgggt aaatga
9969238PRTArtificial SequenceA synthetic chimeric L chain 9Met Glu
Ser Gln Thr His Val Leu Ile Ser Leu Leu Leu Ser Val Ser1 5 10 15Gly
Thr Tyr Gly Asp Ile Ala Ile Thr Gln Ser Pro Ser Ser Val Ala 20 25
30Val Ser Val Gly Glu Thr Val Thr Leu Ser Cys Lys Ser Ser Gln Ser
35 40 45Leu Leu Tyr Ser Glu Asn Gln Lys Asp Tyr Leu Gly Trp Tyr Gln
Gln 50 55 60Lys Pro Gly Gln Thr Pro Lys Pro Leu Ile Tyr Trp Ala Thr
Asn Arg65 70 75 80His Thr Gly Val Pro Asp Arg Phe Thr Gly Ser Gly
Ser Gly Thr Asp 85 90 95Phe Thr Leu Ile Ile Ser Ser Val Gln Ala Glu
Asp Leu Ala Asp Tyr 100 105 110Tyr Cys Gly Gln Tyr Leu Val Tyr Pro
Phe Thr Phe Gly Pro Gly Thr 115 120 125Lys Leu Glu Leu Lys Gln Pro
Lys Ala Ser Pro Ser Val Thr Leu Phe 130 135 140Pro Pro Ser Ser Glu
Glu Leu Gly Ala Asn Lys Ala Thr Leu Val Cys145 150 155 160Leu Ile
Ser Asp Phe Tyr Pro Ser Gly Val Thr Val Ala Trp Lys Ala 165 170
175Ser Gly Ser Pro Val Thr Gln Gly Val Glu Thr Thr Lys Pro Ser Lys
180 185 190Gln Ser Asn Asn Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu
Thr Pro 195 200 205Asp Lys Trp Lys Ser His Ser Ser Phe Ser Cys Leu
Val Thr His Glu 210 215 220Gly Ser Thr Val Glu Lys Lys Val Ala Pro
Ala Glu Cys Ser225 230 23510468PRTArtificial SequenceA synthetic
chimeric H chain 10Met Gly Trp Ser Gln Ile Ile Leu Phe Leu Val Ala
Ala Ala Thr Cys1 5 10 15Val His Ser Gln Val Gln Leu Gln Gln Ser Gly
Ala Glu Leu Val Lys 20 25 30Pro Gly Ser Ser Val Lys Ile Ser Cys Lys
Ala Ser Gly Tyr Thr Phe 35 40 45Thr Ser Asn Phe Met His Trp Val Lys
Gln Gln Pro Gly Asn Gly Leu 50 55 60Glu Trp Ile Gly Trp Ile Tyr Pro
Glu Tyr Gly Asn Thr Lys Tyr Asn65 70 75 80Gln Lys Phe Asp Gly Lys
Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser 85 90 95Thr Ala Tyr Met Gln
Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val 100 105 110Tyr Phe Cys
Ala Ser Glu Glu Ala Val Ile Ser Leu Val Tyr Trp Gly 115 120 125Gln
Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Thr Ala Pro Ser 130 135
140Val Phe Pro Leu Ala Pro Ser Cys Gly Ser Thr Ser Gly Ser Thr
Val145 150 155 160Ala Leu Ala Cys Leu Val Ser Gly Tyr Phe Pro Glu
Pro Val Thr Val 165 170 175Ser Trp Asn Ser Gly Ser Leu Thr Ser Gly
Val His Thr Phe Pro Ser 180 185 190Val Leu Gln Ser Ser Gly Leu Tyr
Ser Leu Ser Ser Thr Val Thr Val 195 200 205Pro Ser Ser Arg Trp Pro
Ser Glu Thr Phe Thr Cys Asn Val Val His 210 215 220Pro Ala Ser Asn
Thr Lys Val Asp Lys Pro Val Pro Lys Glu Ser Thr225 230 235 240Cys
Lys Cys Ile Ser Pro Cys Pro Val Pro Glu Ser Leu Gly Gly Pro 245 250
255Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Ile Leu Arg Ile Thr
260 265 270Arg Thr Pro Glu Ile Thr Cys Val Val Leu Asp Leu Gly Arg
Glu Asp 275 280 285Pro Glu Val Gln Ile Ser Trp Phe Val Asp Gly Lys
Glu Val His Thr 290 295 300Ala Lys Thr Gln Pro Arg Glu Gln Gln Phe
Asn Ser Thr Tyr Arg Val305 310 315 320Val Ser Val Leu Pro Ile Glu
His Gln Asp Trp Leu Thr Gly Lys Glu 325 330 335Phe Lys Cys Arg Val
Asn His Ile Gly Leu Pro Ser Pro Ile Glu Arg 340 345 350Thr Ile Ser
Lys Ala Arg Gly Gln Ala His Gln Pro Ser Val Tyr Val 355 360 365Leu
Pro Pro Ser Pro Lys Glu Leu Ser Ser Ser Asp Thr Val Thr Leu 370 375
380Thr Cys Leu Ile Lys Asp Phe Phe Pro Pro Glu Ile Asp Val Glu
Trp385 390 395 400Gln Ser Asn Gly Gln Pro Glu Pro Glu Ser Lys Tyr
His Thr Thr Ala 405 410 415Pro Gln Leu Asp Glu Asp Gly Ser Tyr Phe
Leu Tyr Ser Lys Leu Ser 420 425 430Val Asp Lys Ser Arg Trp Gln Gln
Gly Asp Thr Phe Thr Cys Ala Val 435 440 445Met His Glu Ala Leu Gln
Asn His Tyr Thr Asp Leu Ser Leu Ser His 450 455 460Ser Pro Gly
Lys46511106PRTHomo sapiens 11Thr Val Ala Ala Pro Ser Val Phe Ile
Phe Pro Pro Ser Asp Glu Gln1 5 10 15Leu Lys Ser Gly Thr Ala Ser Val
Val Cys Leu Leu Asn Asn Phe Tyr 20 25 30Pro Arg Glu Ala Lys Val Gln
Trp Lys Val Asp Asn Ala Leu Gln Ser 35 40 45Gly Asn Ser Gln Glu Ser
Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 50 55 60Tyr Ser Leu Ser Ser
Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys65 70 75 80His Lys Val
Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 85 90 95Val Thr
Lys Ser Phe Asn Arg Gly Glu Cys 100 10512326PRTHomo sapiens 12Ser
Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser1 5 10
15Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe
20 25 30Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
Gly 35 40 45Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr
Ser Leu 50 55 60Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr
Lys Thr Tyr65 70 75 80Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr
Lys Val Asp Lys Arg 85 90 95Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro
Ser Cys Pro Ala Pro Glu 100 105 110Phe Leu Gly Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp 115 120 125Thr Leu Met Ile Ser Arg
Thr Pro Glu Val Thr Cys Val Val Val Asp 130 135 140Val Ser Gln Glu
Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly145 150 155 160Val
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn 165 170
175Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
180 185 190Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly
Leu Pro 195 200 205Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
Gln Pro Arg Glu 210 215 220Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln
Glu Glu Met Thr Lys Asn225 230 235 240Gln Val Ser Leu Thr Cys Leu
Val Lys Gly Phe Tyr Pro Ser Asp Ile 245 250 255Ala Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 260 265 270Thr Pro Pro
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg 275 280 285Leu
Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys 290 295
300Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu305 310 315 320Ser Leu Ser Leu Gly Lys 32513321DNAHomo sapiens
13actgtggctg caccatctgt cttcatcttc ccgccatctg atgagcagtt gaaatctgga
60actgcctctg ttgtgtgcct gctgaataac ttctatccca gagaggccaa agtacagtgg
120aaggtggata acgccctcca atcgggtaac tcccaggaga gtgtcacaga
gcaggacagc 180aaggacagca cctacagcct cagcagcacc ctgacgctga
gcaaagcaga ctacgagaaa 240cacaaagtct acgcctgcga agtcacccat
cagggcctga gctcgcccgt cacaaagagc 300ttcaacaggg gagagtgtta g
32114981DNAHomo sapiens 14tccaccaagg gcccatccgt cttccccctg
gcgccctgct ccaggagcac ctccgagagc 60acagccgccc tgggctgcct ggtcaaggac
tacttccccg aaccggtgac ggtgtcgtgg 120aactcaggcg ccctgaccag
cggcgtgcac accttcccgg ctgtcctaca gtcctcagga 180ctctactccc
tcagcagcgt ggtgaccgtg ccctccagca gcttgggcac gaagacctac
240acctgcaacg tagatcacaa gcccagcaac accaaggtgg acaagagagt
tgagtccaaa 300tatggtcccc catgcccatc atgcccagca cctgagttcc
tggggggacc atcagtcttc 360ctgttccccc caaaacccaa ggacactctc
atgatctccc ggacccctga ggtcacgtgc 420gtggtggtgg acgtgagcca
ggaagacccc gaggtccagt tcaactggta cgtggatggc 480gtggaggtgc
ataatgccaa gacaaagccg cgggaggagc agttcaacag cacgtaccgt
540gtggtcagcg tcctcaccgt cctgcaccag gactggctga acggcaagga
gtacaagtgc 600aaggtctcca acaaaggcct cccgtcctcc atcgagaaaa
ccatctccaa agccaaaggg 660cagccccgag agccacaggt gtacaccctg
cccccatccc aggaggagat gaccaagaac 720caggtcagcc tgacctgcct
ggtcaaaggc ttctacccca gcgacatcgc cgtggagtgg 780gagagcaatg
ggcagccgga gaacaactac aagaccacgc ctcccgtgct ggactccgac
840ggctccttct tcctctacag caggctaacc gtggacaaga gcaggtggca
ggaggggaat 900gtcttctcat gctccgtgat gcatgaggct ctgcacaacc
actacacaca gaagagcctc 960tccctgtctc tgggtaaatg a
98115399DNAArtificial SequenceA synthetic codon-optimized sequence
15atggaatctc aaactcatgt tttgatttca ttacttctga gtgtttccgg aacctacggt
60gatatcgcta tcactcaatc tccctcctct gttgctgtgt ctgtgggcga aaccgttacc
120ctgtcctgca agtccagtca gtctcttctc tactccgaga atcaaaagga
ctacctgggc 180tggtaccaac agaagcccgg ccagacccca aagccactga
tatactgggc aaccaacagg 240cacaccggag tgcccgacag gttcacaggc
agtggatctg gcaccgactt taccttgatc 300atttcaagcg tgcaggctga
agatctggcc gactactact gtggtcagta tctggtgtat 360cctttcactt
tcgggccagg gacaaaattg gaattgaag 39916411DNAArtificial SequenceA
synthetic codon-optimized sequence 16atgggttggt ctcaaattat
cttgtttttg gttgctgcag ccacttgtgt tcattctcag 60gtgcagctgc aacaaagcgg
cgcagaactg gtgaaacctg gcagcagcgt gaaaatatct 120tgtaaggcca
gcggatatac tttcacctcc aatttcatgc attgggtcaa acagcagccc
180ggcaacggac tcgagtggat cggctggatc taccccgagt atggcaacac
aaaatataac 240caaaaatttg atggaaaggc taccctgact gccgataagt
cctccagcac cgcatacatg 300caactctcct ccctgacctc cgaggatagc
gctgtctact tctgtgcttc cgaagaggct 360gtcatatcct tggtctattg
gggccaagga actctggtga ccgtctcatc t 41117318DNAArtificial SequenceA
synthetic codon-optimized sequence 17cagcccaaag cctctcccag
cgtcaccctc ttcccacctt ccagtgagga gctgggggca 60aacaaagcca ctttggtgtg
tctcatctcc gatttttacc cctccggggt cacagtcgca 120tggaaggcct
ccggatcccc tgtgacacag ggagtggaga caacaaaacc tagcaagcag
180agtaacaata agtatgccgc ctcaagctat ctcagcctta ctcctgataa
gtggaagtca 240catagcagtt ttagttgcct cgtaacacat gagggttcaa
ctgtggagaa aaaagtagct 300ccagctgagt gctcatga 31818996DNAArtificial
SequenceA synthetic codon-optimized sequence 18gctagcacaa
ccgctccctc cgtttttccc ctcgccccat cctgcgggtc aaccagcgga 60tccaccgtcg
ctctggcttg tctggtgtca ggatacttcc ccgagcctgt caccgtttct
120tggaatagcg gcagccttac ttccggcgtg cataccttcc ctagcgtgct
tcagtcctcc 180ggtctgtatt ccctcagctc caccgtaact gtcccaagct
caaggtggcc ctctgagaca 240tttacctgca atgtggtcca tcctgcttca
aataccaaag tggacaagcc cgtcccaaaa 300gagtctacct gcaaatgtat
cagtccttgt cccgtgcccg agtctctggg cggaccctca 360gtctttatct
tcccacccaa gccaaaggac atattgcgca ttacacggac acccgaaatc
420acctgtgttg tgttggatct cggccgggaa gatcctgagg tgcagattag
ttggtttgtt 480gatggcaagg aggtgcacac agcaaaaaca cagcccagag
aacagcagtt caacagtact 540tatagagtag tgagtgtgtt gcctatagag
catcaggact ggctgacagg caaagaattc 600aaatgtaggg ttaaccacat
tggcctccct agtccaatcg agaggacaat ctctaaagcc 660cgaggccagg
ctcatcagcc ttctgtgtac gttctgcctc ctagtcctaa ggaactgtct
720tcttcagaca cagtaacact cacttgcctg attaaggact tttttcctcc
agagattgat 780gtggaatggc agtctaacgg gcagccagag ccagaatcta
agtaccacac tactgcacca 840cagctggatg aggatgggtc ttacttcctg
tacagtaagc tgagtgtgga caagtctcga 900tggcagcagg gggatacttt
tacttgcgca gtaatgcacg aagcattgca gaaccactac 960actgacctgt
cacttagtca ctcaccaggg aagtaa 99619717DNAArtificial SequenceA
synthetic codon-optimized sequence 19atggaatctc aaactcatgt
tttgatttca ttacttctga gtgtttccgg aacctacggt 60gatatcgcta tcactcaatc
tccctcctct gttgctgtgt ctgtgggcga aaccgttacc 120ctgtcctgca
agtccagtca gtctcttctc tactccgaga atcaaaagga ctacctgggc
180tggtaccaac agaagcccgg ccagacccca aagccactga tatactgggc
aaccaacagg 240cacaccggag tgcccgacag gttcacaggc agtggatctg
gcaccgactt taccttgatc 300atttcaagcg tgcaggctga agatctggcc
gactactact gtggtcagta tctggtgtat 360cctttcactt tcgggccagg
gacaaaattg gaattgaagc agcccaaagc ctctcccagc 420gtcaccctct
tcccaccttc cagtgaggag ctgggggcaa acaaagccac tttggtgtgt
480ctcatctccg atttttaccc ctccggggtc acagtcgcat ggaaggcctc
cggatcccct 540gtgacacagg gagtggagac aacaaaacct agcaagcaga
gtaacaataa gtatgccgcc 600tcaagctatc tcagccttac tcctgataag
tggaagtcac atagcagttt tagttgcctc 660gtaacacatg agggttcaac
tgtggagaaa aaagtagctc cagctgagtg ctcatga 717201407DNAArtificial
SequenceA synthetic codon-optimized sequence 20atgggttggt
ctcaaattat cttgtttttg gttgctgcag ccacttgtgt tcattctcag 60gtgcagctgc
aacaaagcgg cgcagaactg gtgaaacctg gcagcagcgt gaaaatatct
120tgtaaggcca gcggatatac tttcacctcc aatttcatgc attgggtcaa
acagcagccc 180ggcaacggac tcgagtggat cggctggatc taccccgagt
atggcaacac aaaatataac 240caaaaatttg atggaaaggc taccctgact
gccgataagt cctccagcac cgcatacatg 300caactctcct ccctgacctc
cgaggatagc gctgtctact tctgtgcttc cgaagaggct 360gtcatatcct
tggtctattg gggccaagga actctggtga ccgtctcatc tgctagcaca
420accgctccct ccgtttttcc cctcgcccca tcctgcgggt caaccagcgg
atccaccgtc 480gctctggctt gtctggtgtc aggatacttc cccgagcctg
tcaccgtttc ttggaatagc 540ggcagcctta cttccggcgt gcataccttc
cctagcgtgc ttcagtcctc cggtctgtat 600tccctcagct ccaccgtaac
tgtcccaagc tcaaggtggc cctctgagac atttacctgc 660aatgtggtcc
atcctgcttc aaataccaaa gtggacaagc ccgtcccaaa agagtctacc
720tgcaaatgta tcagtccttg tcccgtgccc gagtctctgg gcggaccctc
agtctttatc 780ttcccaccca agccaaagga catattgcgc attacacgga
cacccgaaat cacctgtgtt 840gtgttggatc tcggccggga agatcctgag
gtgcagatta gttggtttgt tgatggcaag 900gaggtgcaca cagcaaaaac
acagcccaga gaacagcagt tcaacagtac ttatagagta 960gtgagtgtgt
tgcctataga gcatcaggac tggctgacag gcaaagaatt caaatgtagg
1020gttaaccaca ttggcctccc tagtccaatc gagaggacaa tctctaaagc
ccgaggccag 1080gctcatcagc cttctgtgta cgttctgcct cctagtccta
aggaactgtc ttcttcagac 1140acagtaacac tcacttgcct gattaaggac
ttttttcctc cagagattga tgtggaatgg 1200cagtctaacg ggcagccaga
gccagaatct aagtaccaca ctactgcacc acagctggat 1260gaggatgggt
cttacttcct gtacagtaag ctgagtgtgg acaagtctcg atggcagcag
1320ggggatactt ttacttgcgc agtaatgcac gaagcattgc agaaccacta
cactgacctg 1380tcacttagtc actcaccagg gaagtaa 14072120DNAArtificial
SequenceA synthetic primer 21aggatggctc ctagactccc
202220DNAArtificial SequenceA synthetic primer 22agacgatggt
ggcatactcg 202320DNAArtificial SequenceA synthetic primer
23atgagaatgt ttagtgtctt 202424DNAArtificial SequenceA synthetic
primer 24ttatgtctct tcaaattgta tatc 242516DNAArtificial SequenceA
synthetic primer 25gttgatctgt gtgttg 162620DNAArtificial SequenceA
synthetic primer 26cgggacttcc acatgagcat 202717DNAArtificial
SequenceA synthetic primer 27ttttagacag aaagtga 172820DNAArtificial
SequenceA synthetic primer 28gaccagctct tcttggggaa
202929DNAArtificial SequenceA synthetic primer 29ccgctcgaga
tggggagccg gcgggggcc 293030DNAArtificial SequenceA synthetic primer
30cgcggatcct gaggggccac aggccgggtc 303126DNAArtificial SequenceA
synthetic primer 31gaagatctat gagaatgttt agtgtc 263228DNAArtificial
SequenceA synthetic primer 32ggaattctgt ctcttcaaat tgtatatc
283330DNAArtificial SequenceA synthetic primer 33cgcggctagc
atggggagcc ggcgggggcc 303430DNAArtificial SequenceA synthetic
primer 34cgcggatatc cagcccctgc aactggccgc 303530DNAArtificial
SequenceA synthetic primer 35cgcggctagc atgagaatgt ttagtgtctt
303630DNAArtificial SequenceA synthetic primer 36cgcggatatc
agtcctctca cttgctggaa 303712PRTRattus norvegicus 37Gln Ser Leu Leu
Tyr Ser Glu Asn Gln Lys Asp Tyr1 5 10389PRTRattus norvegicus 38Gly
Gln Tyr Leu Val Tyr Pro Phe Thr1 5398PRTRattus norvegicus 39Gly Tyr
Thr Phe Thr Ser Asn Phe1 5408PRTRattus norvegicus 40Ile Tyr Pro Glu
Tyr Gly Asn Thr1 54111PRTRattus norvegicus 41Ala Ser Glu Glu Ala
Val Ile Ser Leu Val Tyr1 5 1042331PRTOvis aries 42Ala Ser Thr Thr
Pro Pro Lys Val Tyr Pro Leu Thr Ser Cys Cys Gly1 5 10 15Asp Thr Ser
Ser Ser Ile Val Thr Leu Gly Cys Leu Val Ser Ser Tyr 20 25 30Met Pro
Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly
Val His Thr Phe Pro Ala Ile Leu Gln Ser Ser Gly Leu Tyr Ser 50 55
60Leu Ser Ser Val Val Thr Val Pro Ala Ser Thr Ser Gly Ala Gln Thr65
70 75 80Phe Ile Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp
Lys 85 90 95Arg Val Glu Pro Gly Cys Pro Asp Pro Cys Lys His Cys Arg
Cys Pro 100 105 110Pro Pro Glu Leu Pro Gly Gly Pro Ser Val Phe Ile
Phe Pro Pro Lys 115 120 125Pro Lys Asp Thr Leu Thr Ile Ser Gly Thr
Pro Glu Val Thr Cys Val 130 135 140Val Val Asp Val Gly Gln Asp Asp
Pro Glu Val Gln Phe Ser Trp Phe145 150 155 160Val Asp Asn Val Glu
Val Arg Thr Ala Arg Thr Lys Pro Arg Glu Glu 165 170 175Gln Phe Asn
Ser Thr Phe Arg Val Val Ser Ala Leu Pro Ile Gln His 180 185 190Gln
Asp Trp Thr Gly Gly Lys Glu Phe Lys Cys Lys Val His Asn Glu 195 200
205Ala Leu Pro Ala Pro Ile Val Arg Thr Ile Ser Arg Thr Lys Gly Gln
210 215 220Ala Arg Glu Pro Gln Val Tyr Val Leu Ala Pro Pro Gln Glu
Glu Leu225 230 235 240Ser Lys Ser Thr Leu Ser Val Thr Cys Leu Val
Thr Gly Phe Tyr Pro 245 250 255Asp Tyr Ile Ala Val Glu Trp Gln Lys
Asn Gly Gln Pro Glu Ser Glu 260 265 270Asp Lys Tyr Gly Thr Thr Thr
Ser Gln Leu Asp Ala Asp Gly Ser Tyr 275 280 285Phe Leu Tyr Ser Arg
Leu Arg Val Asp Lys Asn Ser Trp Gln Glu Gly 290 295 300Asp Thr Tyr
Ala Cys Val Val Met His Glu Ala Leu His Asn His Tyr305 310 315
320Thr Gln Lys Ser Ile Ser Lys Pro Pro Gly Lys 325 33043996DNAOvis
aries 43gcctcaacaa cacccccgaa agtctaccct ctgacttctt gctgcgggga
cacgtccagc 60tccatcgtga ccctgggctg cctggtctcc agctatatgc ccgagccggt
gaccgtgacc 120tggaactctg gtgccctgac cagcggcgtg cacaccttcc
cggccatcct gcagtcctcc 180gggctctact ctctcagcag cgtggtgacc
gtgccggcca gcacctcagg agcccagacc 240ttcatctgca acgtagccca
cccggccagc agcaccaagg tggacaagcg tgttgagccc 300ggatgcccgg
acccatgcaa acattgccga tgcccacccc ctgagctccc cggaggaccg
360tctgtcttca tcttcccacc gaaacccaag gacaccctta caatctctgg
aacgcccgag 420gtcacgtgtg tggtggtgga cgtgggccag gatgaccccg
aggtgcagtt ctcctggttc 480gtggacaacg tggaggtgcg cacggccagg
acaaagccga gagaggagca gttcaacagc 540accttccgcg tggtcagcgc
cctgcccatc cagcaccaag actggactgg aggaaaggag 600ttcaagtgca
aggtccacaa cgaagccctc ccggccccca tcgtgaggac catctccagg
660accaaagggc aggcccggga gccgcaggtg tacgtcctgg ccccacccca
ggaagagctc 720agcaaaagca cgctcagcgt cacctgcctg gtcaccggct
tctacccaga ctacatcgcc 780gtggagtggc agaaaaatgg gcagcctgag
tcggaggaca agtacggcac gaccacatcc 840cagctggacg ccgacggctc
ctacttcctg tacagcaggc tcagggtgga caagaacagc 900tggcaagaag
gagacaccta cgcgtgtgtg gtgatgcacg aggctctgca caaccactac
960acacagaagt cgatctctaa gcctccgggt aaatga 99644329PRTOvis aries
44Ala Ser Thr Thr Ala Pro Lys Val Tyr Pro Leu Thr Ser Cys Cys Gly1
5 10 15Asp Thr Ser Ser Ser Ser Ser Ile Val Thr Leu Gly Cys Leu Val
Ser 20 25 30Ser Tyr Met Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly
Ala Leu 35 40 45Thr Ser Gly Val His Thr Phe Pro Ala Ile Leu Gln Ser
Ser Gly Leu 50 55 60Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ala Ser
Thr Ser Gly Ala65 70 75 80Gln Thr Phe Ile Cys Asn Val Ala His Pro
Ala Ser Ser Ala Lys Val 85 90 95Asp Lys Arg Val Gly Ile Ser Ser Asp
Tyr Ser Lys Cys Ser Lys Pro 100 105 110Pro Cys Val Ser Arg Pro Ser
Val Phe Ile Phe Pro Pro Lys Pro Lys 115 120 125Asp Ser Leu Met Ile
Thr Gly Thr Pro Glu Val Thr Cys Val Val Val 130 135 140Asp Val Gly
Gln Gly Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp145 150 155
160Asn Val Glu Val Arg Thr Ala Arg Thr Lys Pro Arg Glu Glu Gln Phe
165 170 175Asn Ser Thr Phe Arg Val Val Ser Ala Leu Pro Ile Gln His
Asp His 180 185 190Trp Thr Gly Gly Lys Glu Phe Lys Cys Lys Val His
Ser Lys Gly Leu 195 200 205Pro Ala Pro Ile Val Arg Thr Ile Ser Arg
Ala Lys Gly Gln Ala Arg 210 215 220Glu Pro Gln Val Tyr Val Leu Ala
Pro Pro Gln Glu Glu Leu Ser Lys225 230 235 240Ser Thr Leu Ser Val
Thr Cys Leu Val Thr Gly Phe Tyr Pro Asp Tyr 245 250 255Ile Ala Val
Glu Trp Gln Arg Ala Arg Gln Pro Glu Ser Glu Asp Lys 260 265 270Tyr
Gly Thr Thr Thr Ser Gln Leu Asp Ala Asp Gly Ser Tyr Phe Leu 275 280
285Tyr Ser Arg Leu Arg Val Asp Lys Ser Ser Trp Gln Arg Gly Asp Thr
290 295 300Tyr Ala Cys Val Val Met His Glu Ala Leu His Asn His Tyr
Thr Gln305 310 315 320Lys Ser Ile Ser Lys Pro Pro Gly Lys
32545990DNAOvis aries 45gcctccacca cagccccgaa agtctaccct ctgacttctt
gctgcgggga cacgtccagc 60tccagctcca tcgtgaccct gggctgcctg gtctccagct
atatgcccga gccggtgacc 120gtgacctgga actctggtgc cctgaccagc
ggcgtgcaca ccttcccggc catcctgcag 180tcctccgggc tctactctct
cagcagcgtg gtgaccgtgc cggccagcac ctcaggagcc 240cagaccttca
tctgcaacgt agcccacccg gccagcagcg ccaaggtgga caagcgtgtt
300gggatctcca gtgactactc caagtgttct aaaccgcctt gcgtgagccg
accgtctgtc 360ttcatcttcc ccccgaaacc caaggacagc ctcatgatca
caggaacgcc cgaggtcacg 420tgtgtggtgg tggacgtggg ccagggtgac
cccgaggtgc agttctcctg gttcgtggac 480aacgtggagg tgcgcacggc
caggacaaag ccgagagagg agcagttcaa cagcaccttc 540cgcgtggtca
gcgccctgcc catccagcac gaccactgga ctggaggaaa ggagttcaag
600tgcaaggtcc acagcaaagg cctcccggcc cccatcgtga ggaccatctc
cagggccaaa 660gggcaggccc gggagccgca ggtgtacgtc ctggccccac
cccaggaaga gctcagcaaa 720agcacgctca gcgtcacctg cctggtcacc
ggcttctacc cagactacat cgccgtggag 780tggcagagag cgcggcagcc
tgagtcggag gacaagtacg gcacgaccac atcccagctg 840gacgccgacg
gctcctactt cctgtacagc aggctcaggg tggacaagag cagctggcaa
900agaggagaca cctacgcgtg tgtggtgatg cacgaggctc tgcacaacca
ctacacacag 960aagtcgatct ctaagcctcc gggtaaatga 99046102PRTOvis
aries 46Pro Ser Val Phe Leu Phe Lys Pro Ser Glu Glu Gln Leu Arg Thr
Gly1 5 10 15Thr Val Ser Val Val Cys Leu Val Asn Asp Phe Tyr Pro Lys
Asp Ile 20 25 30Asn Val Lys Val Lys Val Asp Gly Val Thr Gln Asn Ser
Asn Phe Gln 35 40 45Asn Ser Phe Thr Asp Gln Asp Ser Lys Lys Ser Thr
Tyr Ser Leu Ser 50 55 60Ser Thr Leu Thr Leu Ser Ser Ser Glu Tyr Gln
Ser His Asn Ala Tyr65 70 75 80Ala Cys Glu Val Ser His Lys Ser Leu
Pro Thr Ala Leu Val Lys Ser 85 90 95Phe Asn Lys Asn Glu Cys
10047309DNAOvis aries 47ccatccgtct tcctcttcaa accatctgag gaacagctga
ggaccggaac tgtctctgtc 60gtgtgcttgg tgaatgattt ctaccccaaa gatatcaatg
tcaaggtgaa agtggatggg 120gttacccaga acagcaactt ccagaacagc
ttcacagacc aggacagcaa gaaaagcacc 180tacagcctca gcagcaccct
gacactgtcc agctcagagt accagagcca taacgcctat 240gcgtgtgagg
tcagccacaa gagcctgccc accgccctcg tcaagagctt caataagaat 300gaatgttag
30948106PRTOvis aries 48Gly Gln Pro Lys Ser Ala Pro Ser Val Thr Leu
Phe Pro Pro Ser Thr1 5 10 15Glu Glu Leu Ser Thr Asn Lys Ala Thr Val
Val Cys Leu Ile Asn Asp 20 25 30Phe Tyr Pro Gly Ser Val Asn Val Val
Trp Lys Ala Asp Gly Ser Thr 35 40 45Ile Asn Gln Asn Val Lys Thr Thr
Gln Ala Ser Lys Gln Ser Asn Ser 50 55 60Lys Tyr Ala Ala Ser Ser Tyr
Leu Thr Leu Thr Gly Ser Glu Trp Lys65 70 75 80Ser Lys Ser Ser Tyr
Thr Cys Glu Val Thr His Glu Gly Ser Thr Val 85 90 95Thr Lys Thr Val
Lys Pro Ser Glu Cys Ser 100 10549321DNAOvis aries 49ggtcagccca
agtccgcacc ctcggtcacc ctgttcccgc cttccacgga ggagctcagt 60accaacaagg
ccaccgtggt gtgtctcatc aacgacttct acccgggtag cgtgaacgtg
120gtctggaagg cagatggcag caccatcaat cagaacgtga agaccaccca
ggcctccaaa 180cagagcaaca gcaagtacgc ggccagcagc tacctgaccc
tgacgggcag cgagtggaag 240tctaagagca gttacacctg cgaggtcacg
cacgagggga gcaccgtgac gaagacagtg 300aagccctcag agtgttctta g
32150328PRTSus scrofa 50Ala Pro Lys Thr Ala Pro Ser Val Tyr Pro Leu
Ala Pro Cys Gly Arg1 5 10 15Asp Thr Ser Gly Pro Asn Val
Ala Leu Gly Cys Leu Ala Ser Ser Tyr 20 25 30Phe Pro Glu Pro Val Thr
Met Thr Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val His Thr Phe
Pro Ser Val Leu Gln Pro Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Met
Val Thr Val Pro Ala Ser Ser Leu Ser Ser Lys Ser65 70 75 80Tyr Thr
Cys Asn Val Asn His Pro Ala Thr Thr Thr Lys Val Asp Lys 85 90 95Arg
Val Gly Thr Lys Thr Lys Pro Pro Cys Pro Ile Cys Pro Gly Cys 100 105
110Glu Val Ala Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp
115 120 125Thr Leu Met Ile Ser Gln Thr Pro Glu Val Thr Cys Val Val
Val Asp 130 135 140Val Ser Lys Glu His Ala Glu Val Gln Phe Ser Trp
Tyr Val Asp Gly145 150 155 160Val Glu Val His Thr Ala Glu Thr Arg
Pro Lys Glu Glu Gln Phe Asn 165 170 175Ser Thr Tyr Arg Val Val Ser
Val Leu Pro Ile Gln His Gln Asp Trp 180 185 190Leu Lys Gly Lys Glu
Phe Lys Cys Lys Val Asn Asn Val Asp Leu Pro 195 200 205Ala Pro Ile
Thr Arg Thr Ile Ser Lys Ala Ile Gly Gln Ser Arg Glu 210 215 220Pro
Gln Val Tyr Thr Leu Pro Pro Pro Ala Glu Glu Leu Ser Arg Ser225 230
235 240Lys Val Thr Val Thr Cys Leu Val Ile Gly Phe Tyr Pro Pro Asp
Ile 245 250 255His Val Glu Trp Lys Ser Asn Gly Gln Pro Glu Pro Glu
Gly Asn Tyr 260 265 270Arg Thr Thr Pro Pro Gln Gln Asp Val Asp Gly
Thr Phe Phe Leu Tyr 275 280 285Ser Lys Leu Ala Val Asp Lys Ala Arg
Trp Asp His Gly Glu Thr Phe 290 295 300Glu Cys Ala Val Met His Glu
Ala Leu His Asn His Tyr Thr Gln Lys305 310 315 320Ser Ile Ser Lys
Thr Gln Gly Lys 32551987DNASus scrofa 51gcccccaaga cggccccatc
ggtctaccct ctggccccct gcggcaggga cacgtctggc 60cctaacgtgg ccttgggctg
cctggcctca agctacttcc ccgagccagt gaccatgacc 120tggaactcgg
gcgccctgac cagtggcgtg cataccttcc catccgtcct gcagccgtca
180gggctctact ccctcagcag catggtgacc gtgccggcca gcagcctgtc
cagcaagagc 240tacacctgca atgtcaacca cccggccacc accaccaagg
tggacaagcg tgttggaaca 300aagaccaaac caccatgtcc catatgccca
ggctgtgaag tggccgggcc ctcggtcttc 360atcttccctc caaaacccaa
ggacaccctc atgatctccc agacccccga ggtcacgtgc 420gtggtggtgg
acgtcagcaa ggagcacgcc gaggtccagt tctcctggta cgtggacggc
480gtagaggtgc acacggccga gacgagacca aaggaggagc agttcaacag
cacctaccgt 540gtggtcagcg tcctgcccat ccagcaccag gactggctga
aggggaagga gttcaagtgc 600aaggtcaaca acgtagacct cccagccccc
atcacgagga ccatctccaa ggctataggg 660cagagccggg agccgcaggt
gtacaccctg cccccacccg ccgaggagct gtccaggagc 720aaagtcaccg
taacctgcct ggtcattggc ttctacccac ctgacatcca tgttgagtgg
780aagagcaacg gacagccgga gccagagggc aattaccgca ccaccccgcc
ccagcaggac 840gtggacggga ccttcttcct gtacagcaag ctcgcggtgg
acaaggcaag atgggaccat 900ggagaaacat ttgagtgtgc ggtgatgcac
gaggctctgc acaaccacta cacccagaag 960tccatctcca agactcaggg taaatga
98752328PRTSus scrofa 52Ala Pro Lys Thr Ala Pro Ser Val Tyr Pro Leu
Ala Pro Cys Gly Arg1 5 10 15Asp Val Ser Gly Pro Asn Val Ala Leu Gly
Cys Leu Ala Ser Ser Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Thr Trp
Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val His Thr Phe Pro Ser Val
Leu Gln Pro Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Met Val Thr Val
Pro Ala Ser Ser Leu Ser Ser Lys Ser65 70 75 80Tyr Thr Cys Asn Val
Asn His Pro Ala Thr Thr Thr Lys Val Asp Lys 85 90 95Arg Val Gly Ile
His Gln Pro Gln Thr Cys Pro Ile Cys Pro Gly Cys 100 105 110Glu Val
Ala Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp 115 120
125Thr Leu Met Ile Ser Gln Thr Pro Glu Val Thr Cys Val Val Val Asp
130 135 140Val Ser Lys Glu His Ala Glu Val Gln Phe Ser Trp Tyr Val
Asp Gly145 150 155 160Val Glu Val His Thr Ala Glu Thr Arg Pro Lys
Glu Glu Gln Phe Asn 165 170 175Ser Thr Tyr Arg Val Val Ser Val Leu
Pro Ile Gln His Gln Asp Trp 180 185 190Leu Lys Gly Lys Glu Phe Lys
Cys Lys Val Asn Asn Val Asp Leu Pro 195 200 205Ala Pro Ile Thr Arg
Thr Ile Ser Lys Ala Ile Gly Gln Ser Arg Glu 210 215 220Pro Gln Val
Tyr Thr Leu Pro Pro Pro Ala Glu Glu Leu Ser Arg Ser225 230 235
240Lys Val Thr Leu Thr Cys Leu Val Ile Gly Phe Tyr Pro Pro Asp Ile
245 250 255His Val Glu Trp Lys Ser Asn Gly Gln Pro Glu Pro Glu Asn
Thr Tyr 260 265 270Arg Thr Thr Pro Pro Gln Gln Asp Val Asp Gly Thr
Phe Phe Leu Tyr 275 280 285Ser Lys Leu Ala Val Asp Lys Ala Arg Trp
Asp His Gly Asp Lys Phe 290 295 300Glu Cys Ala Val Met His Glu Ala
Leu His Asn His Tyr Thr Gln Lys305 310 315 320Ser Ile Ser Lys Thr
Gln Gly Lys 32553987DNASus scrofa 53gcccccaaga cggccccatc
ggtctaccct ctggccccct gcggcaggga cgtgtctggc 60cctaacgtgg ccttgggctg
cctggcctca agctacttcc ccgagccagt gaccgtgacc 120tggaactcgg
gcgccctgac cagtggcgtg cacaccttcc catccgtcct gcagccgtca
180gggctctact ccctcagcag catggtgacc gtgccggcca gcagcctgtc
cagcaagagc 240tacacctgca atgtcaacca cccggccacc accaccaagg
tggacaagcg tgttggaata 300caccagccgc aaacatgtcc catatgccca
ggctgtgaag tggccgggcc ctcggtcttc 360atcttccctc caaaacccaa
ggacaccctc atgatctccc agacccccga ggtcacgtgc 420gtggtggtgg
acgtcagcaa ggagcacgcc gaggtccagt tctcctggta cgtggacggc
480gtagaggtgc acacggccga gacgagacca aaggaggagc agttcaacag
cacctaccgt 540gtggtcagcg tcctgcccat ccagcaccag gactggctga
aggggaagga gttcaagtgc 600aaggtcaaca acgtagacct cccagccccc
atcacgagga ccatctccaa ggctataggg 660cagagccggg agccgcaggt
gtacaccctg cccccacccg ccgaggagct gtccaggagc 720aaagtcacgc
taacctgcct ggtcattggc ttctacccac ctgacatcca tgttgagtgg
780aagagcaacg gacagccgga gccagagaac acataccgca ccaccccgcc
ccagcaggac 840gtggacggga ccttcttcct gtacagcaaa ctcgcggtgg
acaaggcaag atgggaccat 900ggagacaaat ttgagtgtgc ggtgatgcac
gaggctctgc acaaccacta cacccagaag 960tccatctcca agactcaggg taaatga
98754328PRTSus scrofa 54Ala Pro Lys Thr Ala Pro Ser Val Tyr Pro Leu
Ala Pro Cys Ser Arg1 5 10 15Asp Thr Ser Gly Pro Asn Val Ala Leu Gly
Cys Leu Ala Ser Ser Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Thr Trp
Asn Ser Gly Ala Leu Ser Ser 35 40 45Gly Val His Thr Phe Pro Ser Val
Leu Gln Pro Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Met Val Thr Val
Pro Ala Ser Ser Leu Ser Ser Lys Ser65 70 75 80Tyr Thr Cys Asn Val
Asn His Pro Ala Thr Thr Thr Lys Val Asp Lys 85 90 95Arg Val Gly Thr
Lys Thr Lys Pro Pro Cys Pro Ile Cys Pro Ala Cys 100 105 110Glu Ser
Pro Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp 115 120
125Thr Leu Met Ile Ser Arg Thr Pro Gln Val Thr Cys Val Val Val Asp
130 135 140Val Ser Gln Glu Asn Pro Glu Val Gln Phe Ser Trp Tyr Val
Asp Gly145 150 155 160Val Glu Val His Thr Ala Gln Thr Arg Pro Lys
Glu Glu Gln Phe Asn 165 170 175Ser Thr Tyr Arg Val Val Ser Val Leu
Pro Ile Gln His Gln Asp Trp 180 185 190Leu Asn Gly Lys Glu Phe Lys
Cys Lys Val Asn Asn Lys Asp Leu Pro 195 200 205Ala Pro Ile Thr Arg
Ile Ile Ser Lys Ala Lys Gly Gln Thr Arg Glu 210 215 220Pro Gln Val
Tyr Thr Leu Pro Pro His Ala Glu Glu Leu Ser Arg Ser225 230 235
240Lys Val Ser Ile Thr Cys Leu Val Ile Gly Phe Tyr Pro Pro Asp Ile
245 250 255Asp Val Glu Trp Gln Arg Asn Gly Gln Pro Glu Pro Glu Gly
Asn Tyr 260 265 270Arg Thr Thr Pro Pro Gln Gln Asp Val Asp Gly Thr
Tyr Phe Leu Tyr 275 280 285Ser Lys Phe Ser Val Asp Lys Ala Ser Trp
Gln Gly Gly Gly Ile Phe 290 295 300Gln Cys Ala Val Met His Glu Ala
Leu His Asn His Tyr Thr Gln Lys305 310 315 320Ser Ile Ser Lys Thr
Pro Gly Lys 32555987DNASus scrofa 55gcccccaaga cggccccatc
ggtctaccct ctggccccct gcagcaggga cacgtctggc 60cctaacgtgg ccttgggctg
cctggcctca agctacttcc ccgagccagt gaccgtgacc 120tggaactcgg
gcgccctgtc cagtggcgtg cataccttcc catccgtcct gcagccgtca
180gggctctact ccctcagcag catggtgacc gtgccggcca gcagcctgtc
cagcaagagc 240tacacctgca atgtcaacca cccggccacc accaccaagg
tggacaagcg tgttggaaca 300aagaccaaac caccatgtcc catatgccca
gcctgtgaat caccagggcc ctcggtcttc 360atcttccctc caaaacccaa
ggacaccctc atgatctccc ggacacccca ggtcacgtgc 420gtggtggttg
atgtgagcca ggagaacccg gaggtccagt tctcctggta cgtggacggc
480gtagaggtgc acacggccca gacgaggcca aaggaggagc agttcaacag
cacctaccgc 540gtggtcagcg tcctacccat ccagcaccag gactggctga
acgggaagga gttcaagtgc 600aaggtcaaca acaaagacct cccagccccc
atcacaagga tcatctccaa ggccaaaggg 660cagacccggg agccgcaggt
gtacaccctg cccccacacg ccgaggagct gtccaggagc 720aaagtcagca
taacctgcct ggtcattggc ttctacccac ctgacatcga tgtcgagtgg
780caaagaaacg gacagccgga gccagagggc aattaccgca ccaccccgcc
ccagcaggac 840gtggacggga cctacttcct gtacagcaag ttctcggtgg
acaaggccag ctggcagggt 900ggaggcatat tccagtgtgc ggtgatgcac
gaggctctgc acaaccacta cacccagaag 960tctatctcca agactccggg taaatga
98756328PRTSus scrofa 56Ala Pro Lys Thr Ala Pro Leu Val Tyr Pro Leu
Ala Pro Cys Gly Arg1 5 10 15Asp Thr Ser Gly Pro Asn Val Ala Leu Gly
Cys Leu Ala Ser Ser Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Thr Trp
Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val His Thr Phe Pro Ser Val
Leu Gln Pro Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Met Val Thr Val
Pro Ala Ser Ser Leu Ser Ser Lys Ser65 70 75 80Tyr Thr Cys Asn Val
Asn His Pro Ala Thr Thr Thr Lys Val Asp Lys 85 90 95Arg Val Gly Thr
Lys Thr Lys Pro Pro Cys Pro Ile Cys Pro Ala Cys 100 105 110Glu Ser
Pro Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp 115 120
125Thr Leu Met Ile Ser Arg Thr Pro Gln Val Thr Cys Val Val Val Asp
130 135 140Val Ser Gln Glu Asn Pro Glu Val Gln Phe Ser Trp Tyr Val
Asp Gly145 150 155 160Val Glu Val His Thr Ala Gln Thr Arg Pro Lys
Glu Glu Gln Phe Asn 165 170 175Ser Thr Tyr Arg Val Val Ser Val Leu
Pro Ile Gln His Gln Asp Trp 180 185 190Leu Asn Gly Lys Glu Phe Lys
Cys Lys Val Asn Asn Lys Asp Leu Pro 195 200 205Ala Pro Ile Thr Arg
Ile Ile Ser Lys Ala Lys Gly Gln Thr Arg Glu 210 215 220Pro Gln Val
Tyr Thr Leu Pro Pro His Ala Glu Glu Leu Ser Arg Ser225 230 235
240Lys Val Ser Ile Thr Cys Leu Val Ile Gly Phe Tyr Pro Pro Asp Ile
245 250 255Asp Val Glu Trp Gln Arg Asn Gly Gln Pro Glu Pro Glu Gly
Asn Tyr 260 265 270Arg Thr Thr Pro Pro Gln Gln Asp Val Asp Gly Thr
Tyr Phe Leu Tyr 275 280 285Ser Lys Phe Ser Val Asp Lys Ala Ser Trp
Gln Gly Gly Gly Ile Phe 290 295 300Gln Cys Ala Val Met His Glu Ala
Leu His Asn His Tyr Thr Gln Lys305 310 315 320Ser Ile Ser Lys Thr
Pro Gly Lys 32557987DNASus scrofa 57gcccccaaga cggccccatt
ggtctaccct ctggccccct gcggcaggga cacgtctggc 60cctaacgtgg ccttgggctg
cctggcctca agctacttcc ccgagccagt gaccgtgacc 120tggaactcgg
gcgccctgac cagtggcgtg cataccttcc catccgtcct gcagccgtca
180gggctctact ccctcagcag catggtgacc gtgccggcca gcagcctgtc
cagcaagagc 240tacacctgca atgtcaacca cccggccacc accaccaagg
tggacaagcg tgttggaaca 300aagaccaaac caccatgtcc catatgccca
gcctgtgaat cgccagggcc ctcggtcttc 360atcttccctc caaaacccaa
ggacaccctc atgatctccc ggacacccca ggtcacgtgc 420gtggtagttg
atgtgagcca ggagaacccg gaggtccagt tctcctggta cgtggacggc
480gtagaggtgc acacggccca gacgaggcca aaggaggagc agttcaacag
cacctaccgc 540gtggtcagcg tcctgcccat ccagcaccag gactggctga
acgggaagga gttcaagtgc 600aaggtcaaca acaaagacct cccagccccc
atcacaagga tcatctccaa ggccaaaggg 660cagacccggg agccgcaggt
gtacaccctg cccccacacg ccgaggagct gtccaggagc 720aaagtcagca
taacctgcct ggtcattggc ttctacccac ctgacatcga tgtcgagtgg
780caaagaaacg gacagccgga gccagagggc aattaccgca ccaccccgcc
ccagcaggac 840gtggacggga cctacttcct gtacagcaag ttctcggtgg
acaaggccag ctggcagggt 900ggaggcatat tccagtgtgc ggtgatgcac
gaggctctgc acaaccacta cacccagaag 960tctatctcca agactccggg taaatga
98758333PRTSus scrofa 58Ala Tyr Asn Thr Ala Pro Ser Val Tyr Pro Leu
Ala Pro Cys Gly Arg1 5 10 15Asp Val Ser Asp His Asn Val Ala Leu Gly
Cys Leu Val Ser Ser Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Thr Trp
Asn Ser Gly Ala Leu Ser Arg 35 40 45Val Val His Thr Phe Pro Ser Val
Leu Gln Pro Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Met Val Ile Val
Ala Ala Ser Ser Leu Ser Thr Leu Ser65 70 75 80Tyr Thr Cys Asn Val
Tyr His Pro Ala Thr Asn Thr Lys Val Asp Lys 85 90 95Arg Val Asp Ile
Glu Pro Pro Thr Pro Ile Cys Pro Glu Ile Cys Ser 100 105 110Cys Pro
Ala Ala Glu Val Leu Gly Ala Pro Ser Val Phe Leu Phe Pro 115 120
125Pro Lys Pro Lys Asp Ile Leu Met Ile Ser Arg Thr Pro Lys Val Thr
130 135 140Cys Val Val Val Asp Val Ser Gln Glu Glu Ala Glu Val Gln
Phe Ser145 150 155 160Trp Tyr Val Asp Gly Val Gln Leu Tyr Thr Ala
Gln Thr Arg Pro Met 165 170 175Glu Glu Gln Phe Asn Ser Thr Tyr Arg
Val Val Ser Val Leu Pro Ile 180 185 190Gln His Gln Asp Trp Leu Lys
Gly Lys Glu Phe Lys Cys Lys Val Asn 195 200 205Asn Lys Asp Leu Leu
Ser Pro Ile Thr Arg Thr Ile Ser Lys Ala Thr 210 215 220Gly Pro Ser
Arg Val Pro Gln Val Tyr Thr Leu Pro Pro Ala Trp Glu225 230 235
240Glu Leu Ser Lys Ser Lys Val Ser Ile Thr Cys Leu Val Thr Gly Phe
245 250 255Tyr Pro Pro Asp Ile Asp Val Glu Trp Gln Ser Asn Gly Gln
Gln Glu 260 265 270Pro Glu Gly Asn Tyr Arg Thr Thr Pro Pro Gln Gln
Asp Val Asp Gly 275 280 285Thr Tyr Phe Leu Tyr Ser Lys Leu Ala Val
Asp Lys Val Arg Trp Gln 290 295 300Arg Gly Asp Leu Phe Gln Cys Ala
Val Met His Glu Ala Leu His Asn305 310 315 320His Tyr Thr Gln Lys
Ser Ile Ser Lys Thr Gln Gly Lys 325 330591002DNASus scrofa
59gcctacaaca cagctccatc ggtctaccct ctggccccct gtggcaggga cgtgtctgat
60cataacgtgg ccttgggctg ccttgtctca agctacttcc ccgagccagt gaccgtgacc
120tggaactcgg gtgccctgtc cagagtcgtg cataccttcc catccgtcct
gcagccgtca 180gggctctact ccctcagcag catggtgatc gtggcggcca
gcagcctgtc caccctgagc 240tacacgtgca acgtctacca cccggccacc
aacaccaagg tggacaagcg tgttgacatc 300gaacccccca cacccatctg
tcccgaaatt tgctcatgcc cagctgcaga ggtcctggga 360gcaccgtcgg
tcttcctctt ccctccaaaa cccaaggaca tcctcatgat ctcccggaca
420cccaaggtca cgtgcgtggt ggtggacgtg agccaggagg aggctgaagt
ccagttctcc 480tggtacgtgg acggcgtaca gttgtacacg gcccagacga
ggccaatgga ggagcagttc 540aacagcacct accgcgtggt cagcgtcctg
cccatccagc accaggactg gctgaagggg 600aaggagttca agtgcaaggt
caacaacaaa gacctccttt cccccatcac gaggaccatc 660tccaaggcta
cagggccgag ccgggtgccg caggtgtaca ccctgccccc agcctgggaa
720gagctgtcca agagcaaagt cagcataacc tgcctggtca ctggcttcta
cccacctgac 780atcgatgtcg agtggcagag caacggacaa caagagccag
agggcaatta ccgcaccacc 840ccgccccagc aggacgtgga tgggacctac
ttcctgtaca gcaagctcgc ggtggacaag 900gtcaggtggc agcgtggaga
cctattccag tgtgcggtga tgcacgaggc tctgcacaac
960cactacaccc agaagtccat ctccaagact cagggtaaat ga 100260277PRTSus
scrofa 60Thr Phe Pro Ser Val Leu Gln Pro Ser Gly Leu Tyr Ser Leu
Ser Ser1 5 10 15Met Val Thr Val Pro Ala Ser Ser Leu Ser Ser Lys Ser
Tyr Thr Cys 20 25 30Asn Val Asn His Pro Ala Thr Thr Thr Lys Val Asp
Lys Arg Val Gly 35 40 45Thr Lys Thr Lys Pro Pro Cys Pro Ile Cys Pro
Ala Cys Glu Gly Pro 50 55 60Gly Pro Ser Ala Phe Ile Phe Pro Pro Lys
Pro Lys Asp Thr Leu Met65 70 75 80Ile Ser Arg Thr Pro Lys Val Thr
Cys Val Val Val Asp Val Ser Gln 85 90 95Glu Asn Pro Glu Val Gln Phe
Ser Trp Tyr Val Asp Gly Val Glu Val 100 105 110His Thr Ala Gln Thr
Arg Pro Lys Glu Glu Gln Phe Asn Ser Thr Tyr 115 120 125Arg Val Val
Ser Val Leu Pro Ile Gln His Gln Asp Trp Leu Asn Gly 130 135 140Lys
Glu Phe Lys Cys Lys Val Asn Asn Lys Asp Leu Pro Ala Pro Ile145 150
155 160Thr Arg Ile Ile Ser Lys Ala Lys Gly Gln Thr Arg Glu Pro Gln
Val 165 170 175Tyr Thr Leu Pro Pro Pro Thr Glu Glu Leu Ser Arg Ser
Lys Val Thr 180 185 190Leu Thr Cys Leu Val Thr Gly Phe Tyr Pro Pro
Asp Ile Asp Val Glu 195 200 205Trp Gln Arg Asn Gly Gln Pro Glu Pro
Glu Gly Asn Tyr Arg Thr Thr 210 215 220Pro Pro Gln Gln Asp Val Asp
Gly Thr Tyr Phe Leu Tyr Ser Lys Leu225 230 235 240Ala Val Asp Lys
Ala Ser Trp Gln Arg Gly Asp Thr Phe Gln Cys Ala 245 250 255Val Met
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Ile Phe 260 265
270Lys Thr Pro Gly Lys 27561834DNASus scrofa 61accttcccat
ccgtcctgca gccgtcaggg ctctactccc tcagcagcat ggtgaccgtg 60ccggccagca
gcctgtccag caagagctac acctgcaatg tcaaccaccc ggccaccacc
120accaaggtgg acaagcgtgt tggaacaaag accaaaccac catgtcccat
atgcccagcc 180tgtgaagggc ccgggccctc ggccttcatc ttccctccaa
aacccaagga caccctcatg 240atctcccgga cccccaaggt cacgtgcgtg
gtggtagatg tgagccagga gaacccggag 300gtccagttct cctggtacgt
ggacggcgta gaggtgcaca cggcccagac gaggccaaag 360gaggagcagt
tcaacagcac ctaccgcgtg gtcagcgtcc tgcccatcca gcaccaggac
420tggctgaacg ggaaggagtt caagtgcaag gtcaacaaca aagacctccc
agcccccatc 480acaaggatca tctccaaggc caaagggcag acccgggagc
cgcaggtgta caccctgccc 540ccacccaccg aggagctgtc caggagcaaa
gtcacgctaa cctgcctggt cactggcttc 600tacccacctg acatcgatgt
cgagtggcaa agaaacggac agccggagcc agagggcaat 660taccgcacca
ccccgcccca gcaggacgtg gacgggacct acttcctgta cagcaagctc
720gcggtggaca aggccagctg gcagcgtgga gacacattcc agtgtgcggt
gatgcacgag 780gctctgcaca accactacac ccagaagtcc atcttcaaga
ctccgggtaa atga 83462318PRTSus scrofa 62Ala Pro Lys Thr Ala Pro Ser
Val Tyr Pro Leu Ala Pro Cys Gly Arg1 5 10 15Asp Val Ser Gly Pro Asn
Val Ala Leu Gly Cys Leu Ala Ser Ser Tyr 20 25 30Phe Pro Glu Pro Val
Thr Val Thr Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val His Thr
Phe Pro Ser Val Leu Gln Pro Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser
Met Val Thr Val Pro Ala Ser Ser Leu Ser Ser Lys Ser65 70 75 80Tyr
Thr Cys Asn Val Asn His Pro Ala Thr Thr Thr Lys Val Asp Lys 85 90
95Arg Val Gly Ile His Gln Pro Gln Thr Cys Pro Ile Cys Pro Ala Cys
100 105 110Glu Gly Pro Gly Pro Ser Ala Phe Ile Phe Pro Pro Lys Pro
Lys Asp 115 120 125Thr Leu Met Ile Ser Arg Thr Pro Lys Val Thr Cys
Val Val Val Asp 130 135 140Val Ser Gln Glu Asn Pro Glu Val Gln Phe
Ser Trp Tyr Val Asp Gly145 150 155 160Val Glu Val His Thr Ala Gln
Thr Arg Pro Lys Glu Glu Gln Phe Asn 165 170 175Ser Thr Tyr Arg Val
Val Ser Val Leu Leu Ile Gln His Gln Asp Trp 180 185 190Leu Asn Gly
Lys Glu Phe Lys Cys Lys Val Asn Asn Lys Asp Leu Pro 195 200 205Ala
Pro Ile Thr Arg Ile Ile Ser Lys Ala Lys Gly Gln Thr Arg Glu 210 215
220Pro Gln Val Tyr Thr Leu Pro Pro Pro Thr Glu Glu Leu Ser Arg
Ser225 230 235 240Lys Val Thr Leu Thr Cys Leu Val Thr Gly Phe Tyr
Pro Pro Asp Ile 245 250 255Asp Val Glu Trp Gln Arg Asn Gly Gln Pro
Glu Pro Glu Gly Asn Tyr 260 265 270Arg Thr Thr Pro Pro Gln Gln Asp
Val Asp Gly Thr Tyr Phe Leu Tyr 275 280 285Ser Lys Leu Ala Val Asp
Lys Ala Ser Trp Gln Arg Gly Asp Thr Phe 290 295 300Gln Cys Ala Val
Met His Glu Ala Leu His Asn His Tyr Thr305 310 31563955DNASus
scrofa 63gcccccaaga cggccccatc ggtctaccct ctggccccct gcggcaggga
cgtgtctggc 60cctaacgtgg ccttgggctg cctggcctca agctacttcc ccgagccagt
gaccgtgacc 120tggaactcgg gcgccctgac cagtggcgtg cacaccttcc
catccgtcct gcagccgtca 180gggctctact ccctcagcag catggtgacc
gtgccggcca gcagcctgtc cagcaagagc 240tacacctgca atgtcaacca
cccggccacc accaccaagg tggacaagcg tgttggaata 300caccagccgc
aaacatgtcc catatgccca gcctgtgaag ggcccgggcc ctcggccttc
360atcttccctc caaaacccaa ggacaccctc atgatctccc ggacccccaa
ggtcacgtgc 420gtggtggttg atgtgagcca ggagaacccg gaggtccagt
tctcctggta cgtggacggc 480gtagaggtgc acacggccca gacgaggcca
aaggaggagc agttcaacag cacctaccgc 540gtggtcagcg tcctgctcat
ccagcaccag gactggctga acgggaagga gttcaagtgc 600aaggtcaaca
acaaagacct cccagccccc atcacaagga tcatctccaa ggccaaaggg
660cagacccggg agccgcaggt gtacaccctg cccccaccca ccgaggagct
gtccaggagc 720aaagtcacgc taacctgcct ggtcactggc ttctacccac
ctgacatcga tgtcgagtgg 780caaagaaacg gacagccgga gccagagggc
aattaccgca ccaccccgcc ccagcaggac 840gtggacggga cctacttcct
gtacagcaag ctcgcggtgg acaaggccag ctggcagcgt 900ggagacacat
tccagtgtgc ggtgatgcac gaggctctgc acaaccacta caccc 95564323PRTSus
scrofa 64Ala Pro Lys Thr Ala Pro Ser Val Tyr Pro Leu Ala Pro Cys
Ser Arg1 5 10 15Asp Thr Ser Gly Pro Asn Val Ala Leu Gly Cys Leu Val
Ser Ser Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly
Ala Leu Thr Ser 35 40 45Gly Val His Thr Phe Pro Ser Val Leu Gln Pro
Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Met Val Thr Val Pro Ala His
Ser Leu Ser Ser Lys Arg65 70 75 80Tyr Thr Cys Asn Val Asn His Pro
Ala Thr Lys Thr Lys Val Asp Leu 85 90 95Cys Val Gly Arg Pro Cys Pro
Ile Cys Pro Gly Cys Glu Val Ala Gly 100 105 110Pro Ser Val Phe Ile
Phe Pro Pro Lys Pro Lys Asp Ile Leu Met Ile 115 120 125Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp Val Ser Lys Glu 130 135 140His
Ala Glu Val Gln Phe Ser Trp Tyr Val Asp Gly Glu Glu Val His145 150
155 160Thr Ala Glu Thr Arg Pro Lys Glu Glu Gln Phe Asn Ser Thr Tyr
Arg 165 170 175Val Val Ser Val Leu Pro Ile Gln His Glu Asp Trp Leu
Lys Gly Lys 180 185 190Glu Phe Glu Cys Lys Val Asn Asn Glu Asp Leu
Pro Gly Pro Ile Thr 195 200 205Arg Thr Ile Ser Lys Ala Lys Gly Val
Val Arg Ser Pro Glu Val Tyr 210 215 220Thr Leu Pro Pro Pro Ala Glu
Glu Leu Ser Lys Ser Ile Val Thr Leu225 230 235 240Thr Cys Leu Val
Lys Ser Ile Phe Pro Phe Ile His Val Glu Trp Lys 245 250 255Ile Asn
Gly Lys Pro Glu Pro Glu Asn Ala Tyr Arg Thr Thr Pro Pro 260 265
270Gln Glu Asp Glu Asp Arg Thr Tyr Phe Leu Tyr Ser Lys Leu Ala Val
275 280 285Asp Lys Ala Arg Trp Asp His Gly Glu Thr Phe Glu Cys Ala
Val Met 290 295 300His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
Ile Ser Lys Thr305 310 315 320Gln Gly Lys65975DNASus
scrofamisc_feature(748)..(748)n is a, c, g, or t 65gcccccaaga
cggccccatc ggtctaccct ctggccccct gcagcaggga cacgtctggc 60cctaacgtgg
ccttgggctg cctggtctca agctacttcc ccgagccagt gaccgtgacc
120tggaactcgg gcgccctgac cagtggcgtg cacaccttcc catccgtcct
gcagccgtca 180gggctctact ccctcagcag catggtgacc gtgccggccc
acagcttgtc cagcaagcgc 240tatacgtgca atgtcaacca cccagccacc
aaaaccaagg tggacctgtg tgttggacga 300ccatgtccca tatgcccagg
ctgtgaagtg gccgggccct cggtcttcat cttccctcca 360aaacccaagg
acatcctcat gatctcccgg acccccgagg tcacgtgcgt ggtggtggac
420gtcagcaagg agcacgccga ggtccagttc tcctggtacg tggacggcga
agaggtgcac 480acggccgaga cgaggccaaa ggaggagcag ttcaacagca
cctaccgcgt ggtcagcgtc 540ctgcccatcc agcacgagga ctggctgaag
gggaaggagt tcgagtgcaa ggtcaacaac 600gaagacctcc caggccccat
cacgaggacc atctccaagg ccaaaggggt ggtacggagc 660ccggaggtgt
acaccctgcc cccacccgcc gaggagctgt ccaagagcat agtcacgcta
720acctgcctgg tcaaaagcat cttcccgnct ttcatccatg ttgagtggaa
aatcaacgga 780aaaccagagc cagagaacgc atatcgcacc accccgcctc
aggaggacga ggacaggacc 840tacttcctgt acagcaagct cgcggtggac
aaggcaagat gggaccatgg agaaacattt 900gagtgtgcgg tgatgcacga
ggctctgcac aaccactaca cccagaagtc catctccaag 960actcagggta aatga
97566317PRTSus scrofa 66Ala Tyr Asn Thr Ala Pro Ser Val Tyr Pro Leu
Ala Pro Cys Gly Arg1 5 10 15Asp Val Ser Asp His Asn Val Ala Leu Gly
Cys Leu Val Ser Ser Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Thr Trp
Asn Trp Gly Ala Gln Thr Ser 35 40 45Gly Val His Thr Phe Pro Ser Val
Leu Gln Pro Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Thr Val Thr Val
Pro Ala His Ser Leu Ser Ser Lys Cys65 70 75 80Phe Thr Cys Asn Val
Asn His Pro Ala Thr Thr Thr Lys Val Asp Leu 85 90 95Cys Val Gly Lys
Lys Thr Lys Pro Arg Cys Pro Ile Cys Pro Gly Cys 100 105 110Glu Val
Ala Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp 115 120
125Ile Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
130 135 140Val Ser Lys Glu His Ala Glu Val Gln Phe Ser Trp Tyr Val
Asp Gly145 150 155 160Glu Glu Val His Thr Ala Glu Thr Arg Pro Lys
Glu Glu Gln Phe Asn 165 170 175Ser Thr Tyr Arg Val Val Ser Val Leu
Pro Ile Gln His Glu Asp Trp 180 185 190Leu Lys Gly Lys Glu Phe Glu
Cys Lys Val Asn Asn Glu Asp Leu Pro 195 200 205Gly Pro Ile Thr Arg
Thr Ile Ser Lys Ala Lys Gly Val Val Arg Ser 210 215 220Pro Glu Val
Tyr Thr Leu Pro Pro Pro Ala Glu Glu Leu Ser Lys Ser225 230 235
240Ile Val Thr Leu Thr Cys Leu Val Lys Ser Phe Phe Pro Pro Phe Ile
245 250 255His Val Glu Trp Lys Ile Asn Gly Lys Pro Glu Pro Glu Asn
Ala Tyr 260 265 270Arg Thr Thr Pro Pro Gln Glu Asp Glu Asp Gly Thr
Tyr Phe Leu Tyr 275 280 285Ser Lys Phe Ser Val Glu Lys Phe Arg Trp
His Ser Gly Gly Ile His 290 295 300Cys Ala Val Met His Glu Ala Leu
His Asn His Tyr Thr305 310 31567952DNASus scrofa 67gcctacaaca
cagctccatc ggtctaccct ctggccccct gtggcaggga cgtgtctgat 60cataacgtgg
ccttgggctg cctggtctca agctacttcc ccgagccagt gaccgtgacc
120tggaactggg gcgcccagac cagtggcgtg cacaccttcc catccgtcct
gcagccgtca 180gggctctact ccctcagcag cacggtgacc gtgccggccc
acagcttgtc cagcaagtgc 240ttcacgtgca atgtcaacca cccggccacc
accaccaagg tggacctgtg tgttggaaaa 300aagaccaagc ctcgatgtcc
catatgccca ggctgtgaag tggccgggcc ctcggtcttc 360atcttccctc
caaaacccaa ggacatcctc atgatctccc ggacccccga ggtcacgtgc
420gtggtggtgg acgtcagcaa ggagcacgcc gaggtccagt tctcctggta
cgtggacggc 480gaagaggtgc acacggccga gacgagacca aaggaggagc
agttcaacag cacttaccgc 540gtggtcagcg tcctgcccat ccagcacgag
gactggctga aggggaagga gttcgagtgc 600aaggtcaaca acgaagacct
cccaggcccc atcacgagga ccatctccaa ggccaaaggg 660gtggtacgga
gcccggaggt gtacaccctg cccccacccg ccgaggagct gtccaagagc
720atagtcacgc taacctgcct ggtcaaaagc ttcttcccgc ctttcatcca
tgttgagtgg 780aaaatcaacg gaaaaccaga gccagagaac gcataccgca
ccaccccgcc ccaggaggac 840gaggacggga cctacttcct gtacagcaag
ttctcggtgg aaaagttcag gtggcacagt 900ggaggcatcc actgtgcggt
gatgcacgag gctctgcaca accactacac cc 95268314PRTSus scrofa 68Ala Pro
Lys Thr Ala Pro Ser Val Tyr Pro Leu Ala Pro Cys Gly Arg1 5 10 15Asp
Thr Ser Gly Pro Asn Val Ala Leu Gly Cys Leu Ala Ser Ser Tyr 20 25
30Phe Pro Glu Pro Val Thr Leu Thr Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45Gly Val His Thr Phe Pro Ser Val Leu Gln Pro Ser Gly Leu Tyr
Ser 50 55 60Leu Ser Ser Met Val Thr Val Pro Ala Ser Ser Leu Ser Ser
Lys Ser65 70 75 80Tyr Thr Cys Asn Val Asn His Pro Ala Thr Thr Thr
Lys Val Asp Leu 85 90 95Cys Val Gly Arg Pro Cys Pro Ile Cys Pro Ala
Cys Glu Gly Pro Gly 100 105 110Pro Ser Val Phe Ile Phe Pro Pro Lys
Pro Lys Asp Thr Leu Met Ile 115 120 125Ser Arg Thr Pro Gln Val Thr
Cys Val Val Val Asp Val Ser Gln Glu 130 135 140Asn Pro Glu Val Gln
Phe Ser Trp Tyr Val Asp Gly Val Glu Val His145 150 155 160Thr Ala
Gln Thr Arg Pro Lys Glu Ala Gln Phe Asn Ser Thr Tyr Arg 165 170
175Val Val Ser Val Leu Pro Ile Gln His Glu Asp Trp Leu Lys Gly Lys
180 185 190Glu Phe Glu Cys Lys Val Asn Asn Lys Asp Leu Pro Ala Pro
Ile Thr 195 200 205Arg Ile Ile Ser Lys Ala Lys Gly Pro Ser Arg Glu
Pro Gln Val Tyr 210 215 220Thr Leu Ser Pro Ser Ala Glu Glu Leu Ser
Arg Ser Lys Val Ser Ile225 230 235 240Thr Cys Leu Val Thr Gly Phe
Tyr Pro Pro Asp Ile Asp Val Glu Trp 245 250 255Lys Ser Asn Gly Gln
Pro Glu Pro Glu Gly Asn Tyr Arg Thr Thr Pro 260 265 270Pro Gln Gln
Asp Val Asp Gly Thr Tyr Phe Leu Tyr Ser Lys Leu Ala 275 280 285Val
Asp Lys Ala Ser Trp Gln Arg Gly Asp Pro Phe Gln Cys Ala Val 290 295
300Met His Glu Ala Leu His Asn His Tyr Thr305 31069943DNASus scrofa
69gcccccaaga cggccccatc ggtctaccct ctggccccct gcggcaggga cacgtctggc
60cctaacgtgg ccttgggctg cctggcctca agctacttcc ccgagccagt gaccctgacc
120tggaactcgg gcgccctgac cagtggcgtg cataccttcc catccgtcct
gcagccgtca 180gggctctact ccctcagcag catggtgacc gtgccggcca
gcagcctgtc cagcaagagc 240tacacctgca atgtcaacca cccggccacc
accaccaagg tggacctgtg tgttggacga 300ccatgtccca tatgcccagc
ctgtgaaggg cccgggccct cggtcttcat cttccctcca 360aaacccaagg
acaccctcat gatctcccgg acaccccagg tcacgtgcgt ggtggtagat
420gtgagccagg aaaacccgga ggtccagttc tcctggtatg tggacggtgt
agaggtgcac 480acggcccaga cgaggccaaa ggaggcgcag ttcaacagca
cctaccgtgt ggtcagcgtc 540ctgcccatcc agcacgagga ctggctgaag
gggaaggagt tcgagtgcaa ggtcaacaac 600aaagacctcc cagcccccat
cacaaggatc atctccaagg ccaaagggcc gagccgggag 660ccgcaggtgt
acaccctgtc cccatccgcc gaggagctgt ccaggagcaa agtcagcata
720acctgcctgg tcactggctt ctacccacct gacatcgatg tcgagtggaa
gagcaacgga 780cagccggagc cagagggcaa ttaccgcacc accccgcccc
agcaggacgt ggacgggacc 840tacttcctgt acagcaagct cgcggtggac
aaggccagct ggcagcgtgg agacccattc 900cagtgtgcgg tgatgcacga
ggctctgcac aaccactaca ccc 94370320PRTSus scrofa 70Ala Pro Lys Thr
Ala Pro Ser Val Tyr Pro Leu Ala Pro Cys Gly Arg1 5 10 15Asp Thr Ser
Gly Pro Asn Val Ala Leu Gly Cys Leu Ala Ser Ser Tyr 20 25 30Phe Pro
Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly
Val His Thr Phe Pro Ser Val Leu Gln Pro Ser Gly Leu Tyr Ser 50 55
60Leu Ser Ser Thr Val Thr Val Pro Ala Arg Ser Ser Ser Arg Lys Cys65
70 75 80Phe Thr Cys Asn Val Asn His Pro Ala Thr Thr Thr Lys Val Asp
Leu 85 90 95Cys Val Gly Arg Pro Cys Pro Ile Cys Pro Ala Cys Glu Gly
Asn Gly 100
105 110Pro Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
Ile 115 120 125Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser Gln Glu 130 135 140Asn Pro Glu Val Gln Phe Ser Trp Tyr Val Asp
Gly Glu Glu Val His145 150 155 160Thr Ala Glu Thr Arg Pro Lys Glu
Glu Gln Phe Asn Ser Thr Tyr Arg 165 170 175Val Val Ser Val Leu Pro
Ile Gln His Gln Asp Trp Leu Lys Gly Lys 180 185 190Glu Phe Glu Cys
Lys Val Asn Asn Lys Asp Leu Pro Ala Pro Ile Thr 195 200 205Arg Ile
Ile Ser Lys Ala Lys Gly Pro Ser Arg Glu Pro Gln Val Tyr 210 215
220Thr Leu Ser Pro Ser Ala Glu Glu Leu Ser Arg Ser Lys Val Ser
Ile225 230 235 240Thr Cys Leu Val Thr Gly Phe Tyr Pro Pro Asp Ile
Asp Val Glu Trp 245 250 255Lys Ser Asn Gly Gln Pro Glu Pro Glu Gly
Asn Tyr Arg Ser Thr Pro 260 265 270Pro Gln Glu Asp Glu Asp Gly Thr
Tyr Phe Leu Tyr Ser Lys Leu Ala 275 280 285Val Asp Lys Ala Arg Leu
Gln Ser Gly Gly Ile His Cys Ala Val Met 290 295 300His Glu Ala Leu
His Asn His Tyr Thr Gln Lys Ser Ile Ser Lys Thr305 310 315
32071960DNASus scrofa 71gcccccaaga cggccccatc ggtctaccct ctggccccct
gcggcaggga cacgtctggc 60cctaacgtgg ccttgggctg cctggcctca agctacttcc
ccgagccagt gaccgtgacc 120tggaactcgg gcgccctgac cagtggcgtg
cacaccttcc catccgtcct gcagccgtca 180gggctctact ccctcagcag
cacggtgacc gtgccggcca ggagctcgtc cagaaagtgc 240ttcacgtgca
atgtcaacca cccggccacc accaccaagg tggacctgtg tgttggacga
300ccatgtccca tatgcccagc ctgtgaaggg aacgggccct cggtcttcat
cttccctcca 360aaacccaagg acaccctcat gatctcccgg acccccgagg
tcacgtgcgt ggtggtagat 420gtgagccagg aaaacccgga ggtccagttc
tcctggtacg tggacggcga agaggtgcac 480acggccgaga cgaggccaaa
ggaggagcag ttcaacagca cctaccgtgt ggtcagcgtc 540ctgcccatcc
agcaccagga ctggctgaag ggaaaggagt tcgagtgcaa ggtcaacaac
600aaagacctcc cagcccccat cacaaggatc atctccaagg ccaaagggcc
gagccgggag 660ccgcaggtgt acaccctgtc cccatccgcc gaggagctgt
ccaggagcaa agtcagcata 720acctgcctgg tcactggctt ctacccacct
gacatcgatg tcgagtggaa gagcaacgga 780cagccggagc cagagggcaa
ttaccgctcc accccgcccc aggaggacga ggacgggacc 840tacttcctgt
acagcaaact cgcggtggac aaggcgaggt tgcagagtgg aggcatccac
900tgtgcggtga tgcacgaggc tctgcacaac cactacaccc agaagtccat
ctccaagact 96072266PRTBubalus bubalis 72Ser Gly Val His Thr Phe Pro
Ala Val Leu Gln Ser Ser Gly Leu Tyr1 5 10 15Ser Leu Ser Ser Thr Val
Thr Ala Pro Ala Ser Ala Thr Lys Ser Gln 20 25 30Thr Phe Thr Cys Asn
Val Ala His Pro Ala Ser Ser Thr Lys Val Asp 35 40 45Lys Ala Val Val
Pro Pro Cys Arg Pro Lys Pro Cys Asp Cys Cys Pro 50 55 60Pro Pro Glu
Leu Pro Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys65 70 75 80Pro
Lys Asp Thr Leu Thr Ile Ser Gly Thr Pro Glu Val Thr Cys Val 85 90
95Val Val Asp Val Gly His Asp Asp Pro Glu Val Lys Phe Ser Trp Phe
100 105 110Val Asp Asp Val Glu Val Asn Thr Ala Arg Thr Lys Pro Arg
Glu Glu 115 120 125Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Ala Leu
Pro Ile Gln His 130 135 140Asn Asp Trp Thr Gly Gly Lys Glu Phe Lys
Cys Lys Val Tyr Asn Glu145 150 155 160Gly Leu Pro Ala Pro Ile Val
Arg Thr Ile Ser Arg Thr Lys Gly Gln 165 170 175Ala Arg Glu Pro Gln
Val Tyr Val Leu Ala Pro Pro Gln Asp Glu Leu 180 185 190Ser Lys Ser
Thr Val Ser Ile Thr Cys Met Val Thr Gly Phe Tyr Pro 195 200 205Asp
Tyr Ile Ala Val Glu Trp Gln Lys Asp Gly Gln Pro Glu Ser Glu 210 215
220Asp Lys Tyr Gly Thr Thr Pro Pro Gln Leu Asp Ser Asp Gly Ser
Tyr225 230 235 240Phe Leu Tyr Ser Arg Leu Arg Val Asn Lys Asn Ser
Trp Gln Glu Gly 245 250 255Gly Ala Tyr Thr Cys Val Val Met His Glu
260 26573801DNABubalus bubalis 73gagcggcgtg cacaccttcc cggccgtcct
tcagtcctcc gggctctact ctctcagcag 60cacggtgacc gcgcccgcca gcgccacaaa
aagccagacc ttcacctgca acgtagccca 120cccggccagc agcaccaagg
tggacaaggc tgttgttccc ccatgcagac cgaaaccctg 180tgattgctgc
ccaccccctg agctccccgg aggaccctct gtcttcatct tcccaccaaa
240acccaaggac accctcacaa tctctggaac tcctgaggtc acgtgtgtgg
tggtggacgt 300gggccacgat gaccccgagg tgaagttctc ctggttcgtg
gacgatgtgg aggtaaacac 360agccaggacg aagccaagag aggagcagtt
caacagcacc taccgcgtgg tcagcgccct 420gcccatccag cacaacgact
ggactggagg aaaggagttc aagtgcaagg tctacaatga 480aggcctccca
gcccccatcg tgaggaccat ctccaggacc aaagggcagg cccgggagcc
540gcaggtgtac gtcctggccc caccccagga cgagctcagc aaaagcacgg
tcagcatcac 600ttgcatggtc actggcttct acccagacta catcgccgta
gagtggcaga aagatgggca 660gcctgagtca gaggacaaat atggcacgac
cccgccccag ctggacagcg atggctccta 720cttcctgtac agcaggctca
gggtgaacaa gaacagctgg caagaaggag gcgcctacac 780gtgtgtagtg
atgcatgagg c 80174309PRTBulalus bubalis 74Ala Ser Ile Thr Ala Pro
Lys Val Tyr Pro Leu Thr Ser Cys Arg Gly1 5 10 15Glu Thr Ser Ser Ser
Thr Val Thr Leu Gly Cys Leu Val Ser Ser Tyr 20 25 30Met Pro Glu Pro
Val Thr Val Thr Trp Asn Ser Gly Ala Leu Lys Ser 35 40 45Gly Val His
Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser
Ser Thr Val Thr Ala Pro Ala Ser Ala Thr Lys Ser Gln Thr65 70 75
80Phe Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Thr
85 90 95Ala Val Gly Phe Ser Ser Asp Cys Cys Lys Phe Pro Lys Pro Cys
Val 100 105 110Arg Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Pro Lys
Asp Thr Leu 115 120 125Met Ile Thr Gly Asn Pro Glu Val Thr Cys Val
Val Val Asp Val Gly 130 135 140Arg Asp Asn Pro Glu Val Gln Phe Ser
Trp Phe Val Gly Asp Val Glu145 150 155 160Val His Thr Gly Arg Ser
Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr 165 170 175Tyr Arg Val Val
Ser Thr Leu Pro Ile Gln His Asn Asp Trp Thr Gly 180 185 190Gly Lys
Glu Phe Lys Cys Lys Val Asn Asn Lys Gly Leu Pro Ala Pro 195 200
205Ile Val Arg Thr Ile Ser Arg Thr Lys Gly Gln Ala Arg Glu Pro Gln
210 215 220Val Tyr Val Leu Ala Pro Pro Gln Glu Glu Leu Ser Lys Ser
Thr Val225 230 235 240Ser Val Thr Cys Met Val Thr Gly Phe Tyr Pro
Asp Tyr Ile Ala Val 245 250 255Glu Trp His Arg Asp Arg Gln Ala Glu
Ser Glu Asp Lys Tyr Arg Thr 260 265 270Thr Pro Pro Gln Leu Asp Ser
Asp Gly Ser Tyr Phe Leu Tyr Ser Arg 275 280 285Leu Lys Val Asn Lys
Asn Ser Trp Gln Glu Gly Gly Ala Tyr Thr Cys 290 295 300Val Val Met
His Glu30575929DNABubalus bubalis 75gcctccatca cagccccgaa
agtctaccct ctgacttctt gccgcgggga aacgtccagc 60tccaccgtga ccctgggctg
cctggtctcc agctacatgc ccgagccggt gaccgtgacc 120tggaactcgg
gtgccctgaa gagcggcgtg cacaccttcc cggccgtcct tcagtcctct
180gggctctact ctctcagcag cacggtgacc gcgcccgcca gcgccacaaa
aagccagacc 240ttcacctgca acgtagccca cccggccagc agcaccaagg
tggacacggc tgttgggttc 300tccagtgact gctgcaagtt tcctaagcct
tgtgtgaggg gaccatctgt cttcatcttc 360ccgccgaaac ccaaagacac
cctgatgatc acaggaaatc ccgaggtcac atgtgtggtg 420gtggacgtgg
gccgggataa ccccgaggtg cagttctcct ggttcgtggg tgatgtggag
480gtgcacacgg gcaggtcgaa gccgagagag gagcagttca acagcaccta
ccgcgtggtc 540agcaccctgc ccatccagca caatgactgg actggaggaa
aggagttcaa gtgcaaggtc 600aacaacaaag gcctcccagc ccccatcgtg
aggaccatct ccaggaccaa agggcaggcc 660cgggagccgc aggtgtacgt
cctggcccca ccccaggaag agctcagcaa aagcacggtc 720agcgtcactt
gcatggtcac tggcttctac ccagactaca tcgccgtaga gtggcataga
780gaccggcagg ctgagtcgga ggacaagtac cgcacgaccc cgccccagct
ggacagcgat 840ggctcctact tcctgtacag caggctcaag gtgaacaaga
acagctggca agaaggaggc 900gcctacacgt gtgtagtgat gcatgaggc
92976352PRTBubalus bubalis 76Ala Ser Thr Thr Ala Pro Lys Val Tyr
Pro Leu Ala Ser Ser Cys Gly1 5 10 15Asp Thr Ser Ser Ser Thr Val Thr
Leu Gly Cys Leu Val Ser Ser Tyr 20 25 30Met Pro Glu Pro Val Thr Val
Thr Trp Asn Ser Gly Ala Leu Lys Asn 35 40 45Gly Val His Thr Phe Pro
Ala Val Arg Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Met Val
Thr Met Pro Thr Ser Thr Ala Gly Thr Gln Thr65 70 75 80Phe Thr Cys
Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Thr 85 90 95Ala Val
Thr Ala Arg His Pro Val Pro Lys Thr Pro Glu Thr Pro Ile 100 105
110His Pro Val Lys Pro Pro Thr Gln Glu Pro Arg Asp Glu Lys Thr Pro
115 120 125Cys Gln Cys Pro Lys Cys Pro Glu Pro Leu Gly Gly Leu Ser
Val Phe 130 135 140Ile Phe Pro Pro Lys Pro Lys Asp Thr Leu Thr Ile
Ser Gly Thr Pro145 150 155 160Glu Val Thr Cys Val Val Val Asp Val
Gly Gln Asp Asp Pro Glu Val 165 170 175Gln Phe Ser Trp Phe Val Asp
Asp Val Glu Val His Thr Ala Arg Met 180 185 190Lys Pro Arg Glu Glu
Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Ala 195 200 205Leu Pro Ile
Gln His Gln Asp Trp Leu Arg Glu Lys Glu Phe Lys Cys 210 215 220Lys
Val Asn Asn Lys Gly Leu Pro Ala Pro Ile Val Arg Thr Ile Ser225 230
235 240Arg Thr Lys Gly Gln Ala Arg Glu Pro Gln Val Tyr Val Leu Ala
Pro 245 250 255Pro Arg Glu Glu Leu Ser Lys Ser Thr Leu Ser Leu Thr
Cys Leu Ile 260 265 270Thr Gly Phe Tyr Pro Glu Glu Val Asp Val Glu
Trp Gln Arg Asn Gly 275 280 285Gln Pro Glu Ser Glu Asp Lys Tyr His
Thr Thr Pro Pro Gln Leu Asp 290 295 300Ala Asp Gly Ser Tyr Phe Leu
Tyr Ser Arg Leu Arg Val Asn Arg Ser305 310 315 320Ser Trp Gln Glu
Gly Asp His Tyr Thr Cys Ala Val Met His Glu Ala 325 330 335Leu Arg
Asn His Tyr Lys Glu Lys Pro Ile Ser Arg Ser Pro Gly Lys 340 345
350771059DNABubalus bubalis 77gcctccacca cagccccgaa agtctaccct
ctggcatcca gctgcgggga cacgtccagc 60tccaccgtga ccctgggctg cctggtctcc
agctacatgc ccgagccggt gaccgtgacc 120tggaactcgg gtgccctgaa
gaacggcgtg cacaccttcc cggccgtccg gcagtcctcc 180gggctctact
ctctcagcag catggtgacc atgcccacca gcaccgcagg aacccagacc
240ttcacctgca acgtagccca cccggccagc agcaccaagg tggacacggc
tgtcactgca 300aggcatccgg tcccgaagac accagagaca cctatccatc
ctgtaaaacc cccaacccag 360gagcccagag atgaaaagac accctgccag
tgtcccaaat gcccagaacc tctgggagga 420ctgtctgtct tcatcttccc
accgaaaccc aaggacaccc tcacaatctc tggaacgccc 480gaggtcacgt
gtgtggtggt ggacgtgggc caggatgacc ccgaagtgca gttctcctgg
540ttcgtggatg acgtggaggt gcacacagcc aggatgaagc caagagagga
gcagttcaac 600agcacctacc gcgtggtcag cgccctgccc atccagcacc
aggactggct gcgggaaaag 660gagttcaagt gcaaggtcaa caacaaaggc
ctcccggccc ccatcgtgag gaccatctcc 720aggaccaaag ggcaggcccg
ggagccacag gtgtatgtcc tggccccacc ccgggaagag 780ctcagcaaaa
gcacgctcag cctcacctgc ctaatcaccg gcttctaccc agaagaggta
840gacgtggagt ggcagagaaa tgggcagcct gagtcagagg acaagtacca
cacgacccca 900ccccagctgg acgctgacgg ctcctacttc ctgtacagca
ggctcagggt gaacaggagc 960agctggcagg aaggagacca ctacacgtgt
gcagtgatgc atgaagcttt acggaatcac 1020tacaaagaga agcccatctc
gaggtctccg ggtaaatga 105978105PRTBubalus bubalis 78Gln Pro Lys Ser
Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Thr Glu1 5 10 15Glu Leu Ser
Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe 20 25 30Tyr Pro
Gly Ser Met Thr Val Ala Arg Lys Ala Asp Gly Ser Thr Ile 35 40 45Thr
Arg Asn Val Glu Thr Thr Arg Ala Ser Lys Gln Ser Asn Ser Lys 50 55
60Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Gly Ser Glu Trp Lys Ser65
70 75 80Lys Gly Ser Tyr Ser Cys Glu Val Thr His Glu Gly Ser Thr Val
Thr 85 90 95Lys Thr Val Lys Pro Ser Glu Cys Ser 100
10579318DNABubalus buballis 79cagcccaagt ccgcaccctc agtcaccctg
ttcccaccct ccacggagga gctcagcgcc 60aacaaggcca ccctggtgtg tctcatcagc
gacttctacc cgggtagcat gaccgtggcc 120aggaaggcag acggcagcac
catcacccgg aacgtggaga ccacccgggc ctccaaacag 180agcaacagca
agtacgcggc cagcagctac ctgagcctga cgggcagcga gtggaaatcg
240aaaggcagtt acagctgcga ggtcacgcac gaggggagca ccgtgacaaa
gacagtgaag 300ccctcagagt gttcttag 31880229PRTHomo sapiens 80Glu Ser
Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro Glu Phe1 5 10 15Leu
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 20 25
30Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
35 40 45Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly
Val 50 55 60Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe
Asn Ser65 70 75 80Thr Tyr Arg Val Val Ser Val Leu Thr Val Val His
Gln Asp Trp Leu 85 90 95Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys Gly Leu Pro Ser 100 105 110Ser Ile Glu Lys Thr Ile Ser Lys Ala
Lys Gly Gln Pro Arg Glu Pro 115 120 125Gln Val Tyr Thr Leu Pro Pro
Ser Gln Glu Glu Met Thr Lys Asn Gln 130 135 140Val Ser Leu Thr Cys
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala145 150 155 160Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 165 170
175Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu
180 185 190Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser
Cys Ser 195 200 205Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
Lys Ser Leu Ser 210 215 220Leu Ser Leu Gly Lys22581690DNAHomo
sapiens 81gagtccaaat atggtccccc gtgcccatca tgcccagcac ctgagttcct
ggggggacca 60tcagtcttcc tgttcccccc aaaacccaag gacactctca tgatctcccg
gacccctgag 120gtcacgtgcg tggtggtgga cgtgagccag gaagaccccg
aggtccagtt caactggtac 180gtggatggcg tggaggtgca taatgccaag
acaaagccgc gggaggagca gttcaacagc 240acgtaccgtg tggtcagcgt
cctcaccgtc gtgcaccagg actggctgaa cggcaaggag 300tacaagtgca
aggtctccaa caaaggcctc ccgtcctcca tcgagaaaac catctccaaa
360gccaaagggc agccccgaga gccacaggtg tacaccctgc ccccatccca
ggaggagatg 420accaagaacc aggtcagcct gacctgcctg gtcaaaggct
tctaccccag cgacatcgcc 480gtggagtggg agagcaatgg gcagccggag
aacaactaca agaccacgcc tcccgtgctg 540gactccgacg gctccttctt
cctctacagc aggctaaccg tggacaagag caggtggcag 600gaggggaatg
tcttctcatg ctccgtgatg catgaggctc tgcacaacca ctacacgcag
660aagagcctct ccctgtctct gggtaaatga 69082217PRTHomo sapiens 82Ala
Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys1 5 10
15Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
20 25 30Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp
Tyr 35 40 45Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
Glu Glu 50 55 60Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
Val Leu His65 70 75 80Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys 85 90 95Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile
Ser Lys Ala Lys Gly Gln 100 105 110Pro Arg Glu Pro Gln Val Tyr Thr
Leu Pro Pro Ser Gln Glu Glu Met 115 120 125Thr Lys Asn Gln Val Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 130 135 140Ser Asp Ile Ala
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn145 150 155 160Tyr
Lys
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 165 170
175Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val
180 185 190Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
Thr Gln 195 200 205Lys Ser Leu Ser Leu Ser Leu Gly Lys 210
21583654DNAHomo sapiens 83gcacctgagt tcctgggggg accatcagtc
ttcctgttcc ccccaaaacc caaggacact 60ctcatgatct cccggacccc tgaggtcacg
tgcgtggtgg tggacgtgag ccaggaagac 120cccgaggtcc agttcaactg
gtacgtggat ggcgtggagg tgcataatgc caagacaaag 180ccgcgggagg
agcagttcaa cagcacgtac cgtgtggtca gcgtcctcac cgtcctgcac
240caggactggc tgaacggcaa ggagtacaag tgcaaggtct ccaacaaagg
cctcccgtcc 300tccatcgaga aaaccatctc caaagccaaa gggcagcccc
gagagccaca ggtgtacacc 360ctgcccccat cccaggagga gatgaccaag
aaccaggtca gcctgacctg cctggtcaaa 420ggcttctacc ccagcgacat
cgccgtggag tgggagagca atgggcagcc ggagaacaac 480tacaagacca
cgcctcccgt gctggactcc gacggctcct tcttcctcta cagcaagctc
540accgtggaca agagcaggtg gcaggagggg aacgtcttct catgctccgt
gatgcatgag 600gctctgcaca accactacac gcagaagagc ctctccctgt
ctctgggtaa atga 65484329PRTBos taurus 84Ala Ser Thr Thr Ala Pro Lys
Val Tyr Pro Leu Ser Ser Cys Cys Gly1 5 10 15Asp Lys Ser Ser Ser Thr
Val Thr Leu Gly Cys Leu Val Ser Ser Tyr 20 25 30Met Pro Glu Pro Val
Thr Val Thr Trp Asn Ser Gly Ala Leu Lys Ser 35 40 45Gly Val His Thr
Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser
Met Val Thr Val Pro Gly Ser Thr Ser Gly Gln Thr Phe65 70 75 80Thr
Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Ala 85 90
95Val Asp Pro Thr Cys Lys Pro Ser Pro Cys Asp Cys Cys Pro Pro Pro
100 105 110Glu Leu Pro Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys
Pro Lys 115 120 125Asp Thr Leu Thr Ile Ser Gly Thr Pro Glu Val Thr
Cys Val Val Val 130 135 140Asp Val Gly His Asp Asp Pro Glu Val Lys
Phe Ser Trp Phe Val Asp145 150 155 160Asp Val Glu Val Asn Thr Ala
Thr Thr Lys Pro Arg Glu Glu Gln Phe 165 170 175Asn Ser Thr Tyr Arg
Val Val Ser Ala Leu Arg Ile Gln His Gln Asp 180 185 190Trp Thr Gly
Gly Lys Glu Phe Lys Cys Lys Val His Asn Glu Gly Leu 195 200 205Pro
Ala Pro Ile Val Arg Thr Ile Ser Arg Thr Lys Gly Pro Ala Arg 210 215
220Glu Pro Gln Val Tyr Val Leu Ala Pro Pro Gln Glu Glu Leu Ser
Lys225 230 235 240Ser Thr Val Ser Leu Thr Cys Met Val Thr Ser Phe
Tyr Pro Asp Tyr 245 250 255Ile Ala Val Glu Trp Gln Arg Asn Gly Gln
Pro Glu Ser Glu Asp Lys 260 265 270Tyr Gly Thr Thr Pro Pro Gln Leu
Asp Ala Asp Ser Ser Tyr Phe Leu 275 280 285Tyr Ser Lys Leu Arg Val
Asp Arg Asn Ser Trp Gln Glu Gly Asp Thr 290 295 300Tyr Thr Cys Val
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln305 310 315 320Lys
Ser Thr Ser Lys Ser Ala Gly Lys 32585329PRTBos taurus 85Ala Ser Thr
Thr Ala Pro Lys Val Tyr Pro Leu Ser Ser Cys Cys Gly1 5 10 15Asp Lys
Ser Ser Ser Thr Val Thr Leu Gly Cys Leu Val Ser Ser Tyr 20 25 30Met
Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ala Leu Lys Ser 35 40
45Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60Leu Ser Ser Met Val Thr Val Pro Gly Ser Thr Ser Gly Gln Thr
Phe65 70 75 80Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val
Asp Lys Ala 85 90 95Val Asp Pro Thr Cys Lys Pro Ser Pro Cys Asp Cys
Cys Pro Pro Pro 100 105 110Glu Leu Pro Gly Gly Pro Ser Val Phe Ile
Phe Pro Pro Lys Pro Lys 115 120 125Asp Thr Leu Thr Ile Ser Gly Thr
Pro Glu Val Thr Cys Val Val Val 130 135 140Asp Val Gly His Asp Asp
Pro Glu Val Lys Phe Ser Trp Phe Val Asp145 150 155 160Asp Val Glu
Val Asn Thr Ala Thr Thr Lys Pro Arg Glu Glu Gln Phe 165 170 175Asn
Ser Thr Tyr Arg Val Val Ser Ala Leu Arg Ile Gln His Gln Asp 180 185
190Trp Thr Gly Gly Lys Glu Phe Lys Cys Lys Val His Asn Glu Gly Leu
195 200 205Pro Ala Pro Ile Val Arg Thr Ile Ser Arg Thr Lys Gly Pro
Ala Arg 210 215 220Glu Pro Gln Val Tyr Val Leu Ala Pro Pro Gln Glu
Glu Leu Ser Lys225 230 235 240Ser Thr Val Ser Leu Thr Cys Met Val
Thr Ser Phe Tyr Pro Asp Tyr 245 250 255Ile Ala Val Glu Trp Gln Arg
Asn Gly Gln Pro Glu Ser Glu Asp Lys 260 265 270Tyr Gly Thr Thr Pro
Pro Gln Leu Asp Ala Asp Ser Ser Tyr Phe Leu 275 280 285Tyr Ser Lys
Leu Arg Val Asp Arg Asn Ser Trp Gln Glu Gly Asp Thr 290 295 300Tyr
Thr Cys Val Val Met His Glu Ala Leu His Asn His Tyr Thr Gln305 310
315 320Lys Ser Thr Ser Lys Ser Ala Gly Lys 32586329PRTBos taurus
86Ala Ser Thr Thr Ala Pro Lys Val Tyr Pro Leu Ser Ser Cys Cys Gly1
5 10 15Asp Lys Ser Ser Ser Thr Val Thr Leu Gly Cys Leu Val Ser Ser
Tyr 20 25 30Met Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ala Leu
Lys Ser 35 40 45Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly
Leu Tyr Ser 50 55 60Leu Ser Ser Met Val Thr Val Pro Gly Ser Thr Ser
Gly Thr Gln Thr65 70 75 80Phe Thr Cys Asn Val Ala His Pro Ala Ser
Ser Thr Lys Val Asp Lys 85 90 95Ala Val Asp Pro Arg Cys Lys Thr Thr
Cys Asp Cys Cys Pro Pro Pro 100 105 110Glu Leu Pro Gly Gly Pro Ser
Val Phe Ile Phe Pro Pro Lys Pro Lys 115 120 125Asp Thr Leu Thr Ile
Ser Gly Thr Pro Glu Val Thr Cys Val Val Val 130 135 140Asp Val Gly
His Asp Asp Pro Glu Val Lys Phe Ser Trp Phe Val Asp145 150 155
160Asp Val Glu Val Asn Thr Ala Thr Thr Lys Pro Arg Glu Glu Gln Phe
165 170 175Asn Ser Thr Tyr Arg Val Val Ser Ala Leu Arg Ile Gln His
Gln Asp 180 185 190Trp Thr Gly Gly Lys Glu Phe Lys Cys Lys Val His
Asn Glu Gly Leu 195 200 205Pro Ala Pro Ile Val Arg Thr Ile Ser Arg
Thr Lys Gly Pro Ala Arg 210 215 220Glu Pro Gln Val Tyr Val Leu Ala
Pro Pro Gln Glu Glu Leu Ser Lys225 230 235 240Ser Thr Val Ser Leu
Thr Cys Met Val Thr Ser Phe Tyr Pro Asp Tyr 245 250 255Ile Ala Val
Glu Trp Gln Arg Asn Gly Gln Pro Glu Ser Glu Asp Lys 260 265 270Tyr
Gly Thr Thr Pro Pro Gln Leu Asp Ala Asp Gly Ser Tyr Phe Leu 275 280
285Tyr Ser Arg Leu Arg Val Asp Arg Asn Ser Trp Gln Glu Gly Asp Thr
290 295 300Tyr Thr Cys Val Val Met His Glu Ala Leu His Asn His Tyr
Thr Gln305 310 315 320Lys Ser Thr Ser Lys Ser Ala Gly Lys
32587326PRTBos taurus 87Ala Ser Thr Thr Ala Pro Lys Val Tyr Pro Leu
Ala Ser Ser Cys Gly1 5 10 15Asp Thr Ser Ser Ser Thr Val Thr Leu Gly
Cys Leu Val Ser Ser Tyr 20 25 30Met Pro Glu Pro Val Thr Val Thr Trp
Asn Ser Gly Ala Leu Lys Ser 35 40 45Gly Val His Thr Phe Pro Ala Val
Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Met Val Thr Val
Pro Ala Ser Ser Ser Gly Gln Thr Phe65 70 75 80Thr Cys Asn Val Ala
His Pro Ala Ser Ser Thr Lys Val Asp Lys Ala 85 90 95Val Gly Val Ser
Ile Asp Cys Ser Lys Cys His Asn Gln Pro Cys Val 100 105 110Arg Glu
Pro Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Thr Leu 115 120
125Met Ile Thr Gly Thr Pro Glu Val Thr Cys Val Val Val Asn Val Gly
130 135 140His Asp Asn Pro Glu Val Gln Phe Ser Trp Phe Val Asp Asp
Val Glu145 150 155 160Val His Thr Ala Arg Ser Lys Pro Arg Glu Glu
Gln Phe Asn Ser Thr 165 170 175Tyr Arg Val Val Ser Ala Leu Pro Ile
Gln His Gln Asp Trp Thr Gly 180 185 190Gly Lys Glu Phe Lys Cys Lys
Val Asn Asn Lys Gly Leu Ser Ala Pro 195 200 205Ile Val Arg Ile Ile
Ser Arg Ser Lys Gly Pro Ala Arg Glu Pro Gln 210 215 220Val Tyr Val
Leu Asp Pro Pro Lys Glu Glu Leu Ser Lys Ser Thr Leu225 230 235
240Ser Val Thr Cys Met Val Thr Gly Phe Tyr Pro Glu Asp Val Ala Val
245 250 255Glu Trp Gln Arg Asn Arg Gln Thr Glu Ser Glu Asp Lys Tyr
Arg Thr 260 265 270Thr Pro Pro Gln Leu Asp Thr Asp Arg Ser Tyr Phe
Leu Tyr Ser Lys 275 280 285Leu Arg Val Asp Arg Asn Ser Trp Gln Glu
Gly Asp Ala Tyr Thr Cys 290 295 300Val Val Met His Glu Ala Leu His
Asn His Tyr Met Gln Lys Ser Thr305 310 315 320Ser Lys Ser Ala Gly
Lys 32588326PRTBos taurus 88Ala Ser Thr Thr Ala Pro Lys Val Tyr Pro
Leu Ser Ser Cys Cys Gly1 5 10 15Asp Lys Ser Ser Ser Thr Val Thr Leu
Gly Cys Leu Val Ser Ser Tyr 20 25 30Met Pro Glu Pro Val Thr Val Thr
Trp Asn Ser Gly Ala Leu Lys Ser 35 40 45Gly Val His Thr Phe Pro Ala
Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Met Val Thr
Val Pro Gly Ser Thr Ser Gly Gln Thr Phe65 70 75 80Thr Cys Asn Val
Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Ala 85 90 95Val Gly Val
Ser Ser Asp Cys Ser Lys Pro Asn Asn Gln His Cys Val 100 105 110Arg
Glu Pro Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Thr Leu 115 120
125Met Ile Thr Gly Thr Pro Glu Val Thr Cys Val Val Val Asn Val Gly
130 135 140His Asp Asn Pro Glu Val Gln Phe Ser Trp Phe Val Asp Asp
Val Glu145 150 155 160Val His Thr Ala Arg Thr Lys Pro Arg Glu Glu
Gln Phe Asn Ser Thr 165 170 175Tyr Arg Val Val Ser Ala Leu Pro Ile
Gln His Gln Asp Trp Thr Gly 180 185 190Gly Lys Glu Phe Lys Cys Lys
Val Asn Ile Lys Gly Leu Ser Ala Ser 195 200 205Ile Val Arg Ile Ile
Ser Arg Ser Lys Gly Pro Ala Arg Glu Pro Gln 210 215 220Val Tyr Val
Leu Asp Pro Pro Lys Glu Glu Leu Ser Lys Ser Thr Val225 230 235
240Ser Val Thr Cys Met Val Ile Gly Phe Tyr Pro Glu Asp Val Asp Val
245 250 255Glu Trp Gln Arg Asp Arg Gln Thr Glu Ser Glu Asp Lys Tyr
Arg Thr 260 265 270Thr Pro Pro Gln Leu Asp Ala Asp Arg Ser Tyr Phe
Leu Tyr Ser Lys 275 280 285Leu Arg Val Asp Arg Asn Ser Trp Gln Arg
Gly Asp Thr Tyr Thr Cys 290 295 300Val Val Met His Glu Ala Leu His
Asn His Tyr Met Gln Lys Ser Thr305 310 315 320Ser Lys Ser Ala Gly
Lys 32589327PRTBos taurus 89Ala Ser Thr Thr Ala Pro Lys Val Tyr Pro
Leu Ser Ser Cys Cys Gly1 5 10 15Asp Lys Ser Ser Ser Gly Val Thr Leu
Gly Cys Leu Val Ser Ser Tyr 20 25 30Met Pro Glu Pro Val Thr Val Thr
Trp Asn Ser Gly Ala Leu Lys Ser 35 40 45Gly Val His Thr Phe Pro Ala
Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Met Val Thr
Val Pro Ala Ser Ser Ser Gly Thr Gln Thr65 70 75 80Phe Thr Cys Asn
Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys 85 90 95Ala Val Gly
Val Ser Ser Asp Cys Ser Lys Pro Asn Asn Gln His Cys 100 105 110Val
Arg Glu Pro Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Thr 115 120
125Leu Met Ile Thr Gly Thr Pro Glu Val Thr Cys Val Val Val Asn Val
130 135 140Gly His Asp Asn Pro Glu Val Gln Phe Ser Trp Phe Val Asp
Asp Val145 150 155 160Glu Val His Thr Ala Arg Thr Lys Pro Arg Glu
Glu Gln Phe Asn Ser 165 170 175Thr Tyr Arg Val Val Ser Ala Leu Pro
Ile Gln His Gln Asp Trp Thr 180 185 190Gly Gly Lys Glu Phe Lys Cys
Lys Val Asn Ile Lys Gly Leu Ser Ala 195 200 205Ser Ile Val Arg Ile
Ile Ser Arg Ser Lys Gly Pro Ala Arg Glu Pro 210 215 220Gln Val Tyr
Val Leu Asp Pro Pro Lys Glu Glu Leu Ser Lys Ser Thr225 230 235
240Val Ser Leu Thr Cys Met Val Ile Gly Phe Tyr Pro Glu Asp Val Asp
245 250 255Val Glu Trp Gln Arg Asp Arg Gln Thr Glu Ser Glu Asp Lys
Tyr Arg 260 265 270Thr Thr Pro Pro Gln Leu Asp Ala Asp Arg Ser Tyr
Phe Leu Tyr Ser 275 280 285Lys Leu Arg Val Asp Arg Asn Ser Trp Gln
Arg Gly Asp Thr Tyr Thr 290 295 300Cys Val Val Met His Glu Ala Leu
His Asn His Tyr Met Gln Lys Ser305 310 315 320Thr Ser Lys Ser Ala
Gly Lys 32590352PRTBos taurus 90Ala Ser Thr Thr Ala Pro Lys Val Tyr
Pro Leu Ala Ser Ser Cys Gly1 5 10 15Asp Thr Ser Ser Ser Thr Val Thr
Leu Gly Cys Leu Val Ser Ser Tyr 20 25 30Met Pro Glu Pro Val Thr Val
Thr Trp Asn Ser Gly Ala Leu Lys Ser 35 40 45Gly Val His Thr Phe Pro
Ala Val Arg Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Met Val
Thr Val Pro Ala Ser Ser Ser Glu Thr Gln Thr65 70 75 80Phe Thr Cys
Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys 85 90 95Ala Val
Thr Ala Arg Arg Pro Val Pro Thr Thr Pro Lys Thr Thr Ile 100 105
110Pro Pro Gly Lys Pro Thr Thr Pro Lys Ser Glu Val Glu Lys Thr Pro
115 120 125Cys Gln Cys Ser Lys Cys Pro Glu Pro Leu Gly Gly Leu Ser
Val Phe 130 135 140Ile Phe Pro Pro Lys Pro Lys Asp Thr Leu Thr Ile
Ser Gly Thr Pro145 150 155 160Glu Val Thr Cys Val Val Val Asp Val
Gly Gln Asp Asp Pro Glu Val 165 170 175Gln Phe Ser Trp Phe Val Asp
Asp Val Glu Val His Thr Ala Arg Thr 180 185 190Lys Pro Arg Glu Glu
Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Ala 195 200 205Leu Arg Ile
Gln His Gln Asp Trp Leu Gln Gly Lys Glu Phe Lys Cys 210 215 220Lys
Val Asn Asn Lys Gly Leu Pro Ala Pro Ile Val Arg Thr Ile Ser225 230
235 240Arg Thr Lys Gly Gln Ala Arg Glu Pro Gln Val Tyr Val Leu Ala
Pro 245 250 255Pro Arg Glu Glu Leu Ser Lys Ser Thr Leu Ser Leu Thr
Cys Leu Ile 260 265 270Thr Gly Phe Tyr Pro Glu Glu Ile Asp Val Glu
Trp Gln Arg Asn Gly 275 280 285Gln Pro Glu Ser Glu Asp Lys Tyr His
Thr Thr Ala Pro Gln Leu Asp 290 295 300Ala Asp Gly Ser Tyr Phe Leu
Tyr Ser Lys Leu Arg Val Asn Lys Ser305 310 315 320Ser Trp Gln Glu
Gly Asp His Tyr Thr Cys Ala Val Met His Glu Ala
325 330 335Leu Arg Asn His Tyr Lys Glu Lys Ser Ile Ser Arg Ser Pro
Gly Lys 340 345 35091352PRTBos taurus 91Ala Ser Thr Thr Ala Pro Lys
Val Tyr Pro Leu Ala Ser Arg Cys Gly1 5 10 15Asp Thr Ser Ser Ser Thr
Val Thr Leu Gly Cys Leu Val Ser Ser Tyr 20 25 30Met Pro Glu Pro Val
Thr Val Thr Trp Asn Ser Gly Ala Leu Lys Ser 35 40 45Gly Val His Thr
Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser
Met Val Thr Val Pro Ala Ser Thr Ser Glu Thr Gln Thr65 70 75 80Phe
Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys 85 90
95Ala Val Thr Ala Arg Arg Pro Val Pro Thr Thr Pro Lys Thr Thr Ile
100 105 110Pro Pro Gly Lys Pro Thr Thr Gln Glu Ser Glu Val Glu Lys
Thr Pro 115 120 125Cys Gln Cys Ser Lys Cys Pro Glu Pro Leu Gly Gly
Leu Ser Val Phe 130 135 140Ile Phe Pro Pro Lys Pro Lys Asp Thr Leu
Thr Ile Ser Gly Thr Pro145 150 155 160Glu Val Thr Cys Val Val Val
Asp Val Gly Gln Asp Asp Pro Glu Val 165 170 175Gln Phe Ser Trp Phe
Val Asp Asp Val Glu Val His Thr Ala Arg Thr 180 185 190Lys Pro Arg
Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Ala 195 200 205Leu
Arg Ile Gln His Gln Asp Trp Leu Gln Gly Lys Glu Phe Lys Cys 210 215
220Lys Val Asn Asn Lys Gly Leu Pro Ala Pro Ile Val Arg Thr Ile
Ser225 230 235 240Arg Thr Lys Gly Gln Ala Arg Glu Pro Gln Val Tyr
Val Leu Ala Pro 245 250 255Pro Arg Glu Glu Leu Ser Lys Ser Thr Leu
Ser Leu Thr Cys Leu Ile 260 265 270Thr Gly Phe Tyr Pro Glu Glu Ile
Asp Val Glu Trp Gln Arg Asn Gly 275 280 285Gln Pro Glu Ser Glu Asp
Lys Tyr His Thr Thr Ala Pro Gln Leu Asp 290 295 300Ala Asp Gly Ser
Tyr Phe Leu Tyr Ser Arg Leu Arg Val Asn Lys Ser305 310 315 320Ser
Trp Gln Glu Gly Asp His Tyr Thr Cys Ala Val Met His Glu Ala 325 330
335Leu Arg Asn His Tyr Lys Glu Lys Ser Ile Ser Arg Ser Pro Gly Lys
340 345 35092990DNABos taurus 92gcctccacca cagccccgaa agtctaccct
ctgagttctt gctgcgggga caagtccagc 60tccaccgtga ccctgggctg cctggtctcc
agctacatgc ccgagccggt gaccgtgacc 120tggaactcgg gtgccctgaa
gagcggcgtg cacaccttcc cggctgtcct tcagtcctcc 180gggctgtact
ctctcagcag catggtgacc gtgcccggca gcacctcagg acagaccttc
240acctgcaacg tagcccaccc ggccagcagc accaaggtgg acaaggctgt
tgatcccaca 300tgcaaaccat caccctgtga ctgttgccca ccccctgagc
tccccggagg accctctgtc 360ttcatcttcc caccgaaacc caaggacacc
ctcacaatct cgggaacgcc cgaggtcacg 420tgtgtggtgg tggacgtggg
ccacgatgac cccgaggtga agttctcctg gttcgtggac 480gacgtggagg
taaacacagc cacgacgaag ccgagagagg agcagttcaa cagcacctac
540cgcgtggtca gcgccctgcg catccagcac caggactgga ctggaggaaa
ggagttcaag 600tgcaaggtcc acaacgaagg cctcccggcc cccatcgtga
ggaccatctc caggaccaaa 660gggccggccc gggagccgca ggtgtatgtc
ctggccccac cccaggaaga gctcagcaaa 720agcacggtca gcctcacctg
catggtcacc agcttctacc cagactacat cgccgtggag 780tggcagagaa
acgggcagcc tgagtcggag gacaagtacg gcacgacccc gccccagctg
840gacgccgaca gctcctactt cctgtacagc aagctcaggg tggacaggaa
cagctggcag 900gaaggagaca cctacacgtg tgtggtgatg cacgaggccc
tgcacaatca ctacacgcag 960aagtccacct ctaagtctgc gggtaaatga
99093990DNABos taurus 93gcctccacca cagccccgaa agtctaccct ctgagttctt
gctgcgggga caagtccagc 60tccaccgtga ccctgggctg cctggtctcc agctacatgc
ccgagccggt gaccgtgacc 120tggaactcgg gtgccctgaa gagcggcgtg
cacaccttcc cggccgtcct tcagtcctcc 180gggctgtact ctctcagcag
catggtgacc gtgcccggca gcacctcagg acagaccttc 240acctgcaacg
tagcccaccc ggccagcagc accaaggtgg acaaggctgt tgatcccaca
300tgcaaaccat caccctgtga ctgttgccca ccccctgagc tccccggagg
accctctgtc 360ttcatcttcc caccgaaacc caaggacacc ctcacaatct
cgggaacgcc cgaggtcacg 420tgtgtggtgg tggacgtggg ccacgatgac
cccgaggtga agttctcctg gttcgtggac 480gacgtggagg taaacacagc
cacgacgaag ccgagagagg agcagttcaa cagcacctac 540cgcgtggtca
gcgccctgcg catccagcac caggactgga ctggaggaaa ggagttcaag
600tgcaaggtcc acaacgaagg cctcccggcc cccatcgtga ggaccatctc
caggaccaaa 660gggccggccc gggagccgca ggtgtatgtc ctggccccac
cccaggaaga gctcagcaaa 720agcacggtca gcctcacctg catggtcacc
agcttctacc cagactacat cgccgtggag 780tggcagagaa acgggcagcc
tgagtcggag gacaagtacg gcacgacccc gccccagctg 840gacgccgaca
gctcctactt cctgtacagc aagctcaggg tggacaggaa cagctggcag
900gaaggagaca cctacacgtg tgtggtgatg cacgaggccc tgcacaatca
ctacacgcag 960aagtccacct ctaagtctgc gggtaaatga 99094990DNABos
taurus 94gcctccacca cagccccgaa agtctaccct ctgagttctt gctgcgggga
caagtccagc 60tccaccgtga ccctgggctg cctggtctcc agctacatgc ccgagccggt
gaccgtgacc 120tggaactcgg gtgccctgaa gagcggcgtg cacaccttcc
cggccgtcct tcagtcctcc 180gggctctact ctctcagcag catggtgacc
gtgcccggca gcacctcagg aacccagacc 240ttcacctgca acgtagccca
cccggccagc agcaccaagg tggacaaggc tgttgatccc 300agatgcaaaa
caacctgtga ctgttgccca ccgcctgagc tccctggagg accctctgtc
360ttcatcttcc caccgaaacc caaggacacc ctcacaatct cgggaacgcc
cgaggtcacg 420tgtgtggtgg tggacgtggg ccacgatgac cccgaggtga
agttctcctg gttcgtggac 480gacgtggagg taaacacagc cacgacgaag
ccgagagagg agcagttcaa cagcacctac 540cgcgtggtca gcgccctgcg
catccagcac caggactgga ctggaggaaa ggagttcaag 600tgcaaggtcc
acaacgaagg cctcccagcc cccatcgtga ggaccatctc caggaccaaa
660gggccggccc gggagccgca ggtgtatgtc ctggccccac cccaggaaga
gctcagcaaa 720agcacggtca gcctcacctg catggtcacc agcttctacc
cagactacat cgccgtggag 780tggcagagaa atgggcagcc tgagtcagag
gacaagtacg gcacgacccc tccccagctg 840gacgccgacg gctcctactt
cctgtacagc aggctcaggg tggacaggaa cagctggcag 900gaaggagaca
cctacacgtg tgtggtgatg cacgaggccc tgcacaatca ctacacgcag
960aagtccacct ctaagtctgc gggtaaatga 99095981DNABos taurus
95gcctccacca cagccccgaa agtctaccct ctggcatcca gctgcggaga cacatccagc
60tccaccgtga ccctgggctg cctggtgtcc agctacatgc ccgagccggt gaccgtgacc
120tggaactcgg gtgccctgaa gagcggcgtg cacaccttcc cggctgtcct
tcagtcctcc 180gggctctact ctctcagcag catggtgacc gtgcccgcca
gcagctcagg acagaccttc 240acctgcaacg tagcccaccc ggccagcagc
accaaggtgg acaaggctgt tggggtctcc 300attgactgct ccaagtgtca
taaccagcct tgcgtgaggg aaccatctgt cttcatcttc 360ccaccgaaac
ccaaagacac cctgatgatc acaggaacgc ccgaggtcac gtgtgtggtg
420gtgaacgtgg gccacgataa ccccgaggtg cagttctcct ggttcgtgga
tgacgtggag 480gtgcacacgg ccaggtcgaa gccaagagag gagcagttca
acagcacgta ccgcgtggtc 540agcgccctgc ccatccagca ccaggactgg
actggaggaa aggagttcaa gtgcaaggtc 600aacaacaaag gcctctcggc
ccccatcgtg aggatcatct ccaggagcaa agggccggcc 660cgggagccgc
aggtgtatgt cctggaccca cccaaggaag agctcagcaa aagcacgctc
720agcgtcacct gcatggtcac cggcttctac ccagaagatg tagccgtgga
gtggcagaga 780aaccggcaga ctgagtcgga ggacaagtac cgcacgaccc
cgccccagct ggacaccgac 840cgctcctact tcctgtacag caagctcagg
gtggacagga acagctggca ggaaggagac 900gcctacacgt gtgtggtgat
gcacgaggcc ctgcacaatc actacatgca gaagtccacc 960tctaagtctg
cgggtaaatg a 98196981DNABos taurus 96gcctccacca cagccccgaa
agtctaccct ctgagttctt gctgcgggga caagtccagc 60tccaccgtga ccctgggctg
cctggtgtcc agctacatgc ccgagccggt gaccgtgacc 120tggaactcgg
gtgccctgaa gagcggcgtg cacaccttcc cggccgtcct tcagtcctcc
180gggctctact ctctcagcag catggtgacc gtgcccggca gcacctcagg
acagaccttc 240acctgcaacg tagcccaccc ggccagcagc accaaggtgg
acaaggctgt tggggtctcc 300agtgactgct ccaagcctaa taaccagcat
tgcgtgaggg aaccatctgt cttcatcttc 360ccaccgaaac ccaaagacac
cctgatgatc acaggaacgc ccgaggtcac gtgtgtggtg 420gtgaacgtgg
gccacgataa ccccgaggtg cagttctcct ggttcgtgga cgacgtggag
480gtgcacacgg ccaggacgaa gccgagagag gagcagttca acagcacgta
ccgcgtggtc 540agcgccctgc ccatccagca ccaggactgg actggaggaa
aggagttcaa gtgcaaggtc 600aacatcaaag gcctctcggc ctccatcgtg
aggatcatct ccaggagcaa agggccggcc 660cgggagccgc aggtgtatgt
cctggaccca cccaaggaag agctcagcaa aagcacggtc 720agcgtcacct
gcatggtcat cggcttctac ccagaagatg tagacgtgga gtggcagaga
780gaccggcaga ctgagtcgga ggacaagtac cgcacgaccc cgccccagct
ggacgccgac 840cgctcctact tcctgtacag caagctcagg gtggacagga
acagctggca gagaggagac 900acctacacgt gtgtggtgat gcacgaggcc
ctgcacaatc actacatgca gaagtccacc 960tctaagtctg cgggtaaatg a
98197984DNABos taurus 97gcctccacca cagccccgaa agtctaccct ctgagttctt
gctgcgggga caagtccagc 60tcgggggtga ccctgggctg cctggtctcc agctacatgc
ccgagccggt gaccgtgacc 120tggaactcgg gtgccctgaa gagcggcgtg
cacaccttcc cggccgtcct tcagtcctcc 180gggctctact ctctcagcag
catggtgacc gtgcccgcca gcagctcagg aacccagacc 240ttcacctgca
acgtagccca cccggccagc agcaccaagg tggacaaggc tgttggggtc
300tccagtgact gctccaagcc taataaccag cattgcgtga gggaaccatc
tgtcttcatc 360ttcccaccga aacccaaaga caccctgatg atcacaggaa
cgcccgaggt cacgtgtgtg 420gtggtgaacg tgggccacga taaccccgag
gtgcagttct cctggttcgt ggacgacgtg 480gaggtgcaca cggccaggac
gaagccgaga gaggagcagt tcaacagcac gtaccgcgtg 540gtcagcgccc
tgcccatcca gcaccaggac tggactggag gaaaggagtt caagtgcaag
600gtcaacatca aaggcctctc ggcctccatc gtgaggatca tctccaggag
caaagggccg 660gcccgggagc cgcaggtgta tgtcctggac ccacccaagg
aagagctcag caaaagcacg 720gtcagcctca cctgcatggt catcggcttc
tacccagaag atgtagacgt ggagtggcag 780agagaccggc agactgagtc
ggaggacaag taccgcacga ccccgcccca gctggacgcc 840gaccgctcct
acttcctgta cagcaagctc agggtggaca ggaacagctg gcagagagga
900gacacctaca cgtgtgtggt gatgcacgag gccctgcaca atcactacat
gcagaagtcc 960acctctaagt ctgcgggtaa atga 984981059DNABos taurus
98gcctccacca cagccccgaa agtctaccct ctggcatcca gctgcggaga cacatccagc
60tccaccgtga ccctgggctg cctggtctcc agctacatgc ccgagccggt gaccgtgacc
120tggaactcgg gtgccctgaa gagcggcgtg cacaccttcc cggccgtccg
gcagtcctct 180gggctgtact ctctcagcag catggtgact gtgcccgcca
gcagctcaga aacccagacc 240ttcacctgca acgtagccca cccggccagc
agcaccaagg tggacaaggc tgtcactgca 300aggcgtccag tcccgacgac
gccaaagaca actatccctc ctggaaaacc cacaacccca 360aagtctgaag
ttgaaaagac accctgccag tgttccaaat gcccagaacc tctgggagga
420ctgtctgtct tcatcttccc accgaaaccc aaggacaccc tcacaatctc
gggaacgccc 480gaggtcacgt gtgtggtggt ggacgtgggc caggatgacc
ccgaggtgca gttctcctgg 540ttcgtggacg acgtggaggt gcacacggcc
aggacgaagc cgagagagga gcagttcaac 600agcacctacc gcgtggtcag
cgccctgcgc atccagcacc aggactggct gcagggaaag 660gagttcaagt
gcaaggtcaa caacaaaggc ctcccggccc ccattgtgag gaccatctcc
720aggaccaaag ggcaggcccg ggagccgcag gtgtatgtcc tggccccacc
ccgggaagag 780ctcagcaaaa gcacgctcag cctcacctgc ctgatcaccg
gtttctaccc agaagagata 840gacgtggagt ggcagagaaa tgggcagcct
gagtcggagg acaagtacca cacgaccgca 900ccccagctgg atgctgacgg
ctcctacttc ctgtacagca agctcagggt gaacaagagc 960agctggcagg
aaggagacca ctacacgtgt gcagtgatgc acgaagcttt acggaatcac
1020tacaaagaga agtccatctc gaggtctccg ggtaaatga 1059991059DNABos
taurus 99gcctccacca cagccccgaa agtctaccct ctggcatccc gctgcggaga
cacatccagc 60tccaccgtga ccctgggctg cctggtctcc agctacatgc ccgagccggt
gaccgtgacc 120tggaactcgg gtgccctgaa gagtggcgtg cacaccttcc
cggccgtcct tcagtcctcc 180gggctgtact ctctcagcag catggtgacc
gtgcccgcca gcacctcaga aacccagacc 240ttcacctgca acgtagccca
cccggccagc agcaccaagg tggacaaggc tgtcactgca 300aggcgtccag
tcccgacgac gccaaagaca accatccctc ctggaaaacc cacaacccag
360gagtctgaag ttgaaaagac accctgccag tgttccaaat gcccagaacc
tctgggagga 420ctgtctgtct tcatcttccc accgaaaccc aaggacaccc
tcacaatctc gggaacgccc 480gaggtcacgt gtgtggtggt ggacgtgggc
caggatgacc ccgaggtgca gttctcctgg 540ttcgtggacg acgtggaggt
gcacacggcc aggacgaagc cgagagagga gcagttcaac 600agcacctacc
gcgtggtcag cgccctgcgc atccagcacc aggactggct gcagggaaag
660gagttcaagt gcaaggtcaa caacaaaggc ctcccggccc ccattgtgag
gaccatctcc 720aggaccaaag ggcaggcccg ggagccgcag gtgtatgtcc
tggccccacc ccgggaagag 780ctcagcaaaa gcacgctcag cctcacctgc
ctgatcaccg gtttctaccc agaagagata 840gacgtggagt ggcagagaaa
tgggcagcct gagtcggagg acaagtacca cacgaccgca 900ccccagctgg
atgctgacgg ctcctacttc ctgtacagca ggctcagggt gaacaagagc
960agctggcagg aaggagacca ctacacgtgt gcagtgatgc atgaagcttt
acggaatcac 1020tacaaagaga agtccatctc gaggtctccg ggtaaatga
1059100105PRTBos taurus 100Gln Pro Lys Ser Pro Pro Ser Val Thr Leu
Phe Pro Pro Ser Thr Glu1 5 10 15Glu Leu Asn Gly Asn Lys Ala Thr Leu
Val Cys Leu Ile Ser Asp Phe 20 25 30Tyr Pro Gly Ser Val Thr Val Val
Trp Lys Ala Asp Gly Ser Thr Ile 35 40 45Thr Arg Asn Val Glu Thr Thr
Arg Ala Ser Lys Gln Ser Asn Ser Lys 50 55 60Tyr Ala Ala Ser Ser Tyr
Leu Ser Leu Thr Ser Ser Asp Trp Lys Ser65 70 75 80Lys Gly Ser Tyr
Ser Cys Glu Val Thr His Glu Gly Ser Thr Val Thr 85 90 95Lys Thr Val
Lys Pro Ser Glu Cys Ser 100 105101318DNABos taurus 101cagcccaagt
ccccaccctc ggtcaccctg ttcccgccct ccacggagga gctcaacggc 60aacaaggcca
ccctggtgtg tctcatcagc gacttctacc cgggtagcgt gaccgtggtc
120tggaaggcag acggcagcac catcacccgc aacgtggaga ccacccgggc
ctccaaacag 180agcaacagca agtacgcggc cagcagctac ctgagcctga
cgagcagcga ctggaaatcg 240aaaggcagtt acagctgcga ggtcacgcac
gaggggagca ccgtgacgaa gacagtgaag 300ccctcagagt gttcttag
318102328PRTBos taurus 102Ala Ser Thr Thr Ala Pro Lys Val Tyr Pro
Leu Ser Ser Cys Cys Gly1 5 10 15Asp Lys Ser Ser Ser Thr Val Thr Leu
Gly Cys Leu Val Ser Ser Tyr 20 25 30Met Pro Glu Pro Val Thr Val Thr
Trp Asn Ser Gly Ala Leu Lys Ser 35 40 45Gly Val His Thr Phe Pro Ala
Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Met Val Thr
Val Pro Gly Ser Thr Ser Gly Gln Thr Phe65 70 75 80Thr Cys Asn Val
Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Ala 85 90 95Val Asp Pro
Thr Cys Lys Pro Ser Pro Cys Asp Cys Cys Pro Pro Pro 100 105 110Pro
Val Ala Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp 115 120
125Thr Leu Thr Ile Ser Gly Thr Pro Glu Val Thr Cys Val Val Val Asp
130 135 140Val Gly His Asp Asp Pro Glu Val Lys Phe Ser Trp Phe Val
Asp Asp145 150 155 160Val Glu Val Asn Thr Ala Thr Thr Lys Pro Arg
Glu Glu Gln Phe Asn 165 170 175Ser Thr Tyr Arg Val Val Ser Ala Leu
Arg Ile Gln His Gln Asp Trp 180 185 190Thr Gly Gly Lys Glu Phe Lys
Cys Lys Val His Asn Glu Gly Leu Pro 195 200 205Ser Ser Ile Val Arg
Thr Ile Ser Arg Thr Lys Gly Pro Ala Arg Glu 210 215 220Pro Gln Val
Tyr Val Leu Ala Pro Pro Gln Glu Glu Leu Ser Lys Ser225 230 235
240Thr Val Ser Leu Thr Cys Met Val Thr Ser Phe Tyr Pro Asp Tyr Ile
245 250 255Ala Val Glu Trp Gln Arg Asn Gly Gln Pro Glu Ser Glu Asp
Lys Tyr 260 265 270Gly Thr Thr Pro Pro Gln Leu Asp Ala Asp Ser Ser
Tyr Phe Leu Tyr 275 280 285Ser Lys Leu Arg Val Asp Arg Asn Ser Trp
Gln Glu Gly Asp Thr Tyr 290 295 300Thr Cys Val Val Met His Glu Ala
Leu His Asn His Tyr Thr Gln Lys305 310 315 320Ser Thr Ser Lys Ser
Ala Gly Lys 325103987DNABos taurus 103gctagcacaa ctgctcctaa
ggtgtacccc ctgagctctt gctgcggcga caagtctagc 60agcaccgtga ccctcggatg
cctcgtcagc agctatatgc ctgagccagt tacagtgaca 120tggaattctg
gtgcccttaa gtccggcgtc cataccttcc ctgctgtgct gcagtcctct
180ggcctgtaca gtttgtcctc tatggtgaca gtacccggtt ccacctccgg
acagaccttt 240acctgtaatg tggctcatcc cgcctcctcc acaaaggtgg
ataaggctgt tgaccctacc 300tgtaaaccca gtccatgcga ctgctgtccc
ccccctccag ttgccggacc ctcagtcttt 360attttcccac ccaaacccaa
agacaccctg acaatctctg gaacaccaga agtcacctgc 420gtcgtcgtgg
atgtgggcca cgacgatcct gaggtaaaat tctcatggtt cgtcgacgat
480gtggaagtga atacagctac tacaaaacct cgcgaagagc agtttaactc
tacctatcga 540gtggtttctg ctttgcggat tcagcatcag gattggacag
gcggcaaaga gtttaaatgt 600aaagtccata acgagggact tccttctagt
atcgtgcgca ctatcagtag aactaaaggg 660cctgctcggg aacctcaggt
gtacgtcctg gcacctccac aggaagagct gagtaagtct 720acagtttctc
tgacttgtat ggtaacatct ttttatccag attacatcgc agttgaatgg
780cagaggaacg ggcagccaga gagtgaggat aagtacggga ctactccacc
acagctggac 840gcagactcaa gttacttcct gtactcaaag ctgagggttg
acagaaactc atggcaggag 900ggggacactt acacttgcgt agttatgcac
gaggcacttc acaaccacta cactcagaag 960agtacttcaa agagtgcagg gaagtaa
987104318DNAArtificial SequenceA synthetic codon-optimized sequence
104cagcctaaga gtcctccttc tgtaacactc tttcccccct ctaccgagga
actcaacggc 60aataaagcta ccttggtttg ccttatttct gatttctacc ccgggtctgt
gaccgtggtg 120tggaaagctg atgggtccac cattactcgg aatgtggaaa
ccacccgggc
ttctaagcag 180tccaactcta aatacgcagc atcctcctat ttgagtctta
ctagtagtga ctggaagtca 240aagggtagtt acagttgcga agtcacacat
gaaggttcaa cagtgacaaa gacagtcaag 300ccctcagagt gctcatag
318105238PRTArtificial SequenceA synthetic chimeric L chain 105Met
Glu Ser Gln Thr His Val Leu Ile Ser Leu Leu Leu Ser Val Ser1 5 10
15Gly Thr Tyr Gly Asp Ile Ala Ile Thr Gln Ser Pro Ser Ser Val Ala
20 25 30Val Ser Val Gly Glu Thr Val Thr Leu Ser Cys Lys Ser Ser Gln
Ser 35 40 45Leu Leu Tyr Ser Glu Asn Gln Lys Asp Tyr Leu Gly Trp Tyr
Gln Gln 50 55 60Lys Pro Gly Gln Thr Pro Lys Pro Leu Ile Tyr Trp Ala
Thr Asn Arg65 70 75 80His Thr Gly Val Pro Asp Arg Phe Thr Gly Ser
Gly Ser Gly Thr Asp 85 90 95Phe Thr Leu Ile Ile Ser Ser Val Gln Ala
Glu Asp Leu Ala Asp Tyr 100 105 110Tyr Cys Gly Gln Tyr Leu Val Tyr
Pro Phe Thr Phe Gly Pro Gly Thr 115 120 125Lys Leu Glu Leu Lys Gln
Pro Lys Ser Pro Pro Ser Val Thr Leu Phe 130 135 140Pro Pro Ser Thr
Glu Glu Leu Asn Gly Asn Lys Ala Thr Leu Val Cys145 150 155 160Leu
Ile Ser Asp Phe Tyr Pro Gly Ser Val Thr Val Val Trp Lys Ala 165 170
175Asp Gly Ser Thr Ile Thr Arg Asn Val Glu Thr Thr Arg Ala Ser Lys
180 185 190Gln Ser Asn Ser Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu
Thr Ser 195 200 205Ser Asp Trp Lys Ser Lys Gly Ser Tyr Ser Cys Glu
Val Thr His Glu 210 215 220Gly Ser Thr Val Thr Lys Thr Val Lys Pro
Ser Glu Cys Ser225 230 235106465PRTArtificial SequenceA synthetic
chimeric H chain 106Met Gly Trp Ser Gln Ile Ile Leu Phe Leu Val Ala
Ala Ala Thr Cys1 5 10 15Val His Ser Gln Val Gln Leu Gln Gln Ser Gly
Ala Glu Leu Val Lys 20 25 30Pro Gly Ser Ser Val Lys Ile Ser Cys Lys
Ala Ser Gly Tyr Thr Phe 35 40 45Thr Ser Asn Phe Met His Trp Val Lys
Gln Gln Pro Gly Asn Gly Leu 50 55 60Glu Trp Ile Gly Trp Ile Tyr Pro
Glu Tyr Gly Asn Thr Lys Tyr Asn65 70 75 80Gln Lys Phe Asp Gly Lys
Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser 85 90 95Thr Ala Tyr Met Gln
Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val 100 105 110Tyr Phe Cys
Ala Ser Glu Glu Ala Val Ile Ser Leu Val Tyr Trp Gly 115 120 125Gln
Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Thr Ala Pro Lys 130 135
140Val Tyr Pro Leu Ser Ser Cys Cys Gly Asp Lys Ser Ser Ser Thr
Val145 150 155 160Thr Leu Gly Cys Leu Val Ser Ser Tyr Met Pro Glu
Pro Val Thr Val 165 170 175Thr Trp Asn Ser Gly Ala Leu Lys Ser Gly
Val His Thr Phe Pro Ala 180 185 190Val Leu Gln Ser Ser Gly Leu Tyr
Ser Leu Ser Ser Met Val Thr Val 195 200 205Pro Gly Ser Thr Ser Gly
Gln Thr Phe Thr Cys Asn Val Ala His Pro 210 215 220Ala Ser Ser Thr
Lys Val Asp Lys Ala Val Asp Pro Thr Cys Lys Pro225 230 235 240Ser
Pro Cys Asp Cys Cys Pro Pro Pro Pro Val Ala Gly Pro Ser Val 245 250
255Phe Ile Phe Pro Pro Lys Pro Lys Asp Thr Leu Thr Ile Ser Gly Thr
260 265 270Pro Glu Val Thr Cys Val Val Val Asp Val Gly His Asp Asp
Pro Glu 275 280 285Val Lys Phe Ser Trp Phe Val Asp Asp Val Glu Val
Asn Thr Ala Thr 290 295 300Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser
Thr Tyr Arg Val Val Ser305 310 315 320Ala Leu Arg Ile Gln His Gln
Asp Trp Thr Gly Gly Lys Glu Phe Lys 325 330 335Cys Lys Val His Asn
Glu Gly Leu Pro Ser Ser Ile Val Arg Thr Ile 340 345 350Ser Arg Thr
Lys Gly Pro Ala Arg Glu Pro Gln Val Tyr Val Leu Ala 355 360 365Pro
Pro Gln Glu Glu Leu Ser Lys Ser Thr Val Ser Leu Thr Cys Met 370 375
380Val Thr Ser Phe Tyr Pro Asp Tyr Ile Ala Val Glu Trp Gln Arg
Asn385 390 395 400Gly Gln Pro Glu Ser Glu Asp Lys Tyr Gly Thr Thr
Pro Pro Gln Leu 405 410 415Asp Ala Asp Ser Ser Tyr Phe Leu Tyr Ser
Lys Leu Arg Val Asp Arg 420 425 430Asn Ser Trp Gln Glu Gly Asp Thr
Tyr Thr Cys Val Val Met His Glu 435 440 445Ala Leu His Asn His Tyr
Thr Gln Lys Ser Thr Ser Lys Ser Ala Gly 450 455
460Lys465107717DNAArtificial SequenceA synthetic codon-optimized
sequence 107atggaatctc aaactcatgt tttgatttca ttacttctga gtgtttccgg
aacctacggt 60gatatcgcta tcactcaatc tccctcctct gttgctgtgt ctgtgggcga
aaccgttacc 120ctgtcctgca agtccagtca gtctcttctc tactccgaga
atcaaaagga ctacctgggc 180tggtaccaac agaagcccgg ccagacccca
aagccactga tatactgggc aaccaacagg 240cacaccggag tgcccgacag
gttcacaggc agtggatctg gcaccgactt taccttgatc 300atttcaagcg
tgcaggctga agatctggcc gactactact gtggtcagta tctggtgtat
360cctttcactt tcgggccagg gacaaaactc gagctcaaac agcctaagag
tcctccttct 420gtaacactct ttcccccctc taccgaggaa ctcaacggca
ataaagctac cttggtttgc 480cttatttctg atttctaccc cgggtctgtg
accgtggtgt ggaaagctga tgggtccacc 540attactcgga atgtggaaac
cacccgggct tctaagcagt ccaactctaa atacgcagca 600tcctcctatt
tgagtcttac tagtagtgac tggaagtcaa agggtagtta cagttgcgaa
660gtcacacatg aaggttcaac agtgacaaag acagtcaagc cctcagagtg ctcatag
7171081398DNAArtificial SequenceA synthetic codon-optimized
sequence 108atggggtggt cccagattat attgttcctc gtcgccgccg ccacttgcgt
acacagccaa 60gtgcaacttc aacaaagcgg tgcagaactg gtaaagcccg gtagctctgt
gaaaatatcc 120tgtaaagcca gtggctacac atttaccagc aactttatgc
actgggtgaa gcaacagccc 180ggaaatggct tggagtggat tggctggatc
tatcccgaat atggtaacac caagtataat 240cagaagttcg acggtaaggc
caccctcacc gccgataagt catcctccac cgcctatatg 300cagctcagca
gcctgaccag cgaggattcc gctgtgtact tctgtgccag cgaagaggct
360gtgatctcat tggtgtattg gggacagggc accctcgtca ccgtgtccag
cgctagcaca 420actgctccta aggtgtaccc cctgagctct tgctgcggcg
acaagtctag cagcaccgtg 480accctcggat gcctcgtcag cagctatatg
cctgagccag ttacagtgac atggaattct 540ggtgccctta agtccggcgt
ccataccttc cctgctgtgc tgcagtcctc tggcctgtac 600agtttgtcct
ctatggtgac agtacccggt tccacctccg gacagacctt tacctgtaat
660gtggctcatc ccgcctcctc cacaaaggtg gataaggctg ttgaccctac
ctgtaaaccc 720agtccatgcg actgctgtcc cccccctcca gttgccggac
cctcagtctt tattttccca 780cccaaaccca aagacaccct gacaatctct
ggaacaccag aagtcacctg cgtcgtcgtg 840gatgtgggcc acgacgatcc
tgaggtaaaa ttctcatggt tcgtcgacga tgtggaagtg 900aatacagcta
ctacaaaacc tcgcgaagag cagtttaact ctacctatcg agtggtttct
960gctttgcgga ttcagcatca ggattggaca ggcggcaaag agtttaaatg
taaagtccat 1020aacgagggac ttccttctag tatcgtgcgc actatcagta
gaactaaagg gcctgctcgg 1080gaacctcagg tgtacgtcct ggcacctcca
caggaagagc tgagtaagtc tacagtttct 1140ctgacttgta tggtaacatc
tttttatcca gattacatcg cagttgaatg gcagaggaac 1200gggcagccag
agagtgagga taagtacggg actactccac cacagctgga cgcagactca
1260agttacttcc tgtactcaaa gctgagggtt gacagaaact catggcagga
gggggacact 1320tacacttgcg tagttatgca cgaggcactt cacaaccact
acactcagaa gagtacttca 1380aagagtgcag ggaagtaa
139810925DNAArtificial SequenceA synthetic primer 109atgaggatat
atagtgtctt aacat 2511020DNAArtificial SequenceA synthetic primer
110ttacgtctcc tcaaaatgtg 2011125DNAArtificial SequenceA synthetic
primer 111atgaggatat gtagtatctt tacat 2511221DNAArtificial
SequenceA synthetic primer 112ttacgtctcc tcaaattgtg t
2111320DNAArtificial SequenceA synthetic primer 113atgaggatat
atagtgtctt 2011420DNAArtificial SequenceA synthetic primer
114gccactcagg acttggtgat 2011520DNAArtificial SequenceA synthetic
primer 115gggggtttac tgttgcttga 2011619DNAArtificial SequenceA
synthetic primer 116ttacgtctcc tcaaattgt 1911729DNAArtificial
SequenceA synthetic primer 117gaagatctat ggggaccccg cgggcgccg
2911829DNAArtificial SequenceA synthetic primer 118gacccgggga
ggggccagga gcagtgtcc 2911928DNAArtificial SequenceA synthetic
primer 119ccgctcgaga tgaggatata tagtgtct 2812028DNAArtificial
SequenceA synthetic primer 120atcccgggcg tctcctcaaa atgtgtag
2812125DNAArtificial SequenceA synthetic primer 121actaagctta
tggggacccc gcggg 2512226DNAArtificial SequenceA synthetic primer
122actcccgggg aggggccaag agcagt 2612329DNAArtificial SequenceA
synthetic primer 123ccgctcgaga tgaggatatg tagtatctt
2912429DNAArtificial SequenceA synthetic primer 124atcccgggcg
tctcctcaaa ttgtgtatc 2912528DNAArtificial SequenceA synthetic
primer 125gacgctagca tgaggatata tagtgtct 2812628DNAArtificial
SequenceA synthetic primer 126gctctgatat ccctcgtttt tgctggat
2812729DNAArtificial SequenceA synthetic primer 127gacgctagca
tgaggatatg tagtatctt 2912829DNAArtificial SequenceA synthetic
primer 128agcttgatat ccctctttct tgctggatc 2912930DNAArtificial
SequenceA synthetic primer 129cgcggatatc atggattaca cagcgaagtg
3013029DNAArtificial SequenceA synthetic primer 130cggggtaccc
cagagctgtt gctggttat 2913130DNAArtificial SequenceA synthetic
primer 131cgcggctagc atgagaatgt ttagtgtctt 3013249DNAArtificial
SequenceA synthetic primer 132cgcggatatc ttaatggtga tggtgatggt
gagtcctctc acttgctgg 4913332DNAArtificial SequenceA synthetic
primer 133atatgcggcc gcatggggac cccgcgggcg ct 3213430DNAArtificial
SequenceA synthetic primer 134gcgcaagctt tcagaggggc caggagcagt
3013535DNAArtificial SequenceA synthetic primer 135ctagctagca
ccatgaggat atatagtgtc ttaac 3513631DNAArtificial SequenceA
synthetic primer 136caatctcgag ttacagacag aagatgactg c
3113729DNAArtificial SequenceA synthetic primer 137gctagcatga
ggatatatag tgtcttaac 2913826DNAArtificial SequenceA synthetic
primer 138gatatcattc ctcttttttg ctggat 26
* * * * *
References