U.S. patent application number 12/065450 was filed with the patent office on 2009-06-18 for transgenic avian which has foreign gene containing sequence encoding feline-derived protein and method for production thereof.
This patent application is currently assigned to KANEKA CORPORATION. Invention is credited to Tomoko Awa, Tomoyuki Nakaishi, Takuya Shindo.
Application Number | 20090158449 12/065450 |
Document ID | / |
Family ID | 37808857 |
Filed Date | 2009-06-18 |
United States Patent
Application |
20090158449 |
Kind Code |
A1 |
Nakaishi; Tomoyuki ; et
al. |
June 18, 2009 |
TRANSGENIC AVIAN WHICH HAS FOREIGN GENE CONTAINING SEQUENCE
ENCODING FELINE-DERIVED PROTEIN AND METHOD FOR PRODUCTION
THEREOF
Abstract
The present invention has for its object to provide a transgenic
bird with a foreign gene containing a feline-derived
protein-encoding sequence as transferred therein, and a method of
producing the same. The present invention provides a method of
producing a feline-derived protein by using a transgenic bird with
a method which comprises infecting an avian embryo with a
replication defective retrovirus vector containing a foreign gene
by microinjection thereof into the early heart or blood vessel
formed in the embryo and allowing the embryo to hatch.
Inventors: |
Nakaishi; Tomoyuki; (Hyogo,
JP) ; Shindo; Takuya; (Hyogo, JP) ; Awa;
Tomoko; (Hyogo, JP) |
Correspondence
Address: |
SUGHRUE MION, PLLC
2100 PENNSYLVANIA AVENUE, N.W., SUITE 800
WASHINGTON
DC
20037
US
|
Assignee: |
KANEKA CORPORATION
Osaka-shi, Osaka
JP
|
Family ID: |
37808857 |
Appl. No.: |
12/065450 |
Filed: |
August 30, 2006 |
PCT Filed: |
August 30, 2006 |
PCT NO: |
PCT/JP2006/317124 |
371 Date: |
January 13, 2009 |
Current U.S.
Class: |
800/5 ; 530/350;
800/19; 800/25 |
Current CPC
Class: |
C12N 15/8509 20130101;
C12N 2740/10052 20130101; A01K 2267/01 20130101; A61K 38/00
20130101; C12N 2830/008 20130101; A61K 47/60 20170801; A01K 67/0275
20130101; A01K 2217/052 20130101; A01K 2267/02 20130101; C12N
2740/10043 20130101; C07K 14/505 20130101; A01K 2217/05 20130101;
A61K 38/1816 20130101; C12N 7/00 20130101; A01K 2227/30 20130101;
C07K 14/005 20130101; C12N 2015/8518 20130101; C12N 2760/20222
20130101; C12N 2799/027 20130101; A61P 7/06 20180101; C12N 15/86
20130101 |
Class at
Publication: |
800/5 ; 800/19;
800/25; 530/350 |
International
Class: |
A01K 67/027 20060101
A01K067/027; C07K 14/00 20060101 C07K014/00 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 31, 2005 |
JP |
2005-251128 |
Claims
1. A transgenic bird which has a foreign gene containing a sequence
encoding a feline-derived protein.
2. A transgenic bird which has a foreign gene containing a sequence
encoding a feline-derived cytokine protein and/or a protein
substantially identical in biological activity thereto.
3. A transgenic bird which has a foreign gene containing at least a
part of a sequence encoding the feline-derived erythropoietin
identified under SEQ ID NO:1 in the sequence listing and/or a
protein substantially identical in biological activity thereto.
4. A transgenic bird which has a foreign gene containing a sequence
encoding the feline-derived erythropoietin identified under SEQ ID
NO:1 in the sequence listing and/or a protein substantially
identical in biological activity thereto.
5. The transgenic bird according to claim 1, which is produced by
using a replication defective retrovirus vector.
6. The transgenic bird according to claim 5, wherein the
replication defective retrovirus vector contains a sequence derived
from Moloney murine leukemia virus and/or Moloney murine sarcoma
virus.
7. The transgenic bird according to claim 5, wherein the
replication defective retrovirus vector contains a sequence derived
from murine stem cell virus.
8. The transgenic bird according to claim 5, wherein the
replication defective retrovirus vector contains the VSV-G envelope
sequence.
9. The transgenic bird according to claim 5, which is produced by
using a replication defective retrovirus vector containing a
non-tissue-specific promoter gene.
10. The transgenic bird according to claim 9, wherein the
non-tissue-specific promoter gene contains a part or the whole of
the chicken .beta.-actin promoter gene.
11. The transgenic bird according to claim 1, which is produced by
using a replication defective retrovirus vector containing a
tissue-specific promoter gene.
12. The transgenic bird according to claim 11, wherein the
tissue-specific promoter gene contains a part or the whole of an
oviduct-specific promoter gene.
13. The transgenic bird according to claim 12, wherein the
oviduct-specific promoter gene contains at least a part or the
whole of the ovalbumin, ovotransferrin, ovomucoid, ovomutin,
lysozyme, G2 globulin, G3 globulin, ovoinhibitor, ovoglycoprotein,
ovoflavoprotein, ovomacroglobulin, cystatin or avidin promoter gene
or a combination thereof.
14. The transgenic bird according to claim 1, which is produced by
using a replication defective retrovirus vector containing a
transcription enhancer and/or a regulatory element.
15. The transgenic bird according to claim 14, wherein the
regulatory element contains a part or the whole of the woodchuck
posttranscriptional regulatory element sequence.
16. The transgenic bird according to claim 1, which is obtained by
a method comprising infecting an avian embryo with a replication
defective retrovirus vector containing a foreign gene and allowing
the embryo to hatch.
17. The transgenic bird according to claim 16 which is obtained by
a method comprising incubating a fertilized avian egg, infecting
the embryo thereof after the lapse of at least 24 hours following
the start of incubation with a replication defective retrovirus
vector containing a foreign gene and allowing the embryo to
hatch.
18. The transgenic bird according to claim 17, wherein the embryo
is one formed during the period not earlier than 32 hours and not
later than 72 hours after the start of incubation.
19. The transgenic bird according to claim 18, wherein the embryo
is one formed during the period not earlier than 48 hours and not
later than 64 hours after the start of incubation.
20. The transgenic bird according to claim 16, which is obtained by
a method comprising infecting the embryo with the replication
defective retrovirus vector by microinjection thereof into the
heart or blood vessel formed in the embryo.
21. The transgenic bird according to claim 1, wherein said bird is
a poultry bird.
22. The transgenic bird according to claim 21, wherein said bird is
a chicken.
23. The transgenic bird according to claim 1, a descendant thereof,
an egg thereof and spermatozoa thereof.
24. A method of producing the transgenic bird according to claim 1,
which comprises infecting an avian embryo with a replication
defective retrovirus vector containing a foreign gene and allowing
the embryo to hatch.
25. A method of producing a feline-derived protein which comprises
any or a combination of extracting the protein from the blood
and/or somatic cells of the transgenic bird according to claim 1
and/or an egg laid thereby, purifying and activating the same.
26. A polyethylene glycol-modified feline-derived protein which is
obtained by chemically modifying a feline-derived protein produced
by the method according to claim 25 with polyethylene glycol.
27. The polyethylene glycol-modified feline-derived protein
according to claim 26, wherein the feline-derived protein comprises
a feline-derived cytokine protein and/or a protein substantially
identical in biological activity thereto.
28. The polyethylene glycol-modified feline-derived protein
according to claim 26, wherein the feline-derived protein is a
protein containing at least a part of feline-derived erythropoietin
identified under SEQ ID NO: 1 in the sequence listing and/or of a
protein substantially identical in biological activity thereto.
29. The polyethylene glycol-modified feline-derived protein
according to claim 26, wherein the feline-derived protein comprises
feline-derived erythropoietin identified under SEQ ID NO: 1 in the
sequence listing and/or a protein substantially identical in
biological activity thereto.
30. The polyethylene glycol-modified feline-derived protein
according to claim 1, wherein the polyethylene glycol used for the
modification of the feline-derived protein has a weight average
molecular weight of 5 to 40 kDa.
31. The polyethylene glycol-modified feline-derived protein
according to claim 30, wherein the weight average molecular weight
of the polyethylene glycol is 20 kDa.
32. The polyethylene glycol-modified feline-derived protein
according to claim 26, wherein the number of polyethylene glycol
molecules added is 1 or 2 or more and the apparent molecular
weight, per polyethylene glycol-modified molecule, is from 100 kDa
to 900 kDa as determined by gel filtration column chromatography in
an aqueous solvent.
33. The polyethylene glycol-modified feline-derived protein
according to claim 32, wherein the number of polyethylene glycol
molecules added is 1 and the apparent molecular weight, per
polyethylene glycol-modified molecule, is from 100 kDa to 500 kDa
as determined by gel filtration column chromatography in an aqueous
solvent.
34. A polyethylene glycol-modified feline-derived protein
composition which comprises the polyethylene glycol-modified
feline-derived protein according to claim 32.
35. A medicinal composition for feline use which comprises, as an
active ingredient, the polyethylene glycol-modified feline-derived
protein according to claim 26.
36. A medicinal composition for feline use which comprises, as an
active ingredient, the polyethylene glycol-modified feline-derived
protein composition according to claim 34.
37. The medicinal composition according to claim 35 which has
feline erythropoietin activity and prolonged drug efficacy and is
intended for use in the treatment of feline renal anemia.
38. A method of producing the polyethylene glycol-modified
feline-derived protein composition according to claim 34 which
comprises causing a succinimidyl ester derivative of polyethylene
glycol to add to a feline-derived protein.
Description
TECHNICAL FIELD
[0001] The present invention relates to a method of producing a
transgenic bird containing a foreign gene as transferred into the
genome thereof and to the expression of a feline-derived protein in
such transgenic bird. More particularly, it relates to the
expression of feline-derived erythropoietin in such transgenic
bird.
BACKGROUND ART
[0002] In recent years, a number of proteins have come into use as
pharmaceuticals. This is because the gene recombination technology
has been developed for and applied to the introduction or transfer
of a gene coding for a desired protein into microorganisms or
mammalian cells, so that commercial protein production is now
feasible by cultivating the thus-produced genetically modified
organisms. For such a medicinal protein to show the physiological
activity or activities intrinsic therein, posttranscriptional
modifications, for example folding, glycosylation and disulfide
bond formation is necessary as in nature.
[0003] Methods of producing proteins by cultivating microorganisms
are capable of producing proteins at low costs since microorganisms
can grow rapidly and medium compositions therefor are simple.
However, in many cases, due posttranscriptional modifications of
the desired protein are not made properly in microorganisms.
Therefore, it is difficult to obtain a protein having the same
physiological activity as that of the natural counterpart in
sufficient quantities; in the existing circumstances, it is still a
long way to practical use of such protein production methods on a
commercial basis.
[0004] Therefore, it is the mainstream of the art to introduce a
gene for a desired protein into mammalian cells and cultivating the
cells to cause them to product the protein. Such pharmaceutical
proteins as blood coagulation factors, thrombolytic agents and
antibodies for pharmaceutical use as produced using recombinant
mammalian cells are already on the market and used. However, those
methods which use mammalian cells have a problem in that culture
tanks and medium for exclusive use are required and the production
cost is high.
[0005] To overcome these problems, animal factories have now
attracted attention. The technology concerned comprises using
gene-transferred (transgenic) animals to produce desired proteins.
Attempts have been made to produce transgenic mammals using goats,
sheep and cows, among others, and cause the production of the
desired proteins in the milks thereof. Thus, there is a report
describing the expression of an antibody at a level of 10 mg/ml in
milk, although the expression level varies depending on the protein
species (cf. e.g. Non-Patent Document 1). However, this technology
has the BSE (bovine spongiform encephalopathy) problem and other
problems; utilizable mammalian individuals are large-sized and,
therefore, are difficult to produce, raise and handle; a further
problem is that the period from birth to sexual maturation is long,
namely 8 months in goats or sheep, or 15 months in cows.
[0006] Therefore, investigations have been made to use transgenic
birds for the expression of a desired protein in eggs thereof. This
technology has several advantages: the egg-laying productivity is
high, there is no BSE problem, the maturation period is short (5
months in chickens), individuals are small in size and therefore a
large number of individuals can be raised, the technique of
artificial insemination has been established, enabling rapid
raising of large-scale transgenic groups, and the egg inside is
generally sterile by nature.
[0007] As for the methods of producing transgenic birds, the method
using a retrovirus vector, the method using embryonic stem cells,
the method using primordial germ cells and the method comprising
causing a target gene to adhere to spermatozoa for introduction
thereof, among others, are under investigation. Among those
methods, the method using a retrovirus vector is the commonest. So
far, a study in which an avian leukemia virus (ALV)-derived
replication defective retrovirus vector was used has been reported
(cf. e.g. Patent Document 1). The target protein used was
.beta.-lactamase, and the promoter gene used was the
cytomegalovirus (CMV) promoter gene. Transgenic birds were produced
successfully by retrovirus vector introduction into blastoderms at
the stage X just after egg laying. Reportedly, the level of
expression was 0.33 mg/ml (the egg white volume being estimated at
40 ml) as determined by western blot analysis and, when expressed
in terms of .beta.-lactamase activity, it was 0.003 to 0.033 mg/ml.
On that occasion, the frequency of appearance of G0 transgenic
chimeric birds was 20%. The result of an investigation of the
efficiency of introduction into germ cells indicated that about 5%
of male G0 transgenic chimeric birds had the transgene in
spermatozoa. According to another report about a similar
experiment, the transgene expression was about 1.2 .mu.g/ml of egg
white in G2 birds having the transgene introduced in the whole body
(cf. e.g. Non-Patent Document 2). On that occasion, the frequency
of appearance of G1 from G0 was 3/422 (0.71%).
[0008] Further, there are reports about transgenic birds expressing
human interferon or human-derived erythropoietin (cf. e.g. Patent
Document 2 and 3). Interferon is a glycoprotein having a molecular
weight of about 20,000 which is produced and secreted by almost all
animal cells on the occasion of viral infection; it is also known
as virus inhibiting factor. Erythropoietin (EPO) is a sugar
chain-rich polypeptide mainly produced in the kidney and capable of
acting on precursor cells in the hemopoietic tissue to promote the
differentiation thereof into and the growth of erythrocytes.
Currently, recombinant human EPO produced by the recombinant DNA
technology using animal cells as hosts is on the market and is used
mainly as a therapeutic agent for various types of anemia,
typically renal anemia resulting from nephropathy-associated
reduced EPO productivity. When an ALV-derived replication defective
retrovirus vector and the CMV promoter gene or
ovomucoid-ovotransferrin fused promoter gene were used, human
interferon was expressed in serum at a maximum level of 200 ng/ml,
and human-derived erythropoietin in serum and egg white each at a
maximum level of 70 ng/ml.
[0009] In another report, it is reported that high levels of virus
titer, infectivity and expression were obtained using the mouse
stem cell virus (MSCV) vector and VSV-G envelope (cf. e.g. Patent
Document 4). Further, according to that report, high expression
levels were realized by adjusting the time of retrovirus vector
introduction and, when an anti-prion single chain antibody (scFv)
is used as the target protein, high levels of expression of 0.5 to
1 mg/ml in egg white and in egg yolk were realized.
[0010] Cats are animals long loved as pets by humans and recently
have been establishing their position as the so-called "partner,
companion or friend animals" in the human society. On the other
hand, in the fields of medicine, pharmacology, veterinary medicine
and psychology, among others, cats have so far been used as
experimental animals and recently have come into use in testing
pharmaceuticals for safety and efficacy. In view of the
circumstances in which the social importance of cats is increasing,
feline diseases and infections are objects of concern and effective
therapeutic means therefor are desired. In recent years, medicinal
proteins have attracted attention in the treatment of feline
diseases as well and, currently, medicinal proteins for human use
are mainly used in cats as well. However, medicinal proteins for
human use differ in amino acid sequence from in vivo proteins
intrinsic in cats and, therefore, may possibly differ in effect or
efficacy in living cats. Further, the difference in amino acid
sequence may possibly cause an allergic reaction and, in the worst
case, an anaphylactic symptom. Thus, such proteins cannot be used
in high-frequency dosage regimens, so that the development of
medicinal proteins intrinsic in cats is demanded.
[0011] As the feline-derived medicinal proteins so far studied
widely, there may be mentioned cytokines. Cytokines are proteinic
factors which are released from cells and mediate intercellular
interactions in the exertion of immune or inflammatory response
modulating, antiviral, antitumor, and cell proliferation and
differentiation regulating actions. As the feline-derived cytokines
so far reported, there may be mentioned erythropoietin (cf. e.g.
Non-Patent Document 3 and 4) and interleukin 12 (cf. e.g. Patent
Document 5), among others. As regards the production of these,
mammalian cells have so far been used; under the existing
circumstances, any transgenic birds have been used in such
production.
[0012] Patent Document 1: Japanese Kohyo Publication
2001-520009
[0013] Patent Document 2: United States Patent Application
Publication 2004/0019922
[0014] Patent Document 3: United States Patent Application
Publication 2004/0019923
[0015] Patent Document 4: Japanese Kokai Publication
2002-176880
[0016] Patent Document 5: International Publication WO
97/046583
[0017] Non-Patent Document 1: Trends Biotechnol. 1999, September;
17(9):367-74
[0018] Non-Patent Document 2: Nat. Biotechnol. 2002, April;
20(4):396-9
[0019] Non-Patent Document 3: Blood, 1993, Sep. 1;
82(5):1507-16
[0020] Non-Patent Document 4: Vet. Immunol. Immunopathol. 1986,
January; 11(1):1-19
SUMMARY OF THE INVENTION
[0021] No examples have so far been reported of the expression of a
feline-derived protein using transgenic birds. In higher animals,
proteins after translation undergo various modifications such as
folding, glycosylation and disulfide bond formation so that they
may acquire respective specific, physiologically active forms. The
protein modification varies depending on the tissue in one and the
same individual. It is therefore very difficult to obtain a high
level of expression of a foreign gene in animal cells. For example,
on the occasion of producing a medicinal protein by cultivation of
mammalian cells, an appropriate cell line suited for the production
of the medicinal protein from among various animal species and
tissues is to be selected. While human-derived proteins have so far
been produced using transgenic birds, the human and cat
taxonomically belong to different orders; this is a great
difference. Even in the case of human and feline counterpart
proteins having one and the same activity, they differ in amino
acid sequence. In the case of erythropoietin, the amino acid
homology between human and cat is about 83%. Further, since there
is a difference in sugar chain sequence between human and cat, it
is difficult to say that what was possible with a human-derived
protein is also possible with the corresponding feline-derived
protein. Accordingly, it is an object of the present invention to
teach a method producing a feline-derived protein in transgenic
birds.
[0022] The present inventors paid their attention to feline-derived
cytokines as the feline-derived proteins and employed
feline-derived erythropoietin, one of the feline-derived cytokines,
as the target. It is an object of the present invention to teach a
method of producing feline-derived erythropoietin in transgenic
birds, in particular. The human-derived erythropoietin-producing
transgenic birds disclosed in United States Patent Application
Publications 2004/0019922 and 2004/0019923 have a problem in that
the erythropoietin production is low. Accordingly, it is an object
of the present invention to provide a transgenic bird capable of
producing erythropoietin at high concentration levels and a method
of producing the same.
[0023] A characteristic feature of the present invention consists
in a transgenic bird with a foreign gene containing a
feline-derived protein-encoding sequence as transferred therein and
in a method of producing the same. Another characteristic feature
of the invention consists in a transgenic bird having a foreign
gene containing a sequence coding for a feline-derived cytokine
protein and/or a protein substantially identical in biological
activity thereto and in a method of producing the same. A further
characteristic feature of the invention consists in a transgenic
bird having a foreign gene containing at least a part of a sequence
coding for feline-derived erythropoietin identified under SEQ ID
NO:1 in the sequence listing and/or a sequence coding for a protein
substantially identical in biological activity to feline-derived
erythropoietin and in a method of producing the same. A
characteristic feature of the invention consists in a transgenic
bird having a foreign gene containing a sequence coding for
feline-derived erythropoietin identified under SEQ ID NO:1 in the
sequence listing and/or a sequence coding for a protein
substantially identical in biological activity to feline-derived
erythropoietin and in a method of producing the same.
[0024] The present invention is further characterized in that a
replication defective retrovirus vector is used in producing
transgenic birds. The invention is also characterized in that the
replication defective retrovirus vector contains a Moloney murine
leukemia virus- and/or Moloney murine sarcoma virus-derived
sequence. The invention is further characterized in that the
replication defective retrovirus vector contains a murine stem cell
virus (MSCV)-derived sequence, thus enabling use of a virus highly
capable of infecting germ cells and stem cells. The invention is
also characterized in that the replication defective retrovirus
vector contains the VSV-G envelope and, in this respect, a wide
range of mammalian and non-mammalian cells, including cells hardly
allowing transduction, can be infected therewith.
[0025] Further, the invention is characterized in that transgenic
birds are produced using a replication defective retrovirus vector
containing a non-tissue-specific promoter gene. The invention is
further characterized in that the non-tissue-specific promoter gene
contains a part or the whole of the chicken .beta.-actin promoter
gene.
[0026] The invention is further characterized in that transgenic
birds are produced using a replication defective retrovirus vector
containing a tissue-specific promoter gene. The invention is
further characterized in that the replication defective retrovirus
vector contains a tissue-specific promoter gene containing a part
or the whole of an oviduct-specific promoter gene. The invention is
further characterized in that the oviduct-specific promoter gene
comprises at least a part or the whole, or a combination, of the
ovalbumin, ovotransferrin, ovomucoid, ovomutin, lysozyme, G2
globulin, G3 globulin, ovoinhibitor, ovoglycoprotein,
ovoflavoprotein, ovomacroglobulin, cystatin and/or avidin promoter
gene.
[0027] The invention is further characterized in that transgenic
birds are produced using a replication defective retrovirus vector
containing a transcriptional enhancer and/or regulatory element.
The invention is further characterized in that the regulatory
element contains a part or the whole of the woodchuck
posttranscriptional regulatory element sequence.
[0028] The invention is further characterized in that transgenic
birds are produced by a method which comprises infecting avian
embryos with a replication defective retrovirus vector containing a
foreign gene and hatching the embryos. The invention is further
characterized in that transgenic birds are produced by a method
which comprises incubating fertilized avian eggs, infecting the
embryos after at least 24 hours of incubation with a replication
defective retrovirus vector containing a foreign gene and hatching
the embryos. More preferably, it is characterized in that the
embryos to be infected with the replication defective retrovirus
vector containing a foreign gene are those formed not earlier than
32 hours but not later than 72 hours after the start of incubation.
Still more preferably, it is characterized in that the embryos to
be infected with the replication defective retrovirus vector
containing a foreign gene are those formed not earlier than 48
hours but not later than 64 hours after the start of incubation.
The invention is further characterized in that the method of
infecting with a replication defective retrovirus vector containing
a foreign gene comprises microinjection into the heart or blood
vessel formed in the embryo.
[0029] The invention is further characterized in that the
transgenic bird is one derived from a domestic fowl. More
preferably, it is characterized in that the transgenic bird is one
derived from a chicken.
[0030] The invention is further characterized in that it covers
transgenic birds, descendants thereof, eggs thereof and/or
spermatozoa thereof and comprises any of the methods of producing
transgenic birds as mentioned above.
[0031] The invention is further characterized in that it is
directed to a method of producing a foreign gene-derived protein
which comprises the steps of extracting that protein from the
blood, somatic cells and/or eggs of the transgenic bird, purifying
and activating the same, either singly or in combination, and
comprises any of the methods of producing transgenic birds as
mentioned above.
[0032] The present invention also relates to a polyethylene
glycol-modified feline-derived protein obtained by chemically
modifying, with polyethylene glycol, the feline-derived protein
produced in the manner mentioned above.
[0033] Preferably, the feline-derived protein comprises a
feline-derived cytokine and/or a protein substantially identical in
biological activity thereto. More preferably, the feline-derived
protein comprises a protein containing at least a part of
feline-derived erythropoietin identified under SEQ ID NO:1 in the
sequence listing and/or of a protein substantially identical in
biological activity thereto. Still more preferably, the
feline-derived protein comprises feline-derived erythropoietin
identified under SEQ ID NO:1 in the sequence listing and/or a
protein substantially identical in biological activity thereto.
[0034] The invention is further characterized in that the
polyethylene glycol to be used for the modification of the
feline-derived protein has a weight average molecular weight of 5
to 40 kDa. More preferably, the polyethylene glycol has a weight
average molecular weight of 20 kDa.
[0035] The present invention also relates to a polyethylene
glycol-modified feline-derived protein in which the number of
polyethylene glycol molecules added is 1 or 2 or more and which has
an apparent molecular weight of from 100 kDa to 900 kDa per
polyethylene glycol-modified molecule as determined by gel
filtration column chromatography in an aqueous solvent.
[0036] Preferably, the polyethylene glycol addition number is 1 and
the apparent molecular weight per polyethylene glycol-modified
molecule as determined by gel filtration column chromatography in
an aqueous solvent is from 100 kDa to 500 kDa.
[0037] The present invention further relates to a polyethylene
glycol-modified feline-derived protein composition which comprises
the above-mentioned polyethylene glycol-modified feline-derived
protein.
[0038] The invention also relates to a medicinal composition for
feline use which comprises the above-mentioned polyethylene
glycol-modified feline-derived protein or polyethylene
glycol-modified feline-derived protein composition as an active
ingredient. The medicinal composition has feline erythropoietin
activity and prolonged action and is suited for use in the
treatment of feline renal anemia.
[0039] The present invention further relates to a method of
producing a polyethylene glycol-modified feline-derived protein
composition which comprises causing a polyethylene glycol
succinimidyl ester derivative to add to a feline-derived
protein.
DETAILED DESCRIPTION OF THE INVENTION
[0040] In the following, the present invention is described in
detail.
[0041] The term "protein" means a product derived from two or more
amino acids by peptide bonding and generally includes peptides as
well as oligopeptides shorter in chain length. The amino acid or
acids may be modified. For protein secretion, it is preferred that
the protein be provided with a secretory signal sequence. The
secretory signal is not necessarily an autologous sequence.
[0042] The foreign gene is not particularly restricted but includes
not only non-avian genes but also avian ones. Even a sequence
intrinsic in an individual subjected to transgenic production is
referred to as foreign gene since a gene is newly introduced into
the genome intrinsic in that individual.
[0043] In the practice of the invention, the foreign gene contains
a feline-derived protein-encoding sequence and the limits of the
coding sequence are defined by the 5'-terminal initiation codon and
the 3'-terminal termination codon corresponding to the initiation
codon. The vicinity of the 5'-terminal initiation codon preferably
contains Kozak's consensus sequence. Preferably, there is a
ribosome-binding site upstream of the coding sequence.
[0044] The foreign gene may contain a nontranslatable region in
addition to the feline-derived protein-encoding sequence mentioned
above.
[0045] The feline-derived protein is not particularly restricted
but preferably comprises a feline-derived cytokine protein and/or a
protein substantially identical in biological activity thereto. A
cytokine is a proteinic factor released from cells and mediating
intercellular interactions in the exertion of immune or
inflammatory response modulating, antiviral, antitumor, and cell
proliferation and differentiation regulating actions, among others;
as specific examples, there may be mentioned various interleukins,
interferon .alpha., .beta. and .gamma., tumor necrosis factor,
lymphotoxin, colony-stimulating factor and erythropoietin, which
are hematopoietic factors, and epidermal growth factor and
fibroblast growth factor, which are growth factors. In the practice
of the invention, a protein containing at least a part of
feline-derived erythropoietin identified under SEQ ID NO:1 in the
sequence listing and/or of a protein substantially identical in
biological activity thereto is more preferred, and the whole of
feline-derived erythropoietin identified under SEQ ID NO:1 in the
sequence listing and/or of a protein substantially identical in
biological activity thereto is still more preferred.
[0046] In the present specification, the "protein substantially
identical in biological activity", in the case of a cytokine, for
instance, means a protein resulting from deletion, addition or
substitution of 1 to 10 amino acid residues in the amino acid
sequence of the feline-derived cytokine protein and retaining the
physiological activity of the cytokine. When the protein has the
same physiological activity, it is regarded as substantially
identical in biological activity, irrespective of intensity of
activity.
[0047] The region from the first to the 26th amino acid residues in
SEQ ID NO: 1 is the so-called signal peptide and is eliminated by
cleavage on the occasion of secretion. Therefore, it is a region
little influencing the biological activity of feline-derived
erythropoietin. The feline-derived protein obtained in accordance
with the invention may have an amino acid sequence resulting from
deletion, addition or substitution of 1 to 10 amino acid residues
in the amino acid sequence starting from the 27th amino acid
residue in the amino acid sequence shown under SEQ ID NO:1 in the
sequence listing.
[0048] In producing the transgenic bird according to the invention,
a retrovirus vector is preferably used. The retrovirus vector
includes, within the meaning thereof, different forms, namely
plasmid, virus particles and packaging cells. Packaging cells are
cells resulting from introduction thereinto of a gene coding for at
least one of the proteins necessary for the replication of virus
particles.
[0049] From the safety viewpoint, the retrovirus vector to be used
in the practice of the invention is preferably a replication
defective one. The method of rendering the retrovirus replication
defective preferably comprises deleting at least a part or the
whole of each coding sequence or a sequence necessary for the
expression thereof so that one or a combination of the protein
(group specific antigen, gag), which is contained in the internal
core, reverse transcriptase (polymerase, pol) and envelope
glycoprotein (envelope, env), which are necessary for virus
particle replication, may not be expressed, or causing a mutation
or mutations by substitution and/or insertion so that the sequences
mentioned above may not be expressed. Since the length of a gene
that can be inserted into a retrovirus vector is limited depending
on the viral species, mutations by deletion are preferred and, from
the viewpoint of safety and of increased insert fragment length, it
is preferred that a plurality of gag, pol and env be deleted.
Preferably, the retrovirus vector contains a viral packaging signal
(phi) which functions as a landmark of packaging in the virus
particle. Since a part of the gag region may sometimes function as
a viral packaging signal, it is preferred, from the increased virus
titer viewpoint, that the viral vector contain at least a part of
the gag region rendered incapable of being expressed (J. Virol.
1987, May; 61(5):1639-46).
[0050] The retrovirus is not particularly restricted but includes
viruses derived from Moloney murine leukemia virus, Moloney murine
sarcoma virus, avian leukemia virus (ALV) and human
immunodeficiency virus (HIV), among others. While Moloney murine
leukemia virus and/or Moloney murine sarcoma virus is preferred,
viruses highly capable of infecting germ cells and stem cells, such
as murine stem cell virus (MSCV) and murine embryonic stem cell
virus (MESV), are preferably used for the infection of the avian
embryo. MSCV is more preferred.
[0051] The replication defective retrovirus vector to be used in
the practice of the invention preferably contains a sequence
derived from such a virus as mentioned above. For efficient
infection of avian cells with such virus vector, the coat protein
is preferably replaced artificially with the bovine vesicular
stomatitis virus-derived VSV-G envelope protein, although the
retrovirus vector is not limited to this type of virus.
[0052] The replication defective retrovirus vector to be used in
the practice of the invention preferably contains at least a part
or the whole of an appropriate promoter gene for the expression of
the foreign gene in avian cells. The promoter gene is a region on a
DNA or RNA which determines the transcription initiation site on a
gene or directly regulating the frequency thereof.
[0053] The replication defective retrovirus vector to be used in
the practice of the invention preferably contains a
non-tissue-specific promoter gene or a tissue-specific promoter
gene.
[0054] A tissue-specific promoter gene is a promoter gene showing
especially intense activity in a certain specific avian tissue or
cells. By using a tissue-specific promoter gene, it becomes
advantageously possible to reduce or eliminate the possibility of
the expression of a desired protein adversely affecting the
development or survival of birds.
[0055] The tissue-specific promoter gene is not particularly
restricted but includes oviduct-specific promoter genes. The
oviduct tissue becomes active after sexual maturation and therefore
is strongly induced after sexual maturation in many cases.
[0056] As the oviduct-specific promoter gene, there may be
mentioned the ovalbumin, ovotransferrin, ovomucoid, ovomutin,
lysozyme, G2 globulin, G3 globulin, ovoinhibitor, ovoglycoprotein,
ovoflavoprotein, ovomacroglobulin, cystatin and avidin promoter
genes of the avian origin, among others. The use of these
oviduct-specific promoter genes is particularly preferred since the
desired protein can then be expressed in egg white at high
levels.
[0057] The non-tissue-specific promoter gene is a promoter gene
which is not a tissue-specific promoter gene. The
non-tissue-specific promoter gene is not particularly restricted
but includes those active in almost all avian somatic cells. In
that case, the desired protein is expressed in blood as well and
therefore the expression or non-expression thereof can
advantageously be detected in the stage of nestlings.
[0058] The non-tissue-specific promoter gene is not particularly
restricted but includes such virus-derived promoter genes as the
.beta.-actin promoter gene, EF1.alpha. promoter gene, thymidine
kinase promoter gene, simian virus 40 (SV40) promoter gene,
cytomegalovirus (CMV) promoter gene, and Rous sarcoma virus (RSV)
promoter gene. In addition, such a non-tissue-specific inducible
type promoter gene as the tetracycline inducible promoter gene may
also be used. As far as the non-tissue-specific promoter gene is
concerned, the retrovirus vector preferably contains a part or the
whole of the avian .beta.-actin promoter gene.
[0059] The replication defective retrovirus vector to be used in
the practice of the invention may contain a transcription enhancer
and/or regulatory element. The transcription enhancer is a sequence
promoting the transcription from a promoter gene but is a region on
DNA or RNA which by itself cannot cause transcription. A
transcription enhancer, even when connected to a promoter gene
different from the one for which it originally functions, can
function in many instances, so that the combination thereof with
the promoter gene is not limited. The transcription enhancer is not
particularly restricted but includes the SV40, CMV and thymidine
kinase enhancers, steroid responsive element and lysozyme enhancer,
among others. The regulatory element is a region on DNA or RNA
which contributes to transcriptional regulation and RNA
stabilization after transcription but by itself cannot cause
transcription. The regulatory element is not particularly
restricted but includes the woodchuck posttranscriptional
regulatory element (WPRE; U.S. Pat. No. 6,136,597), among
others.
[0060] The retrovirus vector contains at least a part of a long
terminal repeat (LTR) at each of the 5' terminus and 3' terminus.
The LTR contains a transcriptional promoter gene and a polyA
addition signal and therefore can be utilized as a promoter gene or
a terminator gene. In the retrovirus vector, the target
protein-encoding sequence, promoter gene, transcription enhancer
and/or regulatory element are contained between the 5' LTR and 3'
LTR. When a promoter other than LTR is used, the retrovirus vector
preferably has a structure such that the target protein-encoding
sequence is connected to a site downstream from the promoter. For
the retrovirus vector to be transcribed for the construction of
virus particles, it is preferred that no terminator or polyA signal
be contained between the 5' LTR and 3' LTR.
[0061] The retrovirus vector to be used in the practice of the
invention may contain a marker gene. The marker gene is a gene
coding for a protein serving as a landmark in the identification
and isolation of correctly gene-transferred cells. The marker gene
is not particularly restricted but includes genes for fluorescent
proteins such as green fluorescent protein (GFP), cyan fluorescent
protein (CFP) and luciferase; drug resistance genes such as the
neomycin resistance (Neo.sup.r), hygromycin resistance (Hyg.sup.r)
and puromycin resistance (Puro.sup.r) genes; and, further, the
thymidine kinase, dihydrofolate reductase, aminoglycoside
phosphotransferase, chloramphenicol acetyl transferase,
.beta.-lactamase and .beta.-galactosidase genes, among others. The
marker gene is preferably accompanied by a promoter gene and an
element necessary for the expression thereof.
[0062] Now, mention is made of a preferred mode of embodiment of
the method of preparing a replication defective retrovirus vector
suited for use in the practice of the invention.
[0063] The replication defective retrovirus vector to be used in
the practice of the invention is lacking in the gag, pol and env
genes necessary for the replication thereof. A replication
defective retrovirus vector plasmid enabling the expression of the
desired protein and a VSV-G expression plasmid are co-introduced
into packaging cells having the gag and pol genes, and the culture
supernatant is used as a virus-containing fluid. Alternatively and
desirably, a VSV-G expression plasmid is introduced into packaging
cells infected with the above virus-containing fluid, and the
culture supernatant is used as a virus-containing fluid. The
virus-containing fluid is preferably concentrated according to
need. The method of preparing a replication defective retrovirus
vector is not limited to such method, however.
[0064] The titer of the replication defective retrovirus vector in
the virus-containing fluid mentioned above is preferably
1.times.10.sup.8 to 1.times.10.sup.14 cfu/ml, more preferably
1.times.10.sup.9 to 1.times.10.sup.14 cfu/ml.
[0065] The titer of the virus-containing fluid is defined as the
number of infected cells after addition of the virus-containing
fluid to NIH3T3 cells (American Type Culture Collection CRL-1658).
More specifically, 1 ml of a virus solution diluted at a dilution
ratio of 10.sup.2 to 10.sup.6 is added to 5.times.10.sup.4 NIH3T3
cells occurring in each well (base area about 9.4 cm.sup.2) of each
6-well culture plate, and the proportion of cells expressing the
neomycin resistance gene as a marker is determined based on the
resistance to G418 (neomycin). The titer of the virus-containing
fluid is calculated from the data thus obtained.
[0066] How to infect avian embryos with a replication defective
retrovirus vector is now described.
[0067] The transgenic birds according to the present invention can
be adequately obtained by the method which comprises infecting
avian embryos with the replication defective retrovirus vector
containing a foreign gene and allowing the embryos to hatch.
[0068] An embryo is a young animal at the early stage of
development of a multicellular animal, is enveloped in a chorion or
eggshell or is in the mother's body and does not yet take food
independently. Hatching means coming out of the chorion or eggshell
and beginning to take food independently.
[0069] The embryo is desirably infected with the replication
defective retrovirus vector at least 24 hours after the start of
incubation. More desired is an embryo not earlier than 32 hours but
not later than 72 hours after the start of incubation. Still more
desired is an embryo not earlier than 48 hours but not later than
64 hours of incubation. Preferred as the site of infection, namely
the site of introduction of the virus-containing fluid, is the
inside of the heart or blood vessel formed in the embryo. For the
purpose of producing G0 transgenic chimeric avians with high gene
transfer efficiency, it is preferable that the gene transfer be
carried out at the early stage at which the cardiac pulsation can
be observed (within 6 hours after the start of cardiac pulsation).
This is concluded from the viewpoint that the gene is to be
distributed to the whole body by means of blood circulation and
from the viewpoint that the number of cells is small.
[0070] Incubation means that fertilized avian eggs just after egg
laying or stored, immediately following egg laying, in an
environment in which development thereof is impossible are
maintained in an environment in which development thereof is
possible. In the case of chickens, for instance, an optimum
environment for development is such that the incubation temperature
is optimally 37.2 to 37.8.degree. C. in a three-dimensional
incubator (38.9 to 39.4.degree. C. at the upper end of a planar
incubator or the like) and the humidity is optimally about 40 to
70%. The environment to be employed is not limited to such an
environment, however. On the occasion of incubation, eggs are
turned. The egg turning is preferably carried out at an angle of at
least 30.degree. at least twice a day. The conditions are not
restricted to these, however.
[0071] Microinjection is a method of introducing a virus-containing
fluid directly into a specific site using a tapered glass microtube
under a microscope. In this study, the virus-containing fluid is
introduced into such a specific site as the heart or blood vessel
and, therefore, the technique of microinjection is preferred to
other methods of gene transfer, for example the lipofection and
electroporation techniques.
[0072] The bird to be used in the practice of the invention is not
particularly restricted but preferably is a poultry bird utilizable
as a farm animal. As the poultry bird, there may be mentioned
chickens, turkeys, ducks, ostriches, quails and domestic ducks,
among others. Among them, chickens are particularly preferred since
they are readily available and are fecund as egg-laying species,
eggs thereof are large, and the technique of mass rearing has been
established.
[0073] G0 transgenic chimeric birds can be obtained by infecting
avian embryos with a replication defective retrovirus vector
containing a foreign gene and allowing the embryos to hatch, as
mentioned above.
[0074] When G0 transgenic chimeric birds having a foreign gene in
germ cells thereof are mated with wild-type birds, G0 transgenic
chimeric birds or descendants thereof and, after hatching,
nestlings are screened, G1 transgenic birds can be obtained. In G0
transgenic chimeric birds, the probability of introduction of the
foreign gene into all cells is low and, in most cases, they are in
a chimeric state in which there coexist cells different in
genotype, namely cells resulting from foreign gene transfer and
wild type cells. On the other hand, G1 transgenic birds have the
transferred gene uniformly in all somatic cells. The gene transfer
into somatic cells or germ cells can be confirmed by examining DNAs
and RNAs derived from blood, somatic cells, spermatozoa and eggs by
the technique of PCR etc. and can also be confirmed based on the
expression of the desired protein. The expression of the desired
protein can be checked by the ELISA method, the electrophoretic
method and/or the activity measurement of the desired protein, for
instance.
[0075] G2 and the subsequent generations of transgenic birds can be
produced by mating G1 transgenic birds. Conceivable as the mating
types are G1 transgenic males and wild type females, G1 transgenic
females and wild type males, G1 transgenic males and females, for
instance. Further, back crossing of descendants thereof with
parents thereof is also possible. Among them, the mating type
involving G1 males and wild type females is preferred from the
efficiency viewpoint since one G1 male can be mated with a
plurality of wild type females.
[0076] The method of producing a target protein according to the
invention is characterized in that the target protein is recovered
from the above-mentioned transgenic birds. More particularly, the
method is characterized in that the desired protein is recovered
from the blood of the transgenic birds produced, somatic cells
thereof and/or eggs thereof by one or a combination of extraction,
purification and activation. The methods to be used for extraction
and purification are not particularly restricted but include, among
others, methods comprising one and/or a combination of fractional
precipitation, centrifugation, separation into two phases,
ultrafiltration, membrane separation, chromatography,
immunochemical methods and crystallization.
[0077] The feline-derived protein produced in the transgenic birds
according to the invention amounts to about 24 .mu.g/ml in serum or
about 420 .mu.g/ml in egg white, as shown in the example section
given later herein. In the case of the human-derived
erythropoietin-producing transgenic birds disclosed in United
States Patent Application Publications 2004/0019922 and
2004/0019923, the erythropoietin production is about 10 .mu.g/ml.
Therefore, the transgenic birds according to the present invention
can produce the desired protein at higher concentration levels.
[0078] The present invention also relates to a polyethylene glycol
(PEG)-modified feline-derived protein obtainable by chemical
modification, with PEG, of the feline-derived protein produced in
the manner mentioned above. As the feline-derived protein, there
may be mentioned the same ones as those mentioned above.
[0079] The feline-derived protein is preferably purified prior to
addition of PEG. The feline-derived protein can be purified from a
solution prepared from transgenic avian eggs, in particular the egg
white component, by dilution with pure water or equilibrated salt
solution by a column technique or filtration. The dilution is
carried out for the purpose of reducing the viscosity of egg white
and carrying out the column technique smoothly. While a high
dilution ratio is desired for the viscosity reduction, the increase
in volume makes the recovery difficult; therefore, the dilution
ratio is preferably 2 to 10 times, more preferably 5 to 6
times.
[0080] The method of recovering the desired feline-derived protein
from the egg white solution includes, but is not limited to, such
purification methods as salting out, adsorption column
chromatography, ion exchange column chromatography, gel filtration
column chromatography and antibody column technique, employed
either singly or in combination. The adsorption column
chromatography includes Blue Sepharose chromatography and heparin
chromatography, among others, and the ion exchange column
chromatography is, for example, anion exchange chromatography.
[0081] While it is reported that when the hematopoietic effect of
the erythropoietin was checked in such rodents as mice and rats,
the increase in the number of reticulocytes was maximum after 4
days following single administration into blood and no more effect
was observed on the 7th day, it is known that the addition of
polyethylene glycol (PEG), which is a long-chain molecule, results
in inhibition of metabolism in the liver, prolongation of the life
in blood and prolongation of the drug efficacy period and, in a
hematopoietic experiment in rats, the effect lasts for 14 days,
namely twice the duration mentioned above. An N-hydroxysuccinimidyl
active ester derivative of PEG can be bound to a lysine residue and
the N terminus of the protein molecule. The present invention is
characterized by, but is not limited to, the addition of PEG to
feline-derived erythropoietin for prolonging the life thereof in
blood.
[0082] With the increase in molecular weight of PEG added, the life
of the PEG-EPO complex in blood is prolonged. However, the addition
of very high molecular PEG inhibits the hematopoietic effect of EPO
(WO 02/032957), so that the weight average molecular weight of PEG
is preferably 5 to 40 kDa so that the in vivo hematopoietic effect
may be maximized, more preferably 10 to 30 kDa, and still more
preferably 20 kDa. The weight average molecular weight of PEG is
the value determined by MALDI-TOF mass spectrometry.
[0083] Feline-derived erythropoietin has at least three sites where
PEG can bind thereto and, therefore, 1 (mono), 2 (di) or 3 (tri)
PEG molecules can bind to one protein molecule. Since, however, PEG
molecules bound to a plurality of sites inhibit the receptor
binding ability of EPO, leading to decreases in in vivo activity,
the mono-substituted form is preferred.
[0084] The PEG-modified feline-derived protein according to the
invention is preferably one in which the number of PEG molecules
added is 1 or 2 or more and which has an apparent molecular weight,
per PEG-modified molecule, of from 100 kDa to 900 kDa as determined
by gel filtration column chromatography in an aqueous solvent, more
preferably one in which the number of PEG molecules added is 1 and
which has an apparent molecular weight of from 100 kDa to 500 kDa
as determined in the above manner.
[0085] In the present specification, the above-mentioned apparent
molecular weight determination by gel filtration column
chromatography is carried out using the low-pressure chromatograph
AKTA explorer 100 (product of Amersham) and the gel filtration
column Superdex 200 10/300 (product of Amersham).
[0086] The present invention further relates to a PEG-modified
feline-derived protein composition.
[0087] The PEG-modified feline-derived protein composition
according to the invention comprises a mixture of a PEG-modified
feline-derived protein in which the number of PEG molecules added
is 1 or 2 or more and which has an apparent molecular weight, per
PEG-modified molecule, of from 100 kDa to 900 kDa, a PEG-modified
feline-derived protein in which the number of PEG molecules added
is 1 and which has an apparent molecular weight, per PEG-modified
molecule, of from 100 kDa to 500 kDa and/or the non-PEG-modified
feline-derived protein as obtained by the production method
mentioned above and contains at least one PEG-modified
feline-derived protein.
[0088] The invention further relates to a medicinal composition for
feline use which comprises, as an active ingredient, the
above-mentioned PEG-modified feline-derived protein or PEG-modified
feline-derived protein composition.
[0089] The medicinal composition for feline use according to the
invention has feline erythropoietin activity and prolonged drug
activity and therefore can be suitably used in the treatment of
feline renal anemia.
[0090] The medicinal composition for feline use according to the
invention can be administered parenterally. As the route of
parenteral administration, there may be mentioned intravenous
injection, intravenous drip infusion, hypodermal injection,
transmucosal administration (e.g. transpulmonary, transnasal, etc.)
and transdermal administration.
[0091] The dose of the medicinal composition for feline use
according to the invention is not particularly restricted and
cannot be specified without reservation, either, in view of the
differences in responsiveness to erythropoietin in individual
diseased animals. Generally, however, it may be administered
intravenously, for example, twice a week at a dose of about 0.5 to
50 .mu.g per feline as expressed in terms of the weight of the
PEG-modified feline-derived protein.
[0092] The present invention further relates to a method of
producing a PEG-modified feline-derived protein composition which
comprises causing a succinimidyl ester derivative of PEG to add to
the above-mentioned feline-derived protein.
[0093] The succinimidyl ester derivative of PEG is not particularly
restricted but includes, among others, the succinimidylpropionate
ester and succinimidyl-alpha-methylbutanoate ester.
[0094] The mole ratio between the succinimidyl ester derivative of
PEG to be added and the feline-derived protein is preferably 1:1 to
1:10 as expressed in terms of (feline-derived
protein):(succinimidyl ester derivative of PEG).
[0095] The reaction temperature and reaction time are not
particularly restricted but preferably are 4 to 37.degree. C. and
0.5 to 2 hours, respectively.
EFFECT OF THE INVENTION
[0096] According to the present invention, transgenic birds capable
of producing a feline-derived protein and a method of production
thereof are provided. As a result, transgenic birds producing a
feline-derived cytokine and a method of production thereof are
provided. Furthermore, transgenic birds producing a feline-derived
erythropoietin and a method of production thereof are provided.
Transgenic birds producing erythropoietin at higher concentrations
than in the prior art and the production thereof have become
possible.
BEST MODES FOR CARRYING OUT THE INVENTION
[0097] The following examples illustrate the present invention in
detail. These examples are, however, by no means limitative of the
scope of the present invention. Unless otherwise specifically
described, gene manipulation procedures were carried out according
to the typical methods (J. Sambrook, E. F. Fritsch, T. Maniatis;
Molecular Cloning, A Laboratory Manual, 2nd Ed., Cold Spring Harbor
Laboratory). Unless otherwise specifically described, cell cultures
were carried out according to the typical methods (KOYAMA, Hideki
(ed.): "Saibo-Baiyo Labo Manual (Cell Culture Labo Manual)",
Springer Verlag Tokyo, 1st Ed.). When trademarks are given, the
instructions given in the manuals attached were followed, unless
otherwise specifically described.
EXAMPLE 1
Construction of feline-derived erythropoietin gene expression
plasmid pMSCVneobactfEPO
[0098] pMSCVneobact (SEQ ID NO:2) was totally synthesized based on
the relevant prior art document (Gene Ther. 1994, Mar.: 1(2):136-8)
and internet information (http://www.ncbi.nlm.NIH.gov/ etc.) and
inserted into pUC19 (GenBank Accession No. X02514) at a site
between EheI (235) and PvuII (628) (products of Toyobo). The
product was cleaved with HindIII (product of Takara Bio) and, after
treatment with Alkaline Phosphatase BAP (product of Takara Bio),
the desired fragment was purified and recovered using MinElute
Reaction Cleanup Kit (product of QIAGEN). This was electrophoresed
on 1% agarose and the desired fragment was purified and recovered
using MinElute Gel Extraction Kit (product of QIAGEN) (Example 1
vector fragment).
[0099] pUCfEPO (SEQ ID NO:3) encodes, at 911 to 1489 bp, the
feline-derived erythropoietin sequence. A fragment amplified by PCR
using Pyrobest DNA polymerase (product of Takara Bio) as the DNA
polymerase and pUCfEPO as the template, together with two
chemically synthesized oligonucleotides
5'-agccaagcttaccatggggtcgtgcgaatgtcctgccctgctgcttc-3' (SEQ ID NO:4)
and 5'-cgataagcttacgcgttcacctgtctcctcttcggcag-3' (SEQ ID NO:5)
(each underlined portion being a HindIII restriction enzyme site)
as primers was purified and recovered using MinElute PCR
Purification Kit (product of QIAGEN) and cleaved with HindIII
(product of Takara Bio). This was subjected to 1% agarose gel
electrophoresis and the desired fragment was purified and recovered
using MinElute Gel Extraction Kit (product of QIAGEN) (Example 1
insert fragment).
[0100] Example 1 vector fragment and Example 1 insert fragment were
joined together using DNA Ligation Kit Ver. 2.1 (product of Takara
Bio) and the ligation product was used to transform E. coli DH5
alpha Competent Cells (product of Takara Bio). From among
transformant stains obtained, a plasmid having the structure shown
in FIG. 1 was selected and named pMSCVneobactfEPO.
[0101] The marker gene beta-lactamase gene, the virus packaging
signal sequence phi+, the marker gene neomycin resistance gene, the
non-tissue-specific promoter gene beta-actin promoter and the long
repeat 5LTR and 3LTR, shown in FIG. 1, are all derived from
pMSCVneobact.
EXAMPLE 2
Retrovirus Vector Preparation Using pMSCVneobactfEPO and pVSV-G
[0102] Hereinafter, unless otherwise specified, the medium used was
Dulbecco's Modified Eagle Medium (DMEM) (product of Gibco)
containing 10% of fetal bovine serum (FBS) and 50 units/ml each of
penicillin and streptomycin. The cultivation was carried out at
37.degree. C. in the presence of 5% CO.sub.2. The plasmid DNA used
in the retrovirus vector was Endo Free Plasmid Maxi Kit (product of
QIAGEN).
[0103] For retrovirus vector preparation from the plasmid
pMSCVneobactfEPO constructed in Example 1, a collagen-coated
culture dish having a diameter of 100 mm was sowed with GP293
packaging cells having the gag and pol genes (5.times.10.sup.6
cells/dish; 70% confluent) (90% confluent on the next day). On the
next day, the medium was removed, and 7.2 ml of the medium and 10
.mu.l of 25 mM chloroquine (product of Sigma) were added, followed
by further 1 hour of cultivation. A 56-.mu.l portion of
Lipofectamine 2000 (product of Invitrogen) was suspended in 1.4 ml
of Opti-MEMI medium (product of Gibco), and the suspension was
allowed to stand at room temperature for 5 minutes. A 12-.mu.g
portion of pMSCVneobactfEPO and 12 .mu.g of pVSV-G were suspended
in 1.4 ml of Opti-MEMI medium. The Lipofectamine 2000 solution and
plasmid DNA solution were mixed up and the mixture was allowed to
stand at room temperature for 20 minutes. The whole amount of this
was added to the culture dish and cultivation was carried out for 6
hours. After 6 hours, the medium was removed, 9 ml of the medium
and 200 .mu.l of 1 M HEPES Buffer Solution (product of Gibco) were
added, and cultivation was further carried out for 24 hours.
[0104] The culture supernatant was collected in a centrifuge tube
through a 0.45-.mu.m cellulose acetate filter (product of
Advantec). The filtrate was centrifuged at 28,000 rpm
(50,000.times.g) for 1.5 hours using the ultracentrifuge CS100GXL
(product of Hitachi Koki). The supernatant was removed, 20 .mu.l of
TNE buffer (50 mM Tris-HCl (pH 7.8), 130 mM NaCl, 1 mM EDTA) was
added to the sediment, the mixture was allowed to stand at
4.degree. C. overnight and, after thorough suspending, centrifuged
at 12,000 rpm for 1 minute using a small-sized high-speed
centrifuge, and the supernatant was passed through a 0.45-.mu.m
Durapore Ultra-Free filter (product of Advantec) to give a
virus-containing solution.
EXAMPLE 3
Virus Titer Measurement
[0105] The virus titer is defined as the number of infected cells
after addition of the virus-containing fluid to NIH3T3 cells
(American Type Culture Collection CRL-1658). A 1-ml portion of the
virus solution of Example 2 as diluted at a dilution ratio of
10.sup.2 to 10.sup.6 was added to 5.times.10.sup.4 NIH3T3 cells
contained in each well (base area about 9.4 cm.sup.2) of each
6-well culture plate, and the proportion of cells expressing the
neomycin resistance gene as a marker was determined based on the
resistance to G418. If 4 colonies appear at a dilution ratio of
106, the virus titer will be 4.times.10.sup.6 cfu/ml.
[0106] More specifically, 6-well culture plates were sowed with
5.times.10.sup.4 NIH3T3 cells per well on the day before the start
of titer measurement, and the cells were cultured. On the next day,
the cell culture medium was replaced with 900 .mu.l of the medium
containing 9 .mu.g/ml of polybrene, the virus-containing fluid was
diluted to 10.sup.-1 to 10.sup.-5 with the medium and 100-.mu.l
portions of each dilution was added to each well for infection
(final polybrene concentration being 8 .mu.l/ml). After 4 to 6
hours of cultivation, 1 ml of the medium was further added to each
well. On the next day, the medium was replaced with the medium
containing 800 .mu.g/ml of G418 and, thereafter, the
G418-containing medium was exchanged for the old medium at 3- to
4-day intervals. About 2 weeks after infection, the plates were
stained with a methylene blue solution, the colonies obtained were
counted, and the titer was determined. The measurement results are
shown in Table 1.
EXAMPLE 4
Selection of Feline-Derived Erythropoietin Stable Packaging
Cells
[0107] On the day before viral infection, 24-well culture plates
were sowed with 1.5.times.10.sup.4 GP293 cells per well, and the
cells were cultured. On the day of viral infection, 1 ml of the
medium containing 10 .mu.g/ml of polybrene was exchanged for the
medium, followed by infection with the virus-containing fluid
prepared in (Example 2). Thereafter, cells were cloned by the
limiting dilution method. More specifically, on the next day, cells
were suspended in the medium containing 800 .mu.g/ml of G418 and
the suspension was diluted with the same medium to a content of 10
cells/ml. The cell dilution was distributed in 100-.mu.l portions
into the wells of 96-well culture plates (so that one cell might be
contained in each well). A cell line showing a high cell growth
rate and morphologically close to GP293 was selected and, thus, a
feline-derived erythropoietin stable packaging cell clone was
obtained.
EXAMPLE 5
Retrovirus Vector Preparation Using Feline-Derived Erythropoietin
Stable Packaging Cells and pVSV-G
[0108] A collagen-coated culture dish having a diameter of 100 mm
was sowed with the feline-derived erythropoietin stable packaging
cells obtained in Example 4 (5.times.10.sup.6 cells; 70% confluent)
(90% confluent on the next day). On the next day, the medium was
removed, and 7.2 ml of the medium and 10 .mu.l of 25 mM chloroquine
were added, followed by further 1 hour of cultivation. A 56-.mu.l
portion of Lipofectamine 2000 was suspended in 1.4 ml of Opti-MEMI
medium, and the suspension was allowed to stand at room temperature
for 5 minutes. pVSV-G (12 .mu.g) was suspended in 1.4 ml of
Opti-MEMI medium. The Lipofectamine 2000 solution and plasmid DNA
solution were mixed up, and the mixture was allowed to stand at
room temperature for 20 minutes. This was added to the culture
dish, followed by 6 hours of cultivation. The medium was removed, 9
ml of the medium and 200 .mu.l of 1 M HEPES Buffer Solution were
added, and the cultivation was continued for 24 hours. The culture
supernatant was passed through a 0.45-.mu.m cellulose acetate
filter and collected in a centrifuge tube. The filtrate was
centrifuged at 28,000 rpm (50,000.times.g) for 1.5 hours using an
ultracentrifuge. The supernatant was removed, 20 .mu.l of TNE
buffer solution was added to the sediment, the mixture was allowed
to stand at 4.degree. C. overnight and, after thorough suspending,
centrifuged at 12,000 rpm for 1 minute using a small-sized
high-speed centrifuge, and the supernatant was passed through a
0.45-.mu.m Durapore Ultra-Free filter to give a virus-containing
fluid. Thus, a virus-containing fluid having a titer of 10.sup.8
cfu/ml or higher was obtained.
EXAMPLE 6
Construction of WPRE Sequence-Containing Feline-Derived
Erythropoietin Gene Expression Plasmid pMSCVneobactfEPOwpre and
Preparation of Retrovirus Vector
[0109] pMSCVneobactfEPO was cleaved with ClaI (product of Takara
Bio) and, after BAP treatment, purified and recovered. The
purification and recovery were carried out using the product
described in (Example 1). This was subjected to 1% agarose gel
electrophoresis, and the desired fragment was purified and
recovered (Example 6 vector fragment). A fragment amplified from
WPRE sequence-containing pWHV8 (American Type Culture Collection
45097) by PCR using, as primers, two chemically synthesized
oligonucleotides 5'-ccatcgataatcaacctctggattacaaaatttgtga-3' (SEQ
ID NO: 6) and 5'-ccatcgatcaggcggggaggcg-3' (SEQ ID NO:7) (each
underlined portion being a ClaI restriction enzyme site) was
purified, recovered and cleaved with ClaI. This was subjected to 1%
agarose gel electrophoresis, and the desired fragment was purified
and recovered (Example 6 insert fragment).
[0110] Example 6 vector fragment and Example 6 insert fragment were
joined together and the ligation product was used to infect E. coli
DH5 alpha. From among the transformant strains obtained, a plasmid
having structure shown in FIG. 2 was selected and named
pMSCVneobactfEPOwpre. Stable packaging cells and retrovirus vectors
can be prepared in the same manner as in the examples mentioned
above. The use of the WPRE sequence makes it easy to obtain
high-titer stable packaging cells. This is presumably due to
enhancement of the transcriptional activity or stabilization of the
RNA by the use of the WPRE sequence.
EXAMPLE 7
Microinjection of Retrovirus Vector into Chicken Embryos and
Artificial Hatching
[0111] Microinjection and artificial hatching were carried out
under sterile conditions. Fertilized chicken eggs (Shiroyama
Shukeijo (Shiroyama Chicken Farm)) were externally disinfected with
a disinfectant (product of Showa Furanki) and ethanol. An incubator
model P-008(B) (product of Showa Furanki) was adjusted to an
environment of 38.degree. C. and 50 to 60% humidity, and incubation
was carried out from the start of power supply (0 hour) while
thereafter turning eggs at 90.degree. at 15-minute intervals.
[0112] After the lapse of about 55 hours from the start of
incubation, eggs were taken out of the incubator, a circular
portion with a diameter of 3.5 cm was cut off from the sharper end
of each egg using a minirouter (product of Proxxon) equipped with a
diamond edge (edge diameter 20 mm, shaft diameter 2.35 mm). A
circular portion with a diameter of 4.5 cm was cut off from the
sharper end of each double-yolked egg (Shiroyama Shukeijo), the
contents were discarded, the contents of each fertilized egg were
transferred to the remaining eggshell, and the embryo was shifted
upwards by means of the inner cylinder of a syringe. Under the
stereoscopic microscope system SZX12 (product of Olympus), the
virus-containing fluid was poured into FemtoChip II (product of
Eppendorf) and about 2 .mu.l of the virus solution of Example 5 or
6 was microinjected into the embryo using FemtoJet (product of
Eppendorf).
[0113] The circular hole was covered with Saran Wrap (product of
Asahi Chemical Industry) cut to a size of about 8.times.8 cm.sup.2,
using egg white as an adhesive, and the egg was returned to the
incubator for continued incubation. The mode of egg turning was
changed to 30.degree. turning at 30-minute intervals. On the 20th
day after the start of incubation, about 20 holes were bored in the
Saran Wrap using a 20G syringe needle, and oxygen was fed to the
incubator at a rate of 60 cc/min for hatching. When the chick began
pecking, the eggshell was broken to allow hatching. The
hatchability data in this artificial hatching are shown in Table
1.
TABLE-US-00001 TABLE 1 Virus titer Number of Number of Hatchability
(cfu/ml) eggs hatchlings (%) 1st 1.7 .times. 10.sup.8 20 1 5 2nd
1.4 .times. 10.sup.8 22 0 0 3rd 7.3 .times. 10.sup.6 22 0 0 4th 1.7
.times. 10.sup.8 21 1 5 5th 6.8 .times. 10.sup.7 18 6 33 6th 5.0
.times. 10.sup.7 17 7 41 7th 5.7 .times. 10.sup.6 16 5 31 8th 1.9
.times. 10.sup.8 15 2 13 9th 2.7 .times. 10.sup.8 11 2 18 10th 8.7
.times. 10.sup.8 25 2 8 11th 7.8 .times. 10.sup.8 43 7 16 12th 3.8
.times. 10.sup.8 20 3 15 Total 250 36 14
EXAMPLE 8
Confirmation of Expression in Blood and Eggs of Feline-Derived
Erythropoietin Expression Transgenic Chickens
[0114] The nestlings born in (Example 7) were fed for growth. The
feeds used were SX Safety and Neo-Safety 17 (products of Toyohashi
Shiryo (Toyohashi Feedstuff)) for young chicks. Blood was collected
from the transgenic chickens via the vein under the wing. The blood
collected was placed in an Eppendorf tube and, after at least 30
minutes of standing at room temperature, centrifuged at 3,000 rpm
at 4.degree. C. for 5 minutes using a small-sized high-speed
centrifuge to completely separate into serum and blood clot. The
supernatant was employed as serum. On the occasion of extraction
from eggs, egg white and yolk were separated from each other. In
extracting from egg yolk, a syringe was inserted into the middle of
the egg yolk and the egg yolk was drawn out while preventing the
egg white from coming in. The egg white was uniformly homogenized
by ultrasonic or physical means. The samples prepared were stored
frozen at -80.degree. C. until assaying. For avoiding thawing under
freezing, the thawing is preferably carried out rapidly at
37.degree. C.
[0115] The feline-derived erythropoietin activity was determined by
the cell proliferation assay technique (Japanese Kokai Publication
Hei10-94393) using the EPO-dependent cell line BaF/EPOR. In the
cell proliferation assay, a working curve for proliferation was
constructed using Epogin (product of Chugai Pharmaceutical) as a
standard, and the erythropoietin activity of each unknown sample
was determined based on the working curve for proliferation. The
medium used for BaF/EPOR cells was RPMI 1640 liquid medium (product
of Nissui) containing 5% of fetal bovine serum (FBS) and 50
units/ml each of penicillin and streptomycin. In ordinary BaF/EPOR
cell cultivation, Epogin was added to a final concentration of 1
U/ml. In cell proliferation assaying, cells at the logarithmic
growth phase were used.
[0116] In carrying out the cell proliferation assay using BaF/EPOR
cells, the Epogin in the medium was first removed. Thus, the
cultured BaF/EPOR cells were centrifuged at 1,000 rpm for 5
minutes. The supernatant was removed, and 10 ml of the Epogin-free
medium was added to the sediment for suspending the same. The
Epogin in the medium was removed by repeating the above procedure
three times. Cells were counted and diluted to a concentration of
55,555 cells/ml with the Epogin-free medium. The diluted cell
suspension was distributed in 90-.mu.l portions into the wells of
96-well microtiter plates. Thereto were added 10 .mu.l each of
Epogin solutions diluted to 25, 16, 10, 6.4, 4.0, 2.5, 1.6 and 1.0
U/ml and the cells were uniformly suspended therein (the final
erythropoietin concentrations being 2.5, 1.6, 1.0, 0.64, 0.4, 0.25,
0.16 and 0.1 U/ml, respectively). Each sample to be assayed was
diluted serially about 2 to 4 times so that the data might fall
within the assay range of the working curve, and 10 .mu.l of each
dilution was added to the cells sown for attaining uniform
suspension. Three measurements were carried out for each standard
sample and each unknown sample. After 2 days of cultivation, 10
.mu.l of the solution included in Cell Counting Kit-8 (product of
Dojin Kagaku Kenkyusho (Dojindo Laboratories)) was added to each
well. After allowing the color reaction to proceed from 1 to 4
hours, 10 .mu.l of 0.1 mol/l hydrochloric acid was added to
terminate the reaction, and the absorbance at 450 nm was measured
using a microplate reader. An approximate expression was derived
from the measurement results with standard samples by logarithmic
approximation, and the activity of each unknown sample was
calculated based on the expression obtained.
[0117] In the individual showing the maximum expression levels
(Individual No. 10-1), the activity in serum was 5,300 IU/ml and
that in egg white was 92,000 IU/ml. The expression results in G0
transgenics are shown in FIG. 3. After mastering of the injection
technique, the frequency of appearance of G0 transgenics among
hatchings was 100%. However, individuals showing high expression
levels are ready to die. Since the specific activity of
erythropoietin is about 220,000 IU/mg (Eur. J. Biochem. 1990 Dec.
12; 194(2):457-62) although it may vary depending on the extent of
addition of sugar chains, the content in serum can be estimated to
be about 24 .mu.g/ml and that in egg white to be about 420 .mu.g/ml
based on such data. Due to the difference in amino acid sequence
between feline and human erythropoietin species, no correct assays
could be made with the Recombigen EPO kit (product of Mitsubishi
Kagaku Iatron) using the RIA technique.
[0118] Then, the feline-derived erythropoietin levels in blood and
in eggs of Individual No. 6-3 were assayed by western blotting.
Each sample was electrophoresed under denaturing conditions using
12.5% of e-PAGEL (product of Atto Corp.) and, after transfer to a
PVDF membrane, blocking was carried out with PBS containing 10% of
skimmed milk and 0.05% of Tween 20, followed by detection with ECL
Plus Western Blotting Detection System and Hyperfilm ECL (products
of Amersham) using rabbit anti-human EPO antibody (product of G-T
Research Products) as the primary antibody and goat anti-rabbit
IgG-HRP antibody (product of Zymed Laboratories) as the secondary
antibody. The results of the western blotting are shown in FIG.
4.
EXAMPLE 9
Purification of Feline-Derived Erythropoietin from Egg White
[0119] The eggs from each individual for which the feline-derived
erythropoietin activity had been confirmed in egg white in Example
8 were recovered and feline-derived erythropoietin was recovered
and purified from egg white.
[0120] Each sample applied to the column was subjected to syringe
filtration just prior to using Millex-HV (product of Millipore)
with a pore size of 0.45 .mu.m. When the filtration was difficult,
the filtration through Millex was carried out after preliminary
filtration through Puradix 25 (product of Whatman) with a pore
diameter of 2 .mu.m.
[0121] During the purification process, the assaying of
feline-derived erythropoietin content was carried out using Bicore
3000 (product of BIACORE). An assay chip was prepared by subjecting
an anti-human erythropoietin monoclonal antibody (product of
R&D Systems) to NHS immobilization to Sensor Chip CM5 research
grade (product of BIOCORE) using an amine coupling kit (product of
BIOCORE) and, using the same, the concentration was determined
using the assaying program attached to the apparatus, with Epogin
as a standard substance.
[0122] For application to the column, the egg white was subjected
to pretreatment for reducing the viscosity. Each refrigerated egg
was returned to room temperature and then broken and, after
separation into egg yolk and egg white using the eggshell, for
instance, the egg white alone was recovered and weighed. The egg
white was agitated with a stirrer to entangle the dense egg white,
5 volumes of ultrapure water was added, and the mixture was further
stirred. At this point of time, the pH of the egg white had a pH of
about 9.0 to 9.3. The pH was adjusted to 5.0 by addition of an
appropriate amount of 1 N HCl and, after 15 minutes or longer of
stirring, the mixture was centrifuged at 9,500 G at 4.degree. C.
for 30 minutes. The supernatant was adjusted to pH 7.0 by addition
of 1 M NaOH, and 1 M Tris buffer (pH 7.0) was added to a final
concentration of 50 mM. In this step, the maximum recovery of
feline-derived erythropoietin was 95%.
[0123] Then, Blue Sepharose chromatography was carried out. A
500-ml portion of the egg white solution after pretreatment (egg
white of 2 to 3 eggs) was applied to a 50-ml Blue Sepharose 6 Fast
Flow column (product of Amersham) equilibrated with 50 mM Tris, pH
7.0. The column was thoroughly washed with 50 mM Tris, pH 7.0 and
then eluted with 200 ml of 1 M NaCl, 50 mM Tris, pH 7.0. The eluate
fraction was dialyzed overnight against 20 mM MES, pH 6.2 in the
conventional manner in a low-temperature room maintained at
4.degree. C. for buffer exchange. In this step, the maximum
recovery of feline-derived erythropoietin was 98%.
[0124] Then, heparin chromatography was carried out. The Blue
Sepharose eluate fraction (after dialysis) was applied in two
divided portions to a HiPrep 16/10 Heparin FF column (product of
Amersham) equilibrated with 20 mM MES, pH 6.2 and, for each
portion, the column was thoroughly washed with 20 mM MES, pH 6.2,
and then eluted in a gradient manner while increasing the NaCl
concentration to 80 mM. Each time, the column was regenerated with
1 M NaCl and 0.1 M NaOH. The fractions in which feline-derived
erythropoietin was confirmed by means of Biacore were recovered. In
this step, the maximum recovery of feline-derived erythropoietin
was 80%.
[0125] Then, buffer exchange was carried out using a desalting
column. The Heparin Sepharose eluate fraction was concentrated to a
total amount of about 30 to 40 ml by means of Vivaspin 20 (product
of Sartorius) with a cutoff molecular weight of 5,000 and applied,
in 10-ml divided portions, to a HiPrep 26/10 Desalting column
(product of Amersham) equilibrated with 25 mM Tris, pH 7.0, and
eluted with the same buffer to recover a protein-containing
fraction. This was adjusted to pH 9.0 with 1 M NaOH and further
adjusted with 1 M NaCl so that the electric conductivity might
amount to 3.0 to 3.2 mS/cm. In this step, the maximum recovery of
feline-derived erythropoietin was 95%.
[0126] Then, anion exchange column chromatography was carried out.
The sample after buffer exchange was applied, in two divided
portions, to a 5-ml HiTrap DEAE FF column (product of Amersham)
equilibrated with 25 mM Tris (pH 9.0, electric conductivity 3.0 to
3.2 mS/cm) and, for each portion, the pass-through fraction was
recovered. The column was regenerated each time with 1 M NaCl. The
fractions were concentrated to a total volume of about 2 to 3 ml
using Vivaspin 20 with a cutoff molecular weight of 5,000. In this
step, the maximum recovery of feline-derived erythropoietin was
92%.
[0127] Gel filtration was carried out. The sample after
concentration was applied to a Superdex 200 10/300 GL column
(product of Amersham) equilibrated with 50 mM borate buffer, pH 9.0
or some other appropriate buffer, followed by elution with the same
buffer. Fractions in which the occurrence of feline-derived
erythropoietin was confirmed by means of Biacore were recovered and
concentrated to a total amount of about 1 to 2 ml using Vivaspin 6
with a cutoff molecular weight of 5,000. In this step, the maximum
recovery of feline-derived erythropoietin was 93%.
[0128] Each fraction recovered in each purification step was
subjected to SDS-PAGE. Each sample was electrophoresed under
denaturing conditions using 12.5% e-PAGEL, followed by detection
with Bio-Safe Coomassie Stain (product of Bio-Rad). The results of
SDS-PAGE for Individual No. 10-1 are shown in FIG. 5.
[0129] The protein concentration was determined using DC Protein
Assay (product of Bio-Rad) with BSA as a standard. In the
individual in which the expression level was maximum (Individual
No. 10-1), the purification of egg white gave about 3 mg, per egg
white of one egg, of feline-derived erythropoietin.
EXAMPLE 10
Baf/EPOR Cell Proliferation Assay of Purified Feline
Erythropoietin
[0130] The feline-derived erythropoietin purified from egg white in
Example 9 was subjected to Baf/EPOR cell proliferation assay by the
method described in Example 8. As a result, the specific activity
was 160,000-290,000 IU/mg.
EXAMPLE 11
Production of mPEG-Modified Feline-Derived Erythropoietin
[0131] To a solution of feline-derived erythropoietin purified from
egg white (dissolved in phosphate or borate buffer with a pH of 8.0
to 9.0) was added methoxyPEG-SPA (succinimidylpropionate ester) or
methoxyPEG-SMB (succinimidyl-alpha-methylbutanoate ester) (product
of NEKTAR) (the molecular weight of PEG being about 20 kDa), and
the resulting mixture was mixed up by inversion at room temperature
for 0.5 to 1 hour. A 1/10 volume of 100 mM glycine solution was
added and the mixture was mixed up by further inversion at room
temperature for 0.5 hour to deactivate the active ester. The
solution after reaction was subjected to buffer exchange for 50 mM
acetate buffer, pH 4.5, using Prepacked Disposable PD-10 Columns
(product of Amersham) or by dialysis, followed by application to a
1-ml HiTrap SP HP column (product of Amersham) equilibrated with
the same buffer. The unreacted PEG passed through the column, and
mPEG-fEPO and unreacted feline-derived erythropoietin were adsorbed
on the column; these were eluted and recovered by eluting with 500
mM NaCl, 25 mM acetate buffer, pH 4.5. The recovered fraction was
applied to Superdex 200 10/300 GL equilibrated with 150 mM NaCl+20
mM phosphate buffer, pH 7.5, and fractions corresponding to the
number of PEG molecules added of 2 or more, 1 and 0 (unreacted
feline-derived erythropoietin) were recovered. The fractions after
recovery were properly concentrated by means of Vivaspin 6 with a
cutoff molecular weight of 5,000. The activated PEG species,
reaction mixture composition, reaction time and mPEG-fEPO products
ratio data are summarized in Table 2. For each mPEG-fEPO, the
theoretical molecular weight and the molecular weight based on the
separation on Superdex 200 10/300 GL are summarized in Table 3. The
PEG-modified and gel filtration-purified samples were subjected to
SDS-PAGE. The samples were electrophoresed under denaturing
conditions using 12.5% e-PAGEL and detected using Bio-Safe
Coomassie Stain (product of Bio-Rad). The results of SDS-PAGE are
shown in FIG. 6.
TABLE-US-00002 TABLE 2 Products ratio (%) Reaction mixture
composition Reaction time (hr) di- mono- Activated PEG
Buffer.sup.1) EPO concentration EPO:PEG PEG- PEG- Unmodified
species composition (mg/ml) mole ratio fEPO fEPO fEPO mPEG-SPA A
2.3 1:5 1.0 Trace 29 71 B 2.3 1:5 1.0 Trace 31 69 A 2.7 1:10 0.5 6
50 44 mPEG-SMB A 2.3 1:5 1.0 7 61 32 B 2.3 1:5 1.0 11 67 23 C 2.3
1:5 1.0 12 67 21 Note.sup.1): Buffer composition: A. 100 mM
phosphate buffer, pH 8.0 B. 50 mM borate buffer, pH 8.5 C. 50 mM
borate buffer, pH 9.0
TABLE-US-00003 TABLE 3 fEPO or PEG-fEPO species Feline-derived
di-PEG- mono-PEG- erythropoietin Molecular weight (in kDa)
SPA(SMB)-EPO SPA(SMB)-EPO (SEQ ID NO: 1) Theoretical molecular
weight 61 41 21 (excluding sugar chain) Gel filtration molecular
weight 685 377 41
EXAMPLE 12
Baf/EPOR Cell Proliferation Assay of Mono-PEG-Modified
Feline-Derived Erythropoietin
[0132] The mono-mPEG-fEPO PEG-modified and purified in Example 11
was subjected to Baf/EPOR cell proliferation assay by the method
described in Example 10. The mPEG-fEPO was quantitated as the
weight of the protein portion using DC Protein Assay (product of
Bio-Rad) with BSA as a standard. As a result, the specific activity
was 3,200 to 15,000 IU/mg in the case of mono-mPEG-SPA-fEPO or
4,300 to 8,300 IU/mg in the case of mono-mPEG-SMB-fEPO.
EXAMPLE 13
Drug Efficacy Duration Measurement of Mono-PEG-Modified
Feline-Derived Erythropoietin
[0133] Normal Crlj:CD strain male rats (products of Charles River
Japan) were subjected to experiment at the age of 7 weeks. Use was
made of the mono-mPEG-SPA-fEPO PEG-modified and purified in
(Example 11) (specific activity 8,300 IU/mg) was used as a mono-PEG
modified feline-derived erythropoietin, non-PEG-modified
feline-derived erythropoietin purified from Individual No. 10-1 in
Example 9 (specific activity 160,000 IU/mg) as a non-PEG-modified
feline-derived erythropoietin, and Epogin (specific activity
180,000 IU/mg; value described in Interview Form) as CHO-produced
human erythropoietin. These were diluted with physiological saline
containing 0.05% of human serum albumin and 0.05% of Tween 20 and
administered at a single dose of 2 ml/kg. Rats were divided into 12
groups each consisting of 5 rats, and two groups each were
administered with the mono-mPEG-SPA-fEPO diluted to 25, 5, or 1
.mu.g/ml .mu.g (the dose being 50, 10 or 2 .mu.g/kg, respectively)
or the non-PEG-modified feline-derived erythropoietin or Epogin
each diluted to 25 .mu.g/ml (the dose being 50 .mu.g/kg). The
control groups were administered with the solvent alone. The test
substances were prepared in advance and frozen stored at
-20.degree. C. Just before administration, they were thawed at room
temperature and administered into the caudal vein at about the body
temperature using a 27 G injection needle. For lessening the burden
on test animals, blood sampling was carried out alternately in the
two groups given the same dose of the same test substance (group A
and group B, respectively) as follows: group A: 0, 4, 10, and 21
days after administration of the test substance; group B: 2, 7 and
15 days after administration of the test substance. Without
anesthetizing, 0.5-ml blood samples were collected from the common
carotid artery using a 23 G injection needle. For each sample,
reticulocytes were counted using the automated reticulocyte
measuring apparatus R-3000 (product of Sysmex).
[0134] The significances of the differences between the mean
reticulocyte count values in the control group and in the
mono-mPEG-SPA-fEPO group, non-PEG-modified feline-derived
erythropoietin group and Epogin group at each point of measurement
were tested by the Dunnett's multiple comparison method using the
SAS system (product of SAS Institute). The significance levels were
5% on both sides.
[0135] The measurement results are shown in FIG. 7. In the Epogin
(50 .mu.g/kg) group and mono-mPEG-SPA-fEPO groups (50 .mu.g/kg and
10 .mu.g/kg), the reticulocyte count significantly dropped on the
day 2 after administration as compared with the control group and,
on the 4th day, it increased to show the maximum value. On the 7th
day after administration, it again dropped to a level below the
level of the control group. In the mono-mPEG-SPA-fEPO (2 .mu.g/kg)
group and non-PEG-modified feline-derived EPO (50 .mu.g/kg) group,
no significant increases in reticulocyte count were observed. In
the mono-mPEG-SPA-EPO group, the in vivo reticulocyte activity was
high in spite of the decreased specific Baf/EPOR cell proliferating
activity as compared with the non-PEG-modified feline-derived EPO.
Presumably, this was due to an improvement in in vivo stability of
erythropoietin as brought about by modification with PEG.
BRIEF DESCRIPTION OF THE DRAWINGS
[0136] FIG. 1 The figure shows the structure of the feline-derived
erythropoietin expression vector pMSCVneobactfEPO constructed in
Example 1. The symbol phi+ indicates the virus packaging signal
sequence. The packaging signal sequence of Moloney murine leukemia
virus is accompanied by a part of gag with TAG substituting for the
initiation codon (ATG) in gag. fEPO indicates the feline-derived
erythropoietin gene. 5'LTR and 3'LTR indicate the LTR sequences of
MSCV.
[0137] FIG. 2 The figure shows the structure of the feline-derived
erythropoietin expression vector pMSCVneobactfEPOwpre constructed
in Example 6. The symbol phi+ indicates the virus packaging signal
sequence. The packaging signal sequence of Moloney murine leukemia
virus is accompanied by a part of gag with TAG substituting for the
initiation codon (ATG) in gag. fEPO indicates the feline-derived
erythropoietin gene. 5'LTR and 3'LTR indicate the LTR sequences of
MSCV. WPRE indicates the woodchuck hepatitis virus-derived
posttranscriptional regulatory factor.
[0138] FIG. 3 The figure shows the activities in serum and egg
white of transgenic chickens as measured in Example 8. The
activities were measured by cell proliferation assaying using
BaF/EPOR, which is an EPO-dependent cell line, with Epogin as
standard erythropoietin. "Sex unknown" means that the relevant
individual was sacrificed before sex judgment. Chickens marked with
(*) were transgenic chickens produced by using the pMSCVneobactfEPO
vector, and others were transgenic chickens produced by using
pMSCVneobactfEPOwpre. In the individual showing maximum expression
levels (Individual No. 10-1), the activity in serum was 5,300 IU/ml
and that in egg white was 92,000 IU/ml.
[0139] FIG. 4 The figure shows the results of western blot analysis
of the serum and egg white of a transgenic chicken (Individual No.
6-3) as carried out in Example 8 using anti-feline-derived
erythropoietin antibody. The expression level in serum in the
individual No. 6-3 was 620 IU/ml and that in egg white was 6,400
IU/ml. The markers used were molecular weight markers (Dr. Western
markers; product of Oriental Yeast). The molecular weight of
erythropoietin is about 32 to 34 kDa. Epogin was used as a
reference sample. Wild-type samples were derived from a
non-transgenic chicken. Epogin was electrophoresed in an amount of
1.2 U/lane, and the egg white and serum were electrophoresed in
amounts of 0.2 .mu.l/lane and 0.4 .mu.l/lane, respectively. The
bands given by the wild type are bands nonspecific to
erythropoietin. As compared with the wild type, a band due to
serum-derived erythropoietin can be confirmed at the same position
as that of Epogin, although in a slight amount. Egg white-derived
erythropoietin shows a migration distance corresponding to a lower
molecular weight as compared with the ordinary species. This is
presumably due to the difference in sugar chain modification
pattern.
[0140] FIG. 5 The figure shows the results of SDS-PAGE in each step
of purification of feline-derived erythropoietin in the egg white
from a transgenic chicken (Individual No. 10-1) as carried out in
Example 9. The sample size was 2 .mu.g/lane (except for Epogin: 10
.mu.l/lane). From the left, there are shown the egg white (EW),
Blue Sepharose chromatography eluate fraction (BS), heparin
chromatography eluate fraction (HE), anion exchange column
chromatography through-out fraction (DEAE), gel filtration column
chromatography eluate fraction (GPC), and Epogin (Epogen). Used as
the molecular weight markers were Precision Plus Protein Standards
Dual Color (product of Bio-Rad). In agreement with the results of
western blot analysis as shown in FIG. 4, a band due to
feline-derived erythropoietin purified from egg white can be
confirmed at a position corresponding to a lower molecular weight
as compared with Epogin.
[0141] FIG. 6 The figure shows the results of SDS-PAGE of the
reaction mixture resulting from modification of feline-derived
erythropoietin with PEG, and of PEG-fEPO after purification, as
performed in Example 11. Each sample was electrophoresed in an
amount of 10 .mu.l/lane. Lane 1 shows the results obtained with the
PEG modification reaction mixture, Lane 2 with a cation
exchanger-bound fraction, Lane 3 with di-mPEG-SPA-fEPO, Lane 4 with
mono-mPEG-SPA-fEPO, and Lane 5 with unmodified fEPO. In Lane 1,
there is a band unstainable with CBB around 30 to 40 kDa; this
probably corresponds to the unreacted PEG. This band was eliminated
upon cation exchange chromatography (Lane 2).
[0142] FIG. 7 The figure shows the changes in reticulocyte count in
rats following administration of non-PEG-modified feline-derived
erythropoietin, mono-mPEG-SPA-fEPO and Epogen as performed in
Example 13.
Sequence CWU 1
1
71192PRTFelis catus 1Met Gly Ser Cys Glu Cys Pro Ala Leu Leu Leu
Leu Leu Ser Leu Leu1 5 10 15Leu Leu Pro Leu Gly Leu Pro Val Leu Gly
Ala Pro Pro Arg Leu Ile20 25 30Cys Asp Ser Arg Val Leu Glu Arg Tyr
Ile Leu Glu Ala Arg Glu Ala35 40 45Glu Asn Val Thr Met Gly Cys Ala
Glu Gly Cys Ser Phe Ser Glu Asn50 55 60Ile Thr Val Pro Asp Thr Lys
Val Asn Phe Tyr Thr Trp Lys Arg Met65 70 75 80Asp Val Gly Gln Gln
Ala Val Glu Val Trp Gln Gly Leu Ala Leu Leu85 90 95Ser Glu Ala Ile
Leu Arg Gly Gln Ala Leu Leu Ala Asn Ser Ser Gln100 105 110Pro Ser
Glu Thr Leu Gln Leu His Val Asp Lys Ala Val Ser Ser Leu115 120
125Arg Ser Leu Thr Ser Leu Leu Arg Ala Leu Gly Ala Arg Lys Glu
Ala130 135 140Thr Ser Leu Pro Glu Ala Thr Ser Ala Ala Pro Leu Arg
Thr Phe Thr145 150 155 160Val Asp Thr Leu Cys Lys Leu Phe Arg Ile
Tyr Ser Asn Phe Leu Arg165 170 175Gly Lys Leu Thr Leu Tyr Thr Gly
Glu Ala Cys Arg Arg Gly Asp Arg180 185
19025798DNAArtificialRetrovirus vector pMSCVneobact 2aatgaaagac
cccacctgta ggtttggcaa gctagcttaa gtaacgccat tttgcaaggc 60atggaaaata
cataactgag aatagagaag ttcagatcaa ggttaggaac agagagacag
120cagaatatgg gccaaacagg atatctgtgg taagcagttc ctgccccggc
tcagggccaa 180gaacagatgg tccccagatg cggtcccgcc ctcagcagtt
tctagagaac catcagatgt 240ttccagggtg ccccaaggac ctgaaatgac
cctgtgcctt atttgaacta accaatcagt 300tcgcttctcg cttctgttcg
cgcgcttctg ctccccgagc tcaataaaag agcccacaac 360ccctcactcg
gcgcgccagt cctccgatag actgcgtcgc ccgggtaccc gtattcccaa
420taaagcctct tgctgtttgc atccgaatcg tggactcgct gatccttggg
agggtctcct 480cagattgatt gactgcccac ctcgggggtc tttcatttgg
aggttccacc gagatttgga 540gacccctgcc cagggaccac cgaccccccc
gccgggaggt aagctggcca gcggtcgttt 600cgtgtctgtc tctgtctttg
tgcgtgtttg tgccggcatc taatgtttgc gcctgcgtct 660gtactagtta
gctaactagc tctgtatctg gcggacccgt ggtggaactg acgagttctg
720aacacccggc cgcaaccctg ggagacgtcc cagggacttt gggggccgtt
tttgtggccc 780gacctgagga agggagtcga tgtggaatcc gaccccgtca
ggatatgtgg ttctggtagg 840agacgagaac ctaaaacagt tcccgcctcc
gtctgaattt ttgctttcgg tttggaaccg 900aagccgcgcg tcttgtctgc
tgcagcgctg cagcatcgtt ctgtgttgtc tctgtctgac 960tgtgtttctg
tatttgtctg aaaattaggg ccagactgtt accactccct taagtttgac
1020cttaggtcac tggaaagatg tcgagcggat cgctcacaac cagtcggtag
atgtcaagaa 1080gagacgttgg gttaccttct gctctgcaga atggccaacc
tttaacgtcg gatggccgcg 1140agacggcacc tttaaccgag acctcatcac
ccaggttaag atcaaggtct tttcacctgg 1200cccgcatgga cacccagacc
aggtccccta catcgtgacc tgggaagcct tggcttttga 1260cccccctccc
tgggtcaagc cctttgtaca ccctaagcct ccgcctcctc ttcctccatc
1320cgccccgtct ctcccccttg aacctcctcg ttcgaccccg cctcgatcct
ccctttatcc 1380agccctcact ccttctctag gcgccggcgg ccgccgccac
catgggatcg gccattgaac 1440aagatggatt gcacgcaggt tctccggccg
cttgggtgga gaggctattc ggctatgact 1500gggcacaaca gacaatcggc
tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc 1560gcccggttct
ttttgtcaag accgacctgt ccggtgccct gaatgaactg caggacgagg
1620cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg
ctcgacgttg 1680tcactgaagc gggaagggac tggctgctat tgggcgaagt
gccggggcag gatctcctgt 1740catctcacct tgctcctgcc gagaaagtat
ccatcatggc tgatgcaatg cggcggctgc 1800atacgcttga tccggctacc
tgcccattcg accaccaagc gaaacatcgc atcgagcgag 1860cacgtactcg
gatggaagcc ggtcttgtcg atcaggatga tctggacgaa gagcatcagg
1920ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgcg catgcccgac
ggcgaggatc 1980tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat
ggtggaaaat ggccgctttt 2040ctggattcat cgactgtggc cggctgggtg
tggcggaccg ctatcaggac atagcgttgg 2100ctacccgtga tattgctgaa
gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt 2160acggtatcgc
cgctcccgat tcgcagcgca tcgccttcta tcgccttctt gacgagttct
2220tctgagcggc cgcgaattcg tcgacgtgca tgcacgctca ttgcccatcg
ctatccctgc 2280ctctcctgct ggcgctcccc gggaggtgac ttcaagggga
ccgcaggacc acctcggggg 2340tggggggagg gctgcacacg cggaccccgc
tccccctccc caacaaagca ctgtggaatc 2400aaaaaggggg gaggggggat
ggaggggcgc gtcacacccc cgccccacac cctcacctcg 2460aggtgagccc
cacgttctgc ttcactctcc ccatctcccc cccctcccca cccccaattt
2520tgtatttatt tattttttaa ttattttgtg cagcgatggg ggcggggggg
gggggggcgc 2580gcgccaggcg gggcggggcg gggcgagggg cggggcgggg
cgaggcggag aggtgcggcg 2640gcagccaatc agagcggcgc gctccgaaag
tttcctttta tggcgaggcg gcggcggcgg 2700cggccctata aaaagcgaag
cgcgcggcgg gcgggagtcg ctgcgttgtc gacggatcct 2760cgagctgcag
aagctttcgc gaatcgataa aataaaagat tttatttagt ctccagaaaa
2820aggggggaat gaaagacccc acctgtaggt ttggcaagct agcttaagta
acgccatttt 2880gcaaggcatg gaaaatacat aactgagaat agagaagttc
agatcaaggt taggaacaga 2940gagacagcag aatatgggcc aaacaggata
tctgtggtaa gcagttcctg ccccggctca 3000gggccaagaa cagatggtcc
ccagatgcgg tcccgccctc agcagtttct agagaaccat 3060cagatgtttc
cagggtgccc caaggacctg aaatgaccct gtgccttatt tgaactaacc
3120aatcagttcg cttctcgctt ctgttcgcgc gcttctgctc cccgagctca
ataaaagagc 3180ccacaacccc tcactcggcg cgccagtcct ccgatagact
gcgtcgcccg ggtacccgtg 3240tatccaataa accctcttgc agttgcatcc
gacttgtggt ctcgctgttc cttgggaggg 3300tctcctctga gtgattgact
acccgtcagc gggggtcttt caggcctctg cattaatgaa 3360tcggccaacg
cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca
3420ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
tcaaaggcgg 3480taatacggtt atccacagaa tcaggggata acgcaggaaa
gaacatgtga gcaaaaggcc 3540agcaaaaggc caggaaccgt aaaaaggccg
cgttgctggc gtttttccat aggctccgcc 3600cccctgacga gcatcacaaa
aatcgacgct caagtcagag gtggcgaaac ccgacaggac 3660tataaagata
ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
3720tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
ctttctcaat 3780gctcacgctg taggtatctc agttcggtgt aggtcgttcg
ctccaagctg ggctgtgtgc 3840acgaaccccc cgttcagccc gaccgctgcg
ccttatccgg taactatcgt cttgagtcca 3900acccggtaag acacgactta
tcgccactgg cagcagccac tggtaacagg attagcagag 3960cgaggtatgt
aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
4020gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
aaaagagttg 4080gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
tggttttttt gtttgcaagc 4140agcagattac gcgcagaaaa aaaggatctc
aagaagatcc tttgatcttt tctacggggt 4200ctgacgctca gtggaacgaa
aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa 4260ggatcttcac
ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
4320atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
atctcagcga 4380tctgtctatt tcgttcatcc atagttgcct gactccccgt
cgtgtagata actacgatac 4440gggagggctt accatctggc cccagtgctg
caatgatacc gcgagaccca cgctcaccgg 4500ctccagattt atcagcaata
aaccagccag ccggaagggc cgagcgcaga agtggtcctg 4560caactttatc
cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
4620cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
gtgtcacgct 4680cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
atcaaggcga gttacatgat 4740cccccatgtt gtgcaaaaaa gcggttagct
ccttcggtcc tccgatcgtt gtcagaagta 4800agttggccgc agtgttatca
ctcatggtta tggcagcact gcataattct cttactgtca 4860tgccatccgt
aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
4920agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
accgcgccac 4980atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
ttcggggcga aaactctcaa 5040ggatcttacc gctgttgaga tccagttcga
tgtaacccac tcgtgcaccc aactgatctt 5100cagcatcttt tactttcacc
agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg 5160caaaaaaggg
aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
5220attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
gaatgtattt 5280agaaaaataa acaaataggg gttccgcgca catttccccg
aaaagtgcca cctgacgtct 5340aagaaaccat tattatcatg acattaacct
ataaaaatag gcgtatcacg aggccctttc 5400gtctcgcgcg tttcggtgat
gacggtgaaa acctctgaca catgcagctc ccggagacgg 5460tcacagcttg
tctgtaagcg gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg
5520gtgttggcgg gtgtcggggc tggcttaact atgcggcatc agagcagatt
gtactgagag 5580tgcaccatat gcggtgtgaa ataccgcaca gatgcgtaag
gagaaaatac cgcatcaggc 5640aggcctgtta acttcgaacg attagtccaa
tttgttaaag acaggatatc agtggtccag 5700gctctagttt tgactcaaca
atatcaccag ctgaagccta tagagtacga gccatagata 5760aaataaaaga
ttttatttag tctccagaaa aagggggg 579833747DNAArtificialPlasmid vector
pUCfEPO 3tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg
gagacggtca 60cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg
tcagcgggtg 120ttggcgggtg tcggggctgg cttaactatg cggcatcaga
gcagattgta ctgagagtgc 180accataaaat tgtaaacgtt aatattttgt
taaaattcgc gttaaatttt tgttaaatca 240gctcattttt taaccaatag
gccgaaatcg gcaaaatccc ttataaatca aaagaatagc 300ccgagatagg
gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg
360actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta
cgtgaaccat 420cacccaaatc aagttttttg gggtcgaggt gccgtaaagc
actaaatcgg aaccctaaag 480ggagcccccg atttagagct tgacggggaa
agccggcgaa cgtggcgaga aaggaaggga 540agaaagcgaa aggagcgggc
gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa 600ccaccacacc
cgccgcgctt aatgcgccgc tacagggcgc gtactatggt tgctttgacg
660tatgcggtgt gaaataccgc acagatgcgt aaggagaaaa taccgcatca
ggcgccattc 720gccattcagg ctgcgcaact gttgggaagg gcgatcggtg
cgggcctctt cgctattacg 780ccagctggcg aaagggggat gtgctgcaag
gcgattaagt tgggtaacgc cagggttttc 840ccagtcacga cgttgtaaaa
cgacggccag tgaattcgag ctcggtaccc ggggatcctc 900tagagtcgac
atggggtcgt gcgaatgtcc tgccctgctg cttctgctat ctttgctgct
960gcttcccctg ggcctcccag tcctgggcgc cccccctcgc ctcatctgtg
acagccgagt 1020cctggagagg tacattctgg aggccaggga ggccgaaaat
gtcacgatgg gctgtgctga 1080aggctgcagc ttcagtgaga atatcactgt
cccagacacc aaggtcaact tctatacctg 1140gaagaggatg gacgtcgggc
agcaggctgt ggaagtctgg cagggcctcg ccctgctctc 1200agaagccatc
ctgcggggcc aggccctgct ggccaactcc tcccagccat ctgagaccct
1260gcagctgcat gtggataaag ccgtcagcag cctgcgcagc ctcacctccc
tgcttcgggc 1320actgggagcc cggaaggaag ccacctccct tccagaggca
acctctgctg ctccactccg 1380aacattcact gtcgatactt tgtgcaaact
tttccgaatc tactccaact tcctgcgggg 1440aaagctgacg ctgtacacag
gggaggcctg ccgaagagga gacaggtgag tcgacctgca 1500ggcatgcaag
cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc
1560tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg
ggtgcctaat 1620gagtgagcta actcacatta attgcgttgc gctcactgcc
cgctttccag tcgggaaacc 1680tgtcgtgcca gctgcattaa tgaatcggcc
aacgcgcggg gagaggcggt ttgcgtattg 1740ggcgctcttc cgcttcctcg
ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag 1800cggtatcagc
tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
1860gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
gccgcgttgc 1920tggcgttttt ccataggctc cgcccccctg acgagcatca
caaaaatcga cgctcaagtc 1980agaggtggcg aaacccgaca ggactataaa
gataccaggc gtttccccct ggaagctccc 2040tcgtgcgctc tcctgttccg
accctgccgc ttaccggata cctgtccgcc tttctccctt 2100cgggaagcgt
ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
2160ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
tgcgccttat 2220ccggtaacta tcgtcttgag tccaacccgg taagacacga
cttatcgcca ctggcagcag 2280ccactggtaa caggattagc agagcgaggt
atgtaggcgg tgctacagag ttcttgaagt 2340ggtggcctaa ctacggctac
actagaagga cagtatttgg tatctgcgct ctgctgaagc 2400cagttacctt
cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
2460gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
tctcaagaag 2520atcctttgat cttttctacg gggtctgacg ctcagtggaa
cgaaaactca cgttaaggga 2580ttttggtcat gagattatca aaaaggatct
tcacctagat ccttttaaat taaaaatgaa 2640gttttaaatc aatctaaagt
atatatgagt aaacttggtc tgacagttac caatgcttaa 2700tcagtgaggc
acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
2760ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
gctgcaatga 2820taccgcgaga cccacgctca ccggctccag atttatcagc
aataaaccag ccagccggaa 2880gggccgagcg cagaagtggt cctgcaactt
tatccgcctc catccagtct attaattgtt 2940gccgggaagc tagagtaagt
agttcgccag ttaatagttt gcgcaacgtt gttgccattg 3000ctacaggcat
cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
3060aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
agctccttcg 3120gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
atcactcatg gttatggcag 3180cactgcataa ttctcttact gtcatgccat
ccgtaagatg cttttctgtg actggtgagt 3240actcaaccaa gtcattctga
gaatagtgta tgcggcgacc gagttgctct tgcccggcgt 3300caatacggga
taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
3360gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
tcgatgtaac 3420ccactcgtgc acccaactga tcttcagcat cttttacttt
caccagcgtt tctgggtgag 3480caaaaacagg aaggcaaaat gccgcaaaaa
agggaataag ggcgacacgg aaatgttgaa 3540tactcatact cttccttttt
caatattatt gaagcattta tcagggttat tgtctcatga 3600gcggatacat
atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
3660cccgaaaagt gccacctgac gtctaagaaa ccattattat catgacatta
acctataaaa 3720ataggcgtat cacgaggccc tttcgtc
3747447DNAArtificialPrimer for producing pMSCVneobactfEPO
4agccaagctt accatggggt cgtgcgaatg tcctgccctg ctgcttc
47538DNAArtificialPrimer for producing pMSCVneobactfEPO 5cgataagctt
acgcgttcac ctgtctcctc ttcggcag 38637DNAArtificialPrimer for
producing pMSCVneobactfEPOwpre 6ccatcgataa tcaacctctg gattacaaaa
tttgtga 37722DNAArtificialPrimer for producing pMSCVneobactfEPOwpre
7ccatcgatca ggcggggagg cg 22
* * * * *
References