U.S. patent application number 10/770824 was filed with the patent office on 2004-10-07 for novel proteins from actinobacillus pleuropneumoniae.
Invention is credited to Ankenbauer, Robert G., Baarsch, Mary Jo, Campos, Manuel, Keich, Robin, Rosey, Everett, Suiter, Brian, Warren-Stewart, Lynn.
Application Number | 20040198954 10/770824 |
Document ID | / |
Family ID | 31996500 |
Filed Date | 2004-10-07 |
United States Patent
Application |
20040198954 |
Kind Code |
A1 |
Ankenbauer, Robert G. ; et
al. |
October 7, 2004 |
Novel proteins from Actinobacillus pleuropneumoniae
Abstract
The present invention is directed to five novel, low molecular
weight proteins from Actinobacillus pleuropneumoniae (APP), which
are capable of inducing, or contributing to the induction of, a
protective immune response in swine against APP. The present
invention is further directed to polynucleotide molecules having
nucleotide sequences that encode the proteins, as well as vaccines
comprising the proteins or polynucleotide molecules, and methods of
making and using the same.
Inventors: |
Ankenbauer, Robert G.;
(Pawcatuck, CT) ; Baarsch, Mary Jo; (Niantic,
CT) ; Campos, Manuel; (Stonington, CT) ;
Keich, Robin; (Waterford, CT) ; Rosey, Everett;
(Preston, CT) ; Suiter, Brian; (Lincoln, NE)
; Warren-Stewart, Lynn; (Colchester, CT) |
Correspondence
Address: |
SCULLY SCOTT MURPHY & PRESSER, PC
400 GARDEN CITY PLAZA
GARDEN CITY
NY
11530
|
Family ID: |
31996500 |
Appl. No.: |
10/770824 |
Filed: |
February 3, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10770824 |
Feb 3, 2004 |
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09418980 |
Oct 14, 1999 |
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6713071 |
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60105285 |
Oct 22, 1998 |
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Current U.S.
Class: |
530/350 |
Current CPC
Class: |
A61K 39/102 20130101;
A61K 39/00 20130101; G01N 33/56911 20130101; C07K 14/285 20130101;
A61K 2039/52 20130101 |
Class at
Publication: |
530/350 |
International
Class: |
C07K 001/00; C07K
014/00; C07K 017/00 |
Claims
What is claimed is:
1. A substantially purified protein which comprises an amino acid
sequence selected from the group consisting of amino acid residue
20 to amino acid residue 172 of SEQ ID NO:2, amino acid residue 22
to amino acid residue 215 of SEQ ID NO:4, amino acid residue 28 to
amino acid residue 258 of SEQ ID NO:6, amino acid residue 20 to
amino acid residue 364 of SEQ ID NO:8, and amino acid residue 20 to
amino acid residue 369 of SEQ ID NO:10.
2. The protein of claim 1, which comprises an amino acid sequence
selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ
ID NO:6, SEQ ID NO:8, and SEQ ID NO:10.
3. A substantially purified polypeptide that is homologous to a
protein comprising an amino acid sequence selected from the group
consisting of amino acid residue 20 to amino acid residue 172 of
SEQ ID NO:2, amino acid residue 22 to amino acid residue 215 of SEQ
ID NO:4, amino acid residue 28 to amino acid residue 258 of SEQ ID
NO:6, amino acid residue 20 to amino acid residue 364 of SEQ ID
NO:8, and amino acid residue 20 to amino acid residue 369 of SEQ ID
NO:10.
4. The polypeptide of claim 3, which is homologous to a polypeptide
comprising an amino acid sequence selected from the group
consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8,
and SEQ ID NO:10.
5. A substantially purified polypeptide which is a peptide fragment
of: (a) a polypeptide comprising an amino acid sequence selected
from the group consisting of amino acid residue 20 to amino acid
residue 172 of SEQ ID NO:2, amino acid residue 22 to amino acid
residue 215 of SEQ ID NO:4, amino acid residue 28 to amino acid
residue 258 of SEQ ID NO:6, amino acid residue 20 to amino acid
residue 364 of SEQ ID NO:8, and amino acid residue 20 to amino acid
residue 369 of SEQ ID NO:10; or (b) a polypeptide which is
homologous to any of the polypeptides of (a).
6. The polypeptide of claim 5, which is a peptide fragment of a
polypeptide comprising an amino acid sequence selected from the
group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID
NO:8, and SEQ ID NO:10.
7. The polypeptide of claim 6, which comprises an amino acid
sequence selected from the group consisting of amino acid residue 1
to amino acid residue 19 of SEQ ID NO:2, amino acid residue 1 to
amino acid residue 21 of SEQ ID NO:4, amino acid residue 1 to amino
acid residue 27 of SEQ ID NO:6, amino acid residue 1 to amino acid
residue 19 of SEQ ID NO:8, and amino acid residue 1 to amino acid
residue 19 of SEQ ID NO:10.
8. A fusion protein comprising a carrier or fusion partner joined
to: (a) a polypeptide comprising an amino acid sequence selected
from the group consisting of amino acid residue 20 to amino acid
residue 172 of SEQ ID NO:2, amino acid residue 22 to amino acid
residue 215 of SEQ ID NO:4, amino acid residue 28 to amino acid
residue 258 of SEQ ID NO:6, amino acid residue 20 to amino acid
residue 364 of SEQ ID NO:8, and amino acid residue 20 to amino acid
residue 369 of SEQ ID NO:10; (b) a polypeptide that is homologous
to a polypeptide of (a); or 5 (c) a peptide fragment of a
polypeptide of (a) or (b).
9. The fusion protein of claim 8, wherein the fusion partner is
selected from the group consisting of a protective peptide,
.beta.-galactosidase, trpE, maltose-binding protein,
glutathione-S-transferase, and polyhistidine.
10. The fusion protein of claim 9, wherein the fusion partner is a
protective peptide that lb comprises the amino acid sequence
Met-Asn-Thr-Thr-Thr-Thr-Thr-Thr-Ser-Arg (SEQ ID NO:96).
11. An analog or derivative of: (a) a polypeptide comprising an
amino acid sequence selected from the group consisting of amino
acid residue 20 to amino acid residue 172 of SEQ ID NO:2, amino
acid residue 22 to amino acid residue 215 of SEQ ID NO:4, amino
acid residue 15 28 to amino acid residue 258 of SEQ ID NO:6, amino
acid residue 20 to amino acid residue 364 of SEQ ID NO:8, and amino
acid residue 20 to amino acid residue 369 of SEQ ID NO:10; (b) a
polypeptide that is homologous to a polypeptide of (a); (c) a
peptide fragment of a polypeptide of (a) or (b); or (d) a fusion
protein comprising a carrier or fusion partner joined to a
polypeptide of (a) or (b), or a peptide fragment of (c).
12. An isolated polynucleotide molecule comprising a nucleotide
sequence encoding a polypeptide comprising an amino acid sequence
selected from the group consisting of amino acid residue 20 to
amino acid residue 172 of SEQ ID NO:2, amino acid residue 22 to
amino acid residue 215 of SEQ ID NO:4, amino acid residue 28 to
amino acid residue 258 of SEQ ID NO:6, amino acid residue 20 to
amino acid residue 364 of SEQ ID NO:8, and amino acid residue 20 to
amino acid residue 369 of SEQ ID NO:10.
13. The isolated polynucleotide molecule of claim 12 which encodes
the amino acid sequence of amino acid residue 20 to amino acid
residue 172 of SEQ ID NO:2, comprising the nucleotide sequence of
SEQ ID NO:1 from nt 329 to nt 790.
14. The isolated polynucleotide molecule of claim 12, encoding the
amino acid sequence of SEQ ID NO:2.
15. The isolated polynucleotide molecule of claim 14, comprising
the nucleotide sequence of SEQ ID NO:1 from nt 272 to nt 790.
16. The isolated polynucleotide molecule of claim 15, comprising
the, nucleotide sequence of SEQ ID NO:1.
17. The isolated polynucleotide molecule of claim 12 which encodes
the amino acid sequence of amino acid residue 22 to amino acid
residue 215 of SEQ ID NO:4, comprising the nucleotide sequence of
SEQ ID NO:3 from nt 439 to nt 1023.
18. The isolated polynucleotide molecule of claim 12, encoding the
amino acid sequence of SEQ ID NO:4.
19. The isolated polynucleotide molecule of claim 18, comprising
the nucleotide sequence of SEQ ID NO:3 from nt 376 to nt 1023.
20. The isolated polynucleotide molecule of claim 19, comprising
the nucleotide sequence of SEQ ID NO:3.
21. The isolated polynucleotide molecule of claim 12 which encodes
the amino acid sequence of amino acid residue 28 to amino acid
residue 258 of SEQ ID NO:6, comprising the nucleotide sequence of
SEQ ID NO:5 from nt 238 to nt 933.
22. The isolated polynucleotide molecule of claim 12, encoding the
amino acid sequence of SEQ ID NO:6.
23. The isolated polynucleotide molecule of claim 22, comprising
the nucleotide sequence of SEQ ID NO:5 from nt 157 to nt 933.
24. The isolated polynucleotide molecule of claim 23, comprising
the nucleotide sequence of SEQ ID NO:5.
25. The isolated polynucleotide molecule of claim 12 which encodes
the amino acid sequence of amino acid residue 20 to amino acid
residue 364 of SEQ ID NO:8, comprising the nucleotide sequence of
SEQ ID NO:7 from nt 671 to nt 1708.
26. The isolated polynucleotide molecule of claim 12, encoding the
amino acid sequence of SEQ ID NO:8.
27. The isolated polynucleotide molecule of claim 26, comprising
the nucleotide sequence of SEQ ID NO:7 from nt614 to nt 1708.
28. The isolated polynucleotide molecule of claim 27, comprising
the nucleotide sequence of SEQ ID NO:7.
29. The isolated polynucleotide molecule of claim 12 which encodes
the amino acid sequence of amino acid residue 20 to amino acid
residue 369 of SEQ ID NO:10, comprising the nucleotide sequence of
SEQ ID NO:9 from nt 254 to nt 1306.
30. The isolated polynucleotide molecule of claim 12, encoding the
amino acid sequence of SEQ ID NO:10.
31. The isolated polynucleotide molecule of claim 30, comprising
the nucleotide sequence of SEQ ID NO:9 from nt 197 to nt 1306.
32. The isolated polynucleotide molecule of claim 31, comprising
the nucleotide sequence of SEQ ID NO:9.
33. An isolated polynucleotide molecule that is homologous to a
polynucleotide molecule comprising a nucleotide sequence encoding
Omp20, OmpW, Omp27, OmpA1 or OmpA2 from APP.
34. An isolated polynucleotide molecule comprising a nucleotide
sequence that encodes a polypeptide that is homologous to a protein
having an amino acid sequence selected from the group consisting of
amino acid residue 20 to amino acid residue 172 of SEQ ID NO:2,
amino acid residue 22 to amino acid residue 215 of SEQ ID NO:4,
amino acid residue 28 to amino acid residue 258 of SEQ ID NO:6,
amino acid residue 20 to amino acid residue 364 of SEQ ID NO:8, and
amino acid residue 20 to amino acid residue 369 of SEQ ID
NO:10.
35. An isolated polynucleotide molecule consisting of a nucleotide
sequence which is a substantial portion of: (a) a nucleotide
sequence encoding a polypeptide comprising an amino acid sequence
selected from the group consisting of amino acid residue 20 to
amino acid residue 172 of SEQ ID NO:2, amino acid residue 22 to
amino acid residue 215 of SEQ ID NO:4, amino acid residue 28 to
amino acid residue 258 of SEQ ID NO:6, amino acid residue 20 to
amino acid residue 364 of SEQ ID NO:8, and amino acid residue 20 to
amino acid residue 369 of SEQ ID NO:10; (b) a nucleotide sequence
that is homologous to a nucleotide sequence of (a); or (c) a
nucleotide sequence encoding a polypeptide that is homologous to a
polypeptide of (a).
36. An isolated polynucleotide molecule comprising a nucleotide
sequence that encodes an amino acid sequence selected from the
group consisting of amino acid residue 1 to amino acid residue 19
of SEQ ID NO:2, amino acid residue 1 to amino acid residue 21 of
SEQ ID NO:4, amino acid residue 1 to amino acid residue 27 of SEQ
ID NO:6, amino acid residue 1 to amino acid residue 19 of SEQ ID
NO:8, and amino acid residue 1 to amino acid residue 19 of SEQ ID
NO:10.
37. The isolated polynucleotide molecule of claim 36, wherein the
nucleotide sequence is selected from the group consisting of nt 272
to nt 328 of SEQ ID NO:1, nt 376 to nt 438 of SEQ ID NO:3, nt 157
to nt 237 of SEQ ID NO:5, nt 614 to nt 670 of SEQ ID NO:7, and nt
197 to nt 253 of SEQ ID NO:1.
38. An isolated polynucleotide molecule comprising a nucleotide
sequence that encodes a fusion protein comprising a carrier or
fusion partner joined to: (a) a polypeptide comprising an amino
acid sequence selected from the group consisting of amino acid
residue 20 to amino acid residue 172 of SEQ ID NO:2, amino acid
residue 22 to amino acid residue 215 of SEQ ID NO:4, amino acid
residue 28 to amino acid residue 258 of SEQ ID NO:6, amino acid
residue 20 to amino acid residue 364 of SEQ ID NO:8, and amino acid
residue 20 to amino acid residue 369 of SEQ ID NO:10; (b) a
polypeptide that is homologous to a polypeptide of (a); or (c) a
peptide fragment of a polypeptide of (a) or (b).
39. The isolated polynucleotide molecule of claim 38, wherein the
fusion partner is selected from the group consisting of a
protective peptide, .beta.-galactosidase, trpE, maltose-binding
protein, glutathione-S-transferase, and polyhistidine.
40. The isolated polynucleotide molecule of claim 39, wherein the
fusion partner is a protective peptide that comprises the amino
acid sequence Met-Asn-Thr-Thr-Thr-Thr-Thr-Thr-Ser-Arg (SEQ ID
NO:96).
41. An oligonucleotide molecule that can hybridize under highly
stringent conditions to a polynucleotide molecule consisting of a
nucleotide sequence selected from SEQ ID NOS:1, 3, 5, 7, or 9, or
to a polynucleotide molecule consisting of a nucleotide sequence
that is the complement of a nucleotide sequence selected from SEQ
ID NOS:1, 3, 5, 7, or 9.
42. The oligonucleotide molecule of claim 41, comprising a
nucleotide sequence selected from the group consisting of SEQ ID
NOS: 15-47 and 49-93.
43. A recombinant vector, comprising the polynucleotide molecule of
claim 12 or a homologous polynucleotide molecule thereof.
44. The recombinant vector of claim 43, comprising a polynucleotide
molecule comprising a nucleotide sequence of SEQ ID NO:1 from nt
329 to nt 790 or a homologous polynucleotide molecule thereof.
45. The recombinant vector of claim 44, which is a plasmid that is
the same as plasmid pER416 present in host cells of strain Pz416
(ATCC 98926).
46. The recombinant vector of claim 43, comprising a polynucleotide
molecule comprising a nucleotide sequence of SEQ ID NO:3 from nt
439 to nt 1023 or a homologous polynucleotide molecule thereof.
47. The recombinant vector of claim 46, which is a plasmid that is
the same as plasmid pER418 present in host cells of strain Pz418
(ATCC 98928).
48. The recombinant vector of claim 43, comprising a polynucleotide
molecule comprising a nucleotide sequence of SEQ ID NO:5 from nt
238 to nt 933 or a homologous polynucleotide molecule thereof.
49. The recombinant vector of claim 48, which is a plasmid that is
the same as plasmid pER417 present in host cells of strain Pz417
(ATCC 98927).
50. The recombinant vector of claim 43, comprising a polynucleotide
molecule comprising a nucleotide sequence of SEQ ID NO:7 from nt
671 to nt 1708 or a homologous polynucleotide molecule thereof.
51. The recombinant vector of claim 50, which is a plasmid that is
the same as plasmid pER419 present in host cells of strain Pz419
(ATCC 98929).
52. The recombinant vector of claim 43, comprising a polynucleotide
molecule comprising a nucleotide sequence of SEQ ID NO:9 from nt
254 to nt 1306 or a homologous polynucleotide molecule thereof.
53. The recombinant vector of claim 52, which is a plasmid that is
the same as plasmid pER420 present in host cells of strain Pz420
(ATCC 98930).
54. A recombinant vector comprising the polynucleotide molecule of
claim 34, 35, 36 or 38.
55. A transformed host cell, comprising the recombinant vector of
claim 43.
56. A transformed host cell, comprising the recombinant vector of
claim 54.
57. A vaccine against APP, comprising an immunologically effective
amount of an antigen of the present invention selected from the
group consisting of: (a) a polypeptide comprising an amino acid
sequence selected from the group consisting of amino acid residue
20 to amino acid residue 172 of SEQ ID NO:2, amino acid residue 22
to amino acid residue 215 of SEQ ID NO:4, amino acid residue 28 to
amino acid residue 258 of SEQ ID NO:6, amino acid residue 20 to
amino acid residue 364 of SEQ ID NO:8, and amino acid residue 20 to
amino acid residue 369 of SEQ ID NO:10; (b) a polypeptide which is
homologous to the polypeptide of (a); (c) a peptide fragment of the
polypeptide of (a) or (b); (d) a fusion protein comprising a
carrier or fusion partner joined to the polypeptide of (a) or (b)
or the peptide fragment of (c); (e) an analog or derivative of the
polypeptide of (a) or (b), or the peptide fragment of (c), or the
fusion protein of (d); or (f) a polynucleotide molecule encoding
the polypeptide of (a) or (b), the peptide fragment of (c), the
fusion protein of (d), or the analog or derivative of (e); which
antigen is capable of inducing, or contributing to the induction
of, a protective response against APP in swine; and a veterinarily
acceptable carrier or diluent.
58. The vaccine of claim 57, further comprising an immunomodulatory
component.
59. The vaccine of claim 58, wherein the immunomodulatory component
is an adjuvant.
60. The vaccine of claim 57, further comprising an immunologically
effective amount of a different antigen capable of inducing, or
contributing to the induction of, a protective response against a
disease or pathological condition that can afflict swine.
61. A method of preparing a vaccine that can protect swine against
APP, comprising combining an immunologically effective amount of an
antigen of the present invention selected from the group consisting
of: (a) a polypeptide comprising an amino acid sequence selected
from the group consisting of amino acid residue 20 to amino acid
residue 172 of SEQ ID NO:2, amino acid residue 22 to amino acid
residue 215 of SEQ ID NO:4, amino acid residue 28 to amino acid
residue 258 of SEQ ID NO:6, amino acid residue 20 to amino acid
residue 364 of SEQ ID NO:8, and amino acid residue 20 to amino acid
residue 369 of SEQ ID NO:10; (b) a polypeptide which is homologous
to the polypeptide of (a); (c) a peptide fragment of the
polypeptide of (a) or (b); (d) a fusion protein comprising a
carrier or fusion partner joined to the polypeptide of (a) or (b)
or the peptide fragment of (c); (e) an analog or derivative of the
polypeptide of (a) or (b), or the peptide fragment of (c), or the
fusion protein of (d); or (f) a polynucleotide molecule encoding
the polypeptide of (a) or (b), the peptide fragment of (c), the
fusion protein of (d), or the analog or derivative of (e); which
antigen is capable of inducing, or contributing to the induction
of, a protective response against APP in swine; with a veterinarily
acceptable carrier or diluent in a form suitable for administration
to swine.
62. A method of vaccinating swine against APP, comprising
administering the vaccine of claim 57 to a pig.
63. A vaccine kit for vaccinating swine against APP, comprising a
container comprising an immunologically effective amount of one or
more antigens of the vaccine of claim 57.
64. The kit of claim 63, further comprising a second container
comprising a veterinarily acceptable carrier or diluent.
65 An isolated antibody that specifically binds to a polypeptide
comprising an amino acid sequence selected from the group
consisting of amino acid residue 20 to amino acid residue 172 of
SEQ ID NO:2, amino acid residue 22 to amino acid residue 215 of SEQ
ID NO:4, amino acid residue 28 to amino acid residue 258 of SEQ ID
NO:6, amino acid residue 20 to amino acid residue 364 of SEQ ID
NO:8, and amino acid residue 20 to amino acid residue 369 of SEQ ID
NO:10.
66. A diagnostic kit comprising a first container comprising: (a) a
polypeptide comprising an amino acid sequence selected from the
group consisting of amino acid residue 20 to amino acid residue 172
of SEQ ID NO:2, amino acid residue 22 to amino acid residue 215 of
SEQ ID NO:4, amino acid residue 28 to amino acid residue 258 of SEQ
ID NO:6, amino acid residue 20 to amino acid residue 364 of SEQ ID
NO:8, and amino acid residue 20 to amino acid residue 369 of SEQ ID
NO:10; (b) a polypeptide which is homologous to the polypeptide of
(a); (c) a peptide fragment of the polypeptide of (a) or (b); (d) a
fusion protein comprising a carrier or fusion partner joined to the
polypeptide of (a) or (b) or the peptide fragment of (c); or (e) an
analog or derivative of the polypeptide of (a) or (b), or the
peptide fragment of (c); or the fusion protein of (d); that will
specifically bind to porcine antibodies directed against an APP
antigen; and a second container comprising a secondary antibody
directed against porcine antibodies.
67. A diagnostic kit comprising a first container comprising a
primary antibody that binds to an APP protein, said APP protein
comprising an amino acid sequence selected from the group
consisting of amino acid residue 20 to amino acid residue 172 of
SEQ ID NO:2, amino acid residue 22 to amino acid residue 215 of SEQ
ID NO:4, amino acid residue 28 to amino acid residue 258 of SEQ ID
NO:6, amino acid residue 20 to amino acid residue 364 of SEQ ID
NO:8, and amino acid residue 20 to amino acid residue 369 of SEQ ID
NO:10; and a second container comprising a secondary antibody that
binds to a different epitope on the APP protein, or that is
directed against the primary antibody.
68. A diagnostic kit comprising a container comprising a
polynucleotide molecule or oligonucleotide molecule of the present
invention that can specifically hybridize to or amplify an
APP-specific polynucleotide molecule.
Description
[0001] This application claims priority from provisional
application Serial No. 60/105,285, which was filed on Oct. 22,
1998, and which is incorporated by reference in its entirety
herein.
1. FIELD OF THE INVENTION
[0002] The present invention is in the field of animal health, and
is directed to vaccines that protect swine against Actinobacillus
pleuropneumoniae. More particularly, the present invention is
directed to novel antigenic proteins shared by multiple serotypes
of A. pleuropneumoniae, DNA molecules encoding the proteins,
vaccines against APP comprising the proteins, and diagnostic
reagents.
2. BACKGROUND OF THE INVENTION
[0003] A. pleuropneumoniae (hereinafter referred to as "APP") is a
Gram negative coccobacillus recognized as one of the most important
swine pneumonic pathogens (Shope, R. E., 1964, J. Exp. Med.
119:357-368; Sebunya, T. N. K. and Saunders, J. R., 1983, J. Am.
Vet. Med. Assoc. 182:1331-1337). Twelve different serotypes have
been recognized which vary in geographic distribution (Sebunya, T.
N. K. and Saunders, J. R., 1983, above; Nielsen, R., 1985, Proc.
Am. Assoc. Swine Pract. 18-22; Nielsen, R., 1986, Acta. Vet. Scand.
27:453-455). Immune responses to vaccination against APP have been
mainly serotype-specific, suggesting that vaccine-induced immunity
is directed to serotype-specific capsular antigens (MacInnes, J. I.
and Rosendal, S., 1988, Can. Vet. J. 29:572-574; Fedorka-Cray, P.
J., et al., 1994, Comp. Cont. Educ. Pract. Vet. 16:117-125;
Nielsen, R., 1979, Nord. Vet. Med. 31:407-413; Rosendal, S., et
al., 1986; Vet. Microbiol. 12:229-240).
[0004] In contrast, natural immunity to any one serotype seems to
confer significant protection from disease caused by other
serotypes, suggesting that natural exposure induces cross-reactive
immunity to shared antigens (Sebunya, T. N. K. and Saunders, J. R.,
1983, above; MacInnes, J. I. and Rosendal, S., 1988, above;
Fedorka-Cray, P. J., et al., 1994, above; Nielsen, R., 1979, above;
and Rosendal, S.,.et al., 1986, above).
[0005] Virulence factors that might contribute to cross-protection
have been proposed as possible vaccine candidates, including
exotoxins (Apx) (Nakai, T., et al., 1983, Am. J. Vet. Res.
44:344-347; Frey, J., et al., 1993, J. Gen. Microbiol.
139:1723-1728; Fedorka-Cray, P. J., et al., 1993, Vet. Microbiol.
37:85-100); capsular antigens (Rosendal, S., et al., 1986, above);
outer-membrane proteins (OMP) (Denee, H. and Potter, A., 1989,
Infect. Immune 57:798-804; Niven, D. F., et al., 1989, Mol.
Microbiol. 3:1083-1089; Gonzalez, G., et al., 1990, Mol. Microbiol.
4:1173-1179; Gerlach, G. F., et al., 1992, Infect. Immun.
60:3253-3261); and lipopolysaccharides (LPS) (Fenwick, B. W. and
Osborn, B.,. 1986, Infect. Immun. 54:575-582). However, the
patterns of cross-reactivity/cross-protection induced by such
components do not cover all twelve APP serotypes. In addition,
immunization with isolated individual components or combinations of
individual components from APP have so far failed to confer
protection from challenge with some heterologous serotypes
(unpublished observations). Thus, it can be postulated that the
cross-protective responses induced by natural infection are limited
to specific serotype clusters.
[0006] Alternatively, it is possible that some of the antigens
responsible for the cross-protection observed after natural
infection have not yet been identified. Most studies regarding APP
antigens have focused on the characterization of immunodominant
antigens detected in convalescent serum using antibodies. Such an
approach does not allow the identification of possible differences
between the antibody specificities represented during primary
versus secondary responses, nor the identification of dominant
specificities at the infection site that are likely to be
responsible for protection upon secondary encounter with the
pathogen.
[0007] It is generally accepted that lymphocytes are educated
during primary infections so that when there is secondary exposure
to a pathogen the host is better able to prevent disease (MacKay,
C. R., 1993, Adv. Immunol. 53:217-240). Memory cells responsible
for this activity (antigen-experienced T and B lymphocytes) persist
for long periods of time, and are capable of reactivation following
an appropriate subsequent encounter with the antigen. In contrast
to naive cells, they generally show a faster response time,
specialized tissue localization, and more effective antigen
recognition and effector functions (MacKay, C. R., 1993, above;
Linton, P. and Klinman, N. R., 1992, Sem. Immun. 4:3-9; Meeusen, E.
N. T., et al., 1991, Eur. J. Immunol. 21:2269-2272).
[0008] During the generation of a secondary response, the frequency
of precursor cells capable of responding to the particular antigen
is higher than that present during the primary response.
Trafficking patterns of memory cell subsets following secondary
responses are also different from those of naive cells. Naive cells
migrate relatively homogeneously to secondary lymphoid tissues, but
they home poorly to non-lymphoid tissues. By contrast, memory cells
display heterogeneous trafficking and, in some instances, migration
has been shown to be restricted to certain secondary lymphoid
tissues and non-lymphoid sites (MacKay, C. R., 1993, above; Gray,
D., 1993, Ann. Rev. Immunol. 11:49-77; Picker, L. S., et at., 1993,
J. Immunol. 150:1122-1136). Studies in both rodents and sheep have
indicated that lymphocytes from the gut preferentially migrate back
to the gut, whereas cells draining from the skin or from lymph
nodes preferentially migrate back to the skin or lymph nodes (Gray,
D., 1993, above; Picker, L. S., et al., 1993, above). Thus, upon
secondary encounter with a pathogen, specific effector cells for
cell-mediated immunity and antibody secretion can home to infection
sites and local lymph nodes more effectively (Meeusen, E. N. T., et
al., 1991, above). As a result, infiltrating lymphocytes will
rapidly proliferate and their specificities will predominate during
early stages of re-infection.
[0009] Recovery of local B cells from tissues and draining lymph
nodes early after re-infection has allowed some researchers to
obtain antibodies with a narrower specificity range (Meeusen, E. N.
T. and Brandon, M., 1994, J. Immunol. Meth. 172:71-76). Such
antibodies have been successfully used to identify potential
protective antigens to several pathogens (Meeusen, E. N. T. and
Brandon, M., 1994, above; Meeusen, E. N. T. and Brandon, M., 1994,
Eur. J. Immunol. 24:469-474; Bowles, V. M., et at., 1995, Immunol.
84:669-674). The invention disclosed herein below is based on a
modification of this approach, in which antibody-secreting cell
(ASC) probes were recovered that were associated with local memory
responses elicited after homologous and heterologous APP challenge.
Antibodies obtained from bronchial lymph node (BLN) cultures after
heterologous challenge recognized four previously unrecognized
proteins present in all twelve APP serotypes. Partial amino acid
sequences for each protein were obtained and used to generate PCR
primers that allowed the identification of five novel APP proteins
and polynucleotide molecules that encode them.
3. SUMMARY OF THE INVENTION
[0010] The present invention provides five novel, low molecular
weight proteins isolated from APP, which are designated herein,
respectively, as "Omp20," "OmpW," "Omp27," "OmpA1," and "OmpA2".
These "APP proteins" and the polynucleotide molecules that encode
them are useful either as antigenic components in a vaccine to
protect swine against APP, or as diagnostic reagents to identify
swine that are, or have been, infected with APP, or that have been
vaccinated with a vaccine of the present invention.
[0011] The amino acid sequence of Omp20 is encoded by the
Omp20-encoding ORF of plasmid pER416 which is present in host cells
of strain Pz416 (ATCC 98926), and its deduced amino acid sequence
is presented as SEQ ID NO:2, which comprises a signal sequence from
amino acid residues 1 to 19. The amino acid sequence of OmpW is
encoded by the OmpW-encoding ORF of plasmid pER418 which is present
in host cells of strain Pz418 (ATCC 98928); and its deduced amino
acid sequence is presented as SEQ ID NO:4, which comprises a signal
sequence from amino acid residues 1 to 21. The amino acid sequence
of Omp27 is encoded by the Omp27-encoding ORF of plasmid pER417
which is present in host cells of strain Pz417 (ATCC 98927), and
its deduced amino acid sequence is presented as SEQ ID NO:6, which
comprises a signal sequence from amino acid residues 1 to 27. The
amino acid sequence of OmpA1 is encoded by the OmpA1-encoding ORF
of plasmid pER419 which is present in host cells of strain Pz419
(ATCC 98929), and its deduced amino acid sequence is presented as
SEQ ID NO:8, which comprises a signal sequence from amino acid
residues 1 to 19. The amino acid sequence of OmpA2 is encoded by
the OmpA2-encoding ORF of plasmid pER420 which is present in host
cells of strain Pz420 (ATCC 98930), and its deduced amino acid
sequence is presented as SEQ ID NO:10, which comprises a signal
sequence from amino acid residues 1 to 19. Each of these APP
proteins, in substantially purified form, is provided by the
present invention.
[0012] The present invention further provides substantially
purified polypeptides that are homologous to any of the
aforementioned APP proteins of the present invention. The present
invention further provides peptide fragments of any of the APP
proteins or homologous polypeptides of the present invention. The
present invention further provides fusion proteins comprising an
APP protein, homologous polypeptide, or peptide fragment of the
present invention joined to a carrier or fusion partner. The
present invention further provides analogs and derivatives of an
APP protein, homologous polypeptide, peptide fragment, or fusion
protein of the present invention.
[0013] The present invention further provides an isolated
polynucleotide molecule comprising a nucleotide sequence encoding
the APP protein, Omp20, with or without signal sequence. In a
preferred embodiment, the isolated Omp20-encoding polynucleotide
molecule of the present invention comprises the nucleotide sequence
of SEQ ID NO:1 from about nt 329 to about nt 790. In a more
preferred embodiment, the isolated Omp20-encoding polynucleotide
molecule of the present invention comprises the nucleotide sequence
of SEQ ID NO:1 from about nt 272 to about nt 790.
[0014] The present invention further provides an isolated
polynucleotide molecule comprising a nucleotide sequence encoding
the APP protein, OmpW, with or without signal sequence. In a
preferred embodiment, the isolated OmpW-encoding polynucleotide
molecule of the present invention comprises the nucleotide sequence
of SEQ ID NO:3 from about nt 439 to about nt 1023. In a more
preferred embodiment, the isolated OmpW-encoding polynucleotide
molecule of the present invention comprises the nucleotide sequence
of SEQ ID NO:3 from about nt 376 to about nt 1023.
[0015] The present invention further provides an isolated
polynucleotide molecule comprising a nucleotide sequence encoding
the APP protein, Omp27, with or without signal sequence. In a
preferred embodiment, the isolated Omp27-encoding polynucleotide
molecule of the present invention comprises the nucleotide sequence
of SEQ ID NO:5 from about nt 238 to about nt 933. In a more
preferred embodiment, the isolated Omp27-encoding polynucleotide
molecule of the present invention comprises the nucleotide sequence
of SEQ ID NO:5 from about nt 157 to about nt 933.
[0016] The present invention further provides an isolated
polynucleotide molecule comprising a nucleotide sequence encoding
the APP protein, OmpA1, with or without signal sequence. In a
preferred embodiment, the isolated OmpA1-encoding polynucleotide
molecule of the present invention comprises the nucleotide sequence
of SEQ ID NO:7 from about nt 671 to about nt 1708. In a more
preferred embodiment, the isolated OmpA1-encoding polynucleotide
molecule of the present invention comprises the nucleotide sequence
of SEQ ID NO:7 from about nt 614 to about nt 1708.
[0017] The present invention further provides an isolated
polynucleotide molecule comprising a nucleotide sequence encoding
the APP protein, OmpA2, with or without signal sequence. In a
preferred embodiment, the isolated OmpA2-encoding polynucleotide
molecule of the present invention comprises the nucleotide sequence
of SEQ ID NO:9 from about nt 254 to about nt 1306. In a more
preferred embodiment, the isolated OmpA2-encoding polynucleotide
molecule of the present invention comprises the nucleotide sequence
of SEQ ID NO:9 from about nt 197 to about nt 1306.
[0018] The present invention further provides an isolated
polynucleotide molecule that is homologous to any of the
aforementioned polynucleotide molecules of the present invention.
The present invention further provides an isolated polynucleotide
molecule comprising a nucleotide sequence that encodes a
polypeptide that is homologous to any of the APP proteins of the
present invention. The present invention further provides an
isolated polynucleotide molecule consisting of a nucleotide
sequence that is a substantial portion of any of the aforementioned
polynucleotide molecules of the present invention. In a
non-limiting embodiment, the substantial portion of a
polynucleotide molecule of the present invention encodes a peptide
fragment of an APP protein or homologous polypeptide of the present
invention. The present invention further provides a polynucleotide
molecule comprising a nucleotide sequence that encodes a fusion
protein comprising an APP protein, homologous polypeptide, or
peptide fragment of the present invention joined to a carrier or
fusion partner.
[0019] The present invention further provides oligonucleotide
molecules that are useful as primers for amplifying any of the
polynucleotide molecules of the present invention, or as diagnostic
reagents. Specific though non-limiting embodiments of such
oligonucleotide molecules include oligonucleotide molecules having
nucleotide sequences selected from the group consisting of any of
SEQ ID NOS:1547 and 49-93.
[0020] The present invention further provides compositions and
methods for cloning and expressing any of the polynucleotide
molecules of the present invention, including recombinant cloning
vectors and recombinant expression vectors comprising a
polynucleotide molecule of the present invention, host cells
transformed with any of said vectors, and cell lines derived
therefrom.
[0021] The present invention further provides a
recombinantly-expressed APP protein, homologous polypeptide,
peptide fragment, or fusion protein encoded by a polynucleotide
molecule of the present invention.
[0022] The present invention further provides a vaccine for
protecting swine against APP, comprising an immunologically
effective amount of one or more antigens of the present invention
selected from the group consisting of an APP protein, homologous
polypeptide, peptide fragment, fusion protein, analog, derivative,
or polynucleotide molecule of the present invention capable of
inducing, or contributing to the induction of, a protective
response against APP in swine; and a veterinarily acceptable
carrier or diluent. The vaccine of the present invention can
further comprise an adjuvant or other immunomodulatory component.
In a non-limiting embodiment, the vaccine of the present invention
can be a combination vaccine for protecting swine against APP and,
optionally, one or more other diseases or pathological conditions
that can afflict swine, which combination vaccine has a first
component comprising an immunologically effective amount of one or
more antigens of the present invention selected from the group
consisting of an APP protein, homologous polypeptide, peptide
fragment, fusion protein, analog, derivative, or polynucleotide
molecule of the present invention capable of inducing, or
contributing to the induction of, a protective response against APP
in swine; a second component comprising an immunologically
effective amount of a different antigen capable of inducing, or
contributing to the induction of, a protective response against a
disease or pathological condition that can afflict swine; and a
veterinarily acceptable carrier or diluent.
[0023] The present invention further provides a method of preparing
a vaccine that can protect swine against APP, comprising combining
an immunologically effective amount of one or more antigens of the
present invention selected from the group consisting of an APP
protein, homologous polypeptide, peptide fragment, fusion protein,
analog, derivative, or polynucleotide molecule of the present
invention capable of inducing, or contributing to the induction of,
a protective response against APP in swine, with a veterinarily
acceptable carrier or diluent, in a form suitable for
administration to swine.
[0024] The present invention further provides a method of
vaccinating swine against APP, comprising administering a vaccine
of the present invention to a pig.
[0025] The present invention further provides a vaccine kit for
vaccinating swine against APP, comprising a container comprising an
immunologically effective amount of one or more antigens of the
present invention selected from the group consisting of an APP
protein, homologous polypeptide, peptide fragment, fusion protein,
analog, derivative, or polynucleotide molecule of the present
invention capable of inducing, or contributing to the induction of,
a protective response against APP in swine. The kit can further
comprise a second container comprising a veterinarily acceptable
carrier or diluent.
[0026] The present invention further provides antibodies that
specifically bind to an APP protein of the present invention.
[0027] The present invention further provides diagnostic kits. In a
non-limiting embodiment, the diagnostic kit comprises a first
container comprising an APP protein, homologous polypeptide,
peptide fragment, fusion protein, analog, or derivative of the
present invention that will specifically bind to porcine antibodies
directed against an APP protein; and a second container comprising
a secondary antibody directed against the porcine anti-APP
antibodies. The secondary antibody preferably comprises a
detectable label. Such a diagnostic kit is useful to detect pigs
that currently are, or have previously been, infected with APP, or
that have seroconverted as a result of vaccination with a vaccine
of the present invention. In a different non-limiting embodiment,
the diagnostic kit comprises a first container comprising a primary
antibody that binds to an APP protein of the present invention; and
a second container comprising a secondary antibody that binds to a
different epitope on the APP protein, or that binds to the primary
antibody. The secondary antibody preferably comprises a detectable
label. In a different non-limiting embodiment, the diagnostic kit
comprises a container comprising a polynucleotide molecule or
oligonucleotide molecule of the present invention that is useful to
specifically amplify an APP-specific polynucleotide molecule of the
present invention. These latter two diagnostic kits are useful to
detect pigs that are currently infected with APP.
4. BRIEF DESCRIPTION OF THE DRAWINGS
[0028] FIG. 1. Western blot analysis of antibodies present in (a)
serum, and (b) bronchial lymph node (BLN) tissue explant
supernatants from pig No. 803 challenged with APP serotype-5 and
heterologously rechallenged with APP serotype-7. All BLN tissue
explant supernatants collected after 24 or 48 hr of incubation
contained antibodies that specifically recognized APP proteins. The
antibodies from the BLN tissue explant supernatants highlighted
several low molecular weight proteins present in APP serotypes-1,
5, and 7.
[0029] FIG. 2. Western blot analysis of cross-reactivity of
antibodies present in BLN tissue explant supernatants from pig No.
803 against whole bacterial cell antigens from each of the twelve
different APP serotypes, demonstrating that at least three of the
low molecular weight proteins recognized by antibodies induced by
heterologous rechallenge were present in all twelve APP serotypes.
Antibodies present in this particular BLN supernatant also
recognized other protein bands.
[0030] FIG. 3. Western blot analysis demonstrating that reactivity
of antibodies in BLN tissue explant supernatants from pig No. 803
against the low molecular weight proteins is restricted to proteins
present in the cell pellets (cells) rather than bacterial cell
supernatants (Sups).
[0031] FIG. 4. Western blot analysis of reactivity of (a) BLN
tissue explant supernatant and (b) serum from pig No. 803, against
proteins purified from APP serotype-7 by continuous flow
electrophoresis. Four protein bands with molecular weights of about
19-20, about 23, about 27, and about 29 kDa, respectively, were
identified using this procedure.
[0032] FIG. 5. Alignment of deduced amino acid sequences of APP
OmpA1 and APP OmpA2 proteins. The two proteins share 73.1% amino
acid identity.
[0033] FIG. 6. Alignment of OmpW protein from Vibrio cholerae and
OmpW protein from APP. The two proteins share 44.9% amino acid
identity.
5. DETAILED DESCRIPTION OF THE INVENTION
5.1. Novel Proteins Shared by Multiple APP Serotypes
[0034] The present invention is based on the discovery of five
novel, low molecular weight proteins from APP (referred to
hereinafter as "APP proteins"). These APP proteins are designated
herein, respectively, as "Omp20," "OmpW," "Omp27," "OmpA1," and
"OmpA2."
[0035] The amino acid sequence of Omp20 is encoded by the
Omp20-encoding ORF of plasmid pER416 which is present in host cells
of strain Pz416 (ATCC 98926). The deduced amino acid sequence of
Omp20 is presented as SEQ ID NO:2. The first 19 amino acids of the
protein shown in SEQ ID NO:2 represent a signal sequence, and the
present invention encompasses both an Omp20 protein having only
amino acid residues 20 to 172 of SEQ ID NO:2 (i.e., lacking the
signal sequence), and an Omp20 protein having the sequence of SEQ
ID NO:2 (i.e., including the signal sequence). The present
invention thus provides a substantially purified protein comprising
the amino acid sequence of amino acid residue 20 to amino acid
residue 172 of SEQ ID NO:2. The present invention further provides
a substantially purified protein comprising the amino acid sequence
of SEQ ID NO:2.
[0036] The amino acid sequence of OmpW is encoded by the
OmpW-encoding ORF of plasmid pER418 which is present in host cells
of strain Pz418 (ATCC 98928). The deduced amino acid sequence of
OmpW is presented as SEQ ID NO:4. The first 21 amino acids of the
protein shown in SEQ ID NO:4 represent a signal sequence, and the
present invention encompasses both an OmpW protein having only
amino acid residues 22 to 215 of SEQ ID NO:4 (i.e., lacking the
signal sequence), and an OmpW protein having the sequence of SEQ ID
NO:4 (i.e., including the signal sequence). The present invention
thus provides a substantially purified protein comprising the amino
acid sequence of amino acid residue 22 to amino acid residue 215 of
SEQ ID NO:4. The present invention further provides a substantially
purified protein comprising the amino acid sequence of SEQ ID
NO:4.
[0037] The amino acid sequence of Omp27 is encoded by the
Omp27-encoding ORF of plasmid pER417 which is present in host cells
of strain Pz417 (ATCC 98927). The deduced amino acid sequence of
Omp27 is presented as SEQ ID NO:6. The first 27 amino acids of the
protein shown in SEQ ID NO:6 represent a signal sequence, and the
present invention encompasses both an Omp27 protein having only
amino acid residues 28 to 258 of SEQ ID NO:6 (i.e., lacking the
signal sequence), and an Omp27 protein having the sequence of SEQ
ID NO:6 (i.e., including the signal sequence). The present
invention thus provides a substantially purified protein comprising
the amino acid sequence of amino acid residue 28 to amino acid
residue 258 of SEQ ID NO:6. The present invention further provides
a substantially purified protein comprising the amino acid sequence
of SEQ ID NO:6.
[0038] The amino acid sequence of OmpA1 is encoded by the
OmpA1-encoding ORF of plasmid pER419 which is present in host cells
of strain Pz419 (ATCC 98929). The deduced amino acid sequence of
OmpA1 is presented as SEQ ID NO:8. The first 19 amino acids of the
protein shown in SEQ ID NO:8 represent a signal sequence, and the
present invention encompasses both an OmpA1 protein having only
amino acid residues 20 to 364 of SEQ ID NO:8 (i.e., lacking the
signal sequence), and an OmpA1 protein having the sequence of SEQ
ID NO:8 (i.e., including the signal sequence). The present
invention thus provides a substantially purified protein comprising
the amino acid sequence of amino acid residue 20 to amino acid
residue 364 of SEQ ID NO:8. The present invention further provides
a substantially purified protein comprising the amino acid sequence
of SEQ ID NO:8.
[0039] The amino acid sequence of OmpA2 is encoded by the
OmpA2-encoding ORF of plasmid pER420 which is present in host cells
of strain Pz420 (ATCC 98930). The deduced amino acid sequence of
OmpA2 is presented as SEQ ID NO:10. The first 19 amino acids of the
protein shown in SEQ ID NO:10 represent a signal sequence, and the
present invention encompasses both an OmpA2 protein having only
amino acid residues 20 to 369 of SEQ ID NO:10 (i.e., lacking the
signal sequence), and an OmpA2 protein having the sequence of SEQ
ID NO:10 (i.e., including the signal sequence). The present
invention thus provides a substantially purified protein comprising
the amino acid sequence of amino acid residue 20 to amino acid
residue 369 of SEQ ID NO:10. The present invention further provides
a substantially purified protein comprising the amino acid sequence
of SEQ ID NO:10.
[0040] The APP proteins of the present invention, i.e., Omp20,
OmpW, Omp27, OmpA1, and OmpA2, have molecular weights of about
19-20, about 23, about 27, about 29 and about 29 kDa, respectively,
as based on their electrophoretic mobility; and about 20, about 23,
about 27, about 35 and about 35 kDa, respectively, as based on
their deduced amino acid sequences without signal sequences.
[0041] The present invention further provides polypeptides that are
homologous to an APP protein of the present invention. As used
herein to refer to polypeptides, the term "homologous" refers to a
polypeptide otherwise having an amino acid sequence selected from
the group of amino acid sequences consisting of SEQ ID NOS: 2, 4,
6, 8, and 10, or those same amino acid sequences but without their
native signal sequences, in which one or more amino acid residues
have been conservatively substituted with a different amino acid
residue, wherein the homologous polypeptide has an amino acid
sequence that has about 70%, more preferably about 80%, and most
preferably about 90% sequence identity, as determined by any
standard amino acid sequence analysis algorithm (such as one of the
BlastP algorithms of GENBANK), to a polypeptide having an amino
acid sequence selected from the group of amino acid sequences
consisting of SEQ ID NOS: 2, 4, 6, 8, and 10, and wherein the
resulting homologous polypeptide is useful in practicing the
present invention. Conservative amino acid substitutions are
well-known in the art. Rules for making such substitutions include
those described by Dayhof, M. D., 1978, Nat. Biomed. Res. Found.,
Washington, D.C., Vol. 5, Sup. 3, among others. More specifically,
conservative amino acid substitutions are those that generally take
place within a family of amino acids that are related in acidity or
polarity. Genetically encoded amino acids are generally divided
into four groups: (1) acidic=aspartate, glutamate; (2)
basic=lysine, arginine, histidine; (3) non-polar=alanine, valine,
leucine, isoleucine, proline, phenylalanine, methionine,
tryptophan; and (4) uncharged polar=glycine, asparagine, glutamine,
cysteine, serine, threonine, tyrosine. Phenylalanine, tryptophan
and tyrosine are also jointly classified as aromatic amino acids.
One or more replacements within any particular group, e.g., of a
leucine with an isoleucine or valine, or of an aspartate with a
glutamate, or of a threonine with a serine, or of any other amino
acid residue with a structurally related amino acid residue, e.g.,
an amino acid residue with similar acidity, polarity, or with
similarity in some combination thereof, will generally have an
insignificant effect on the function or immunogenicity of the
polypeptide.
[0042] As used herein, a polypeptide is "useful in practicing the
present invention" where the polypeptide: (a) is immunogenic, i.e.,
can be used in a vaccine composition, either alone to induce a
protective response in swine against APP, or in combination with
other antigens of the present invention to contribute to the
induction of a protective response in swine against APP; or (b) can
be used to induce the production of APP-specific antibodies when
administered to a member of a mammalian species, which antibodies
are useful as diagnostic reagents; or (c) can be used as a
diagnostic reagent to detect the presence of anti-APP antibodies in
a blood or serum sample from a pig resulting either from infection
with APP or vaccination with a vaccine of the present
invention.
[0043] The present invention further provides peptide fragments of
an APP protein or homologous polypeptide of the present invention.
As used herein, a "peptide fragment" means a polypeptide consisting
of less than the complete amino acid sequence of the corresponding
full-length APP protein, either with or without signal sequence, or
homologous polypeptide thereof, but comprising a sub-sequence of at
least about 10, more preferably at least about 20, and most
preferably at least about 30 amino acid residues of the amino acid
sequence thereof, and that is useful in practicing the present
invention, as usefulness is defined above for polypeptides. A
peptide fragment of the present invention can comprise more than
one sub-sequence of a full-length APP protein or homologous
polypeptide of the present invention. For example, two or more
different sub-sequences from the full-length APP protein or
homologous polypeptide can be brought together and made contiguous
to each other in the peptide fragment where they were
non-contiguous in the APP protein or homologous polypeptide. In a
preferred embodiment, a peptide fragment of the present invention
comprises one or more sub-sequences representing one or more
epitopes of the APP protein or homologous polypeptide, or multiple
copies of an epitope, against which antibodies can be raised.
[0044] In a non-limiting embodiment, the present invention provides
a peptide fragment of an APP protein of the present invention,
which peptide fragment comprises the native signal sequence of the
APP protein. In a preferred embodiment, the peptide fragment
consists of an amino acid sequence selected from the group
consisting of amino acid residue 1 to amino acid residue 19 of SEQ
ID NO:2 (Omp20), amino acid residue 1 to amino acid residue 21 of
SEQ ID NO:4 (OmpW), amino acid residue 1 to amino acid residue 27
of SEQ ID NO:6 (Omp27), amino acid residue 1 t6 amino acid residue
19 of SEQ ID NO:8 (OmpA1), and amino acid residue 1 to amino acid
residue 19 of SEQ ID NO:10 (OmpA1). Such signal sequences, and the
polynucleotide molecules that encode-them, are useful for a variety
of purposes, including to direct the cellular trafficking of
recombinant proteins expressed in APP or other bacterial host
cells, or as diagnostic probes for detecting an APP-specific
polynucleotide molecule in a fluid or tissue sample from an
infected animal.
[0045] The present invention further provides full-length APP
proteins or homologous polypeptides in which sub-sequences thereof
are arranged in a different relative order to each other compared
to that found in the native molecule so as to increase, alter, or
otherwise improve the antigenicity of the polypeptide.
[0046] As used herein, the terms "antigen," "antigenic," and the
like, refer to a molecule containing one or more epitopes that
stimulate a host's immune system to make a humoral and/or cellular
antigen-specific response. The term is also used interchangeably
with "immunogen." As used herein, the term "epitope" or "epitopic
region" refers to a site on an antigen or hapten to which a
specific antibody molecule binds. The term is also used
interchangeably with "antigenic determinant."
[0047] The present invention further provides fusion proteins
comprising an APP protein partner with or without its native signal
sequence, a homologous polypeptide thereof, or a peptide fragment
of the present invention, joined to a carrier or fusion partner,
which fusion proteins are useful in practicing the present
invention, as usefulness is defined above for polypeptides. See
Section 5.4.1. below for examples of fusion partners. Fusion
proteins are useful for a variety of reasons, including to increase
the stability of recombinantly-expressed APP proteins, as antigenic
components in an APP vaccine, to raise antisera against the
particular APP protein partner, to study the biochemical properties
of the APP protein partner, to engineer APP proteins with different
or enhanced antigenic properties, to serve as diagnostic reagents
or to aid in the identification or purification of the expressed
APP protein partner as described, e.g., in Section 5.4.1 below.
[0048] Fusion proteins of the present invention can be further
engineered using standard techniques to contain specific protease
cleavage sites so that the particular APP protein partner can be
released from the carrier or fusion partner by treatment with a
specific protease. For example, a fusion protein of the present
invention can comprise a thrombin or factor Xa cleavage site, among
others.
[0049] The present invention further provides analogs and
derivatives of an APP protein, homologous polypeptide, peptide
fragment or fusion protein of the present invention, where such
analogs and derivatives are useful in practicing the present
invention, as usefulness is defined above for polypeptides.
Manipulations that result in the production of analogs can be
carried out either at the gene level or at the protein level, or
both, to improve or otherwise alter the biological or immunological
characteristics of the particular polypeptide from which the analog
is prepared. For example, at the gene level, a cloned DNA molecule
encoding an APP, protein of the present invention can be modified
by one or more known strategies to encode an analog of that
protein. Such modifications include, but are not limited to,
endonuclease digestion, and mutations that create or destroy
translation, initiation or termination sequences, or that create
variations in the coding region, or a combination thereof. Such
techniques are described, among other places, in Maniatis et al.,
1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y.;. Ausubel et al., 1989,
Current Protocols In Molecular Biology, Greene Publishing
Associates & Wiley Interscience, NY; Sambrook et al., 1989,
Molecular, Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y.; Innis et al. (eds),
1995, PCR Strategies, Academic Press, Inc., San Diego; and Erlich
(ed), 1992, PCR Technology, Oxford University Press, New York, all
of which are incorporated herein by reference.
[0050] Alternatively or additionally, an analog of the present
invention can be prepared by modification of an APP protein or
other polypeptide of the present invention at the protein level.
One or more chemical modifications of the protein can be carried
out using known techniques, including but not limited to one or
more of the following: substitution of one or more L-amino acids of
the protein with corresponding D-amino acids, amino acid analogs,
or amino acid mimics, so as to produce, e.g., carbazates or
tertiary centers; or specific chemical modification, such as
proteolytic cleavage with, e.g., trypsin, chymotrypsin, papain or
V8 protease, or treatment with NaBH.sub.4 or cyanogen bromide, or
acetylation, formylation, oxidation or reduction, etc.
[0051] An APP protein or other polypeptide of the present invention
can be derivatized by conjugation thereto of one or more chemical
groups, including but not limited to acetyl groups, sulfur bridging
groups, glycosyl groups, lipids, and phosphates, and/or a second
APP protein or other polypeptide of the present invention, or
another protein, such as, e.g., serum albumin, keyhole limpet
hemocyanin, or commercially activated BSA, or a polyamino acid
(e.g., polylysine), or a polysaccharide, (e.g., sepharose, agarose,
or modified or unmodified celluloses), among others. Such
conjugation is preferably by covalent linkage at amino acid side
chains and/or at the N-terminus or C-terminus of the APP protein.
Methods for carrying out such conjugation reactions are well-known
in the field of protein chemistry.
[0052] Derivatives useful in practicing the claimed invention also
include those in which a water-soluble polymer, such as, e.g.,
polyethylene glycol, is conjugated to an APP protein or other
polypeptide of the present invention, or to an analog thereof,
thereby providing additional desirable properties while retaining,
at least in part, or improving the immunogenicity of the APP
protein. These additional desirable properties include, e.g.,
increased solubility in aqueous solutions, increased stability in
storage, increased resistance to proteolytic degradation, and
increased in vivo half-life. Water-soluble polymers suitable for
conjugation to an APP protein or other polypeptide of the present
invention include but are not limited to polyethylene glycol
homopolymers, polypropylene glycol homopolymers, copolymers of
ethylene glycol with propylene glycol, wherein said homopolymers
and copolymers are unsubstituted or substituted at one end with an
alkyl group, polyoxyethylated polyols, polyvinyl alcohol,
polysaccharides, polyvinyl ethyl ethers, and
.alpha.,.beta.-poly[2-hydroxyethyl]-DL-aspartamide. Polyethylene
glycol is particularly preferred. Methods for making water-soluble
polymer conjugates of polypeptides are known in the art and are
described in, among other places, U.S. Pat. No. 3,788,948; U.S.
Pat. No. 3,960,830; U.S. Pat. No. 4,002,531; U.S. Pat. No.
4,055,635; U.S. Pat. No. 4,179,337; U.S. Pat. No. 4,261,973; U.S.
Pat. No. 4,412,989; U.S. Pat. No. 4,414,147; U.S. Pat. No.
4,415,665; U.S. Pat. No. 4,609,546; U.S. Pat. No. 4,732,863; U.S.
Pat. No. 4,745,180; European Patent (EP) 152,847; EP 98,110; and
Japanese Patent (JP) 5,792,435, which patents are incorporated
herein by reference.
[0053] All subsequent references to "APP proteins" and the like are
intended to include the APP proteins, homologous polypeptides,
peptide fragments, fusion proteins, analogs, and derivatives of the
present invention, as these terms are defined above, unless
otherwise indicated.
5.2. Polynucleotide Molecules Encoding Novel APP Proteins
[0054] The present invention further provides isolated
polynucleotide molecules comprising a nucleotide sequence encoding
an APP protein. As used herein, the terms "polynucleotide
molecule," "polynucleotide sequence," "coding sequence,"
"open-reading frame (ORF)," and the like, are intended to refer to
both DNA and RNA molecules, which can either be single-stranded or
double-stranded; that can include one or more prokaryotic
sequences, cDNA sequences, genomic DNA sequences (including exons
and introns), or chemically synthesized DNA and RNA sequences; and
that can include both sense and anti-sense strands. As used herein,
the term "ORF" refers to the minimal nucleotide sequence required
to encode a particular APP protein of the present invention without
any intervening termination codons. The boundaries of the
polynucleotide coding sequence are generally determined by the
presence of a start codon at the 5' (amino) terminus and a
translation stop codon at the 3' (carboxy) terminus.
[0055] Production and manipulation of the polynucleotide molecules
and oligonucleotide molecules disclosed herein below are within the
skill in the art and can be carried out according to recombinant
techniques described, among other places, in Maniatis et al., 1989,
above; Ausubel et a., 1989, above; Sambrook et a., 1989, above;
Innis et al., 1995, above; and Erlich, 1992, above.
5.2.1. Polynucleotide Molecules Encoding Omp20
[0056] References herein below to nucleotide sequences from SEQ ID
NO:1, and to selected and substantial portions thereof, are
intended to also refer to the corresponding Omp20-related
nucleotide sequences of plasmid pER416 which is present in host
cells of strain Pz416 (ATCC 98926), unless otherwise indicated. In
addition, references herein below to the amino acid sequences shown
in SEQ ID NO:2, and to peptide fragments thereof, are intended to
also refer to the corresponding amino acid sequences encoded by the
Omp20-related nucleotide sequence of plasmid pER416, unless
otherwise indicated.
[0057] The present invention provides an isolated polynucleotide
molecule comprising a nucleotide sequence encoding the APP protein,
Omp20, with or without signal sequence. In a preferred embodiment,
the isolated Omp20-encoding polynucleotide molecule of the present
invention comprises the nucleotide sequence of SEQ ID NO:1 from nt
329 to nt 790. In a more preferred embodiment, the isolated
Omp20-encoding polynucleotide molecule of the present invention
comprises the nucleotide sequence of SEQ ID NO:1 from nt 272 to nt
790. In a non-limiting embodiment, the isolated Omp20-encoding
polynucleotide molecule of the present invention comprises the
nucleotide sequence of SEQ ID NO:1.
[0058] The present invention further provides an isolated
polynucleotide molecule that is homologous to an APP Omp20-encoding
polynucleotide molecule of the present invention. The term
"homologous" when used to refer to an Omp20-encoding polynucleotide
molecule means a polynucleotide molecule having a nucleotide
sequence: (a) that encodes the same amino acid sequence as the
nucleotide sequence of SEQ ID NO:1 from nt 329 to nt 790, but that
includes one or more silent changes to the nucleotide sequence
according to the degeneracy of the genetic code; or (b) that
hybridizes to the complement of a polynucleotide molecule having a
nucleotide sequence that encodes amino acid residues 20 to 172 of
SEQ ID NO:2 under moderately stringent conditions, i.e.,
hybridization to filter-bound DNA in 0.5 M NaHPO.sub.4, 7% sodium
dodecyl sulfate (SDS), 1 mM EDTA at 65.degree. C., and washing in
0.2.times.SSC/0.1% SDS at 42.degree. C. (see Ausubel et al. (eds.),
1989, Current Protocols in Molecular Biology, Vol. I, Green
Publishing Associates, Inc., and John Wiley & Sons, Inc., New
York, at p. 2.10.3), and that is useful in practicing the present
invention. In a preferred embodiment, the homologous polynucleotide
molecule hybridizes to the complement of a polynucleotide molecule
having a nucleotide sequence that encodes amino acid residues 20 to
172 of SEQ ID NO:2 under highly stringent conditions, i.e.,
hybridization to filter-bound DNA in 0.5 M NaHPO.sub.4, 7% SDS, 1
mM EDTA at 65.degree. C., and washing in 0.1.times.SSC/0.1% SDS at
68.degree. C. (Ausubel et al., 1989, above), and is useful in
practicing the present invention. In a more preferred embodiment,
the homologous polynucleotide molecule hybridizes under highly
stringent conditions to the complement of a polynucleotide molecule
consisting of the nucleotide sequence of SEQ ID NO:1 from nt 329 to
nt 790, and is useful in practicing the present invention.
[0059] As used herein, a polynucleotide molecule is "useful in
practicing the present invention" where: (a) the polynucleotide
molecule encodes a polypeptide which can be used in a vaccine
composition either to induce by itself, or to contribute in
combination with one or more other antigens to the induction of, a
protective response in swine against APP; or (b) the polynucleotide
molecule can be used directly in a DNA vaccine composition to
induce by itself, or to contribute in combination with one or more
other polynucleotide molecules or one or more other antigens to the
induction of, a protective response in swine against APP; or (c)
the polynucleotide molecule encodes a polypeptide that can be used
to induce the production of APP-specific antibodies when
administered to a member of a mammalian species, which antibodies
are useful as diagnostic reagents; or (d) the polynucleotide
molecule encodes a polypeptide that can be used as a diagnostic
reagent to detect the presence of APP-specific antibodies in a
blood or serum sample from a pig; or (e) the polynucleotide
molecule can be used as a diagnostic reagent to detect the presence
of an APP-specific polynucleotide molecule in a fluid or tissue
sample from an APP-infected pig.
[0060] The present invention further provides an isolated
polynucleotide molecule comprising a nucleotide sequence that
encodes a polypeptide that is homologous to the Omp20 protein of
the present invention, as "homologous polypeptides" are defined
above in Section 5.1.
[0061] The present invention further provides a polynucleotide
molecule consisting of a nucleotide sequence that is a substantial
portion of any of the aforementioned Omp20-related polynucleotide
molecules of the present invention. As used herein, a "substantial
portion" of an Omp20-related polynucleotide molecules means a
polynucleotide molecule consisting of less than the complete
nucleotide sequence of the particular full-length Omp20-related
polynucleotide molecule, but comprising at least about 10%, and
more preferably at least about 20%, of the nucleotide sequence of
the particular full-length Omp20-related polynucleotide molecule,
and that is useful in practicing the present invention, as
"usefulness" is defined above for polynucleotide molecules. In a
non-limiting embodiment, the substantial portion of the
Omp20-related polynucleotide molecule encodes a peptide fragment of
any of the aforementioned Omp20-related proteins or polypeptides of
the present invention, as the term "peptide fragment" is defined
above.
[0062] The present invention further provides a polynucleotide
molecule comprising a nucleotide sequence, which encodes the native
Omp20 signal sequence from amino acid residue 1 to amino acid
residue 19 of SEQ ID NO:2. In a preferred though non-limiting
embodiment, the Omp20 signal sequence-encoding polynucleotide
molecule comprises from nt 272 to nt 328 of SEQ ID NO:1.
[0063] The present invention further provides a polynucleotide
molecule comprising a nucleotide sequence that encodes a fusion
protein comprising the Omp20 protein, homologous polypeptide, or
peptide fragment, fused to a carrier or fusion partner.
5.2.2. Polynucleotide Molecules Encoding OmpW
[0064] References herein below to nucleotide sequences from SEQ ID
NO:3, and to selected and substantial portions thereof, are
intended to also refer to the corresponding OmpW-related nucleotide
sequences of plasmid pER418 which is present in host cells of
strain Pz418 (ATCC 98928), unless otherwise indicated. In addition,
references herein below to the amino acid sequences shown in SEQ ID
NO:4, and to peptide fragments thereof, are intended to also refer
to the corresponding amino acid sequences encoded by the
OmpW-related nucleotide sequence of plasmid pER418, unless
otherwise indicated.
[0065] The present invention provides an isolated polynucleotide
molecule comprising a nucleotide sequence encoding the APP protein,
OmpW, with or without signal sequence. In a preferred embodiment,
the isolated OmpW-encoding polynucleotide molecule of the present
invention comprises the nucleotide sequence of SEQ ID Nb:3 from nt
439 to nt 1023. In a more preferred embodiment, the isolated
OmpW-encoding polynucleotide molecule of the present invention
comprises the nucleotide sequence of SEQ ID NO:3 from nt 376 to nt
1023. In a non-limiting embodiment, the isolated OmpW-encoding
polynucleotide molecule of the present invention comprises the
nucleotide sequence of SEQ ID NO:3.
[0066] The present invention further provides an isolated
polynucleotide molecule that is homologous to an APP OmpW-encoding
polynucleotide molecule of the present invention. The term
"homologous" when used to refer to an OmpW-encoding polynucleotide
molecule means a polynucleotide molecule having a nucleotide
sequence: (a) that encodes the same amino acid sequence as the
nucleotide sequence of SEQ ID NO:3 from nt 439 to nt 1023, but that
includes one or more silent changes to the nucleotide sequence
according to the degeneracy of the genetic code; or (b) that
hybridizes to the complement of a polynucleotide molecule having a
nucleotide sequence that encodes amino acid residues 22 to 215 of
SEQ ID NO:4 under moderately stringent conditions, i.e.,
hybridization to filter-bound. DNA in 0.5 M NaHPO.sub.4, 7% SDS, 1
mM EDTA at 65.degree. C., and washing in 0.2.times.SSC/0.1% SDS at
42.degree. C. (Ausubel et al., 1989, above), and that is useful in
practicing the present invention, as "usefulness" is defined above
for polynucleotide molecules. In a preferred embodiment, the
homologous polynucleotide molecule hybridizes to the complement of
a polynucleotide molecule having a nucleotide sequence that encodes
amino acid residues 22 to 215 of SEQ ID NO:4 under highly stringent
conditions, i.e., hybridization to filter-bound DNA in 0.5 M
NaHPO.sub.4, 7% SDS, 1 mM EDTA at 65.degree. C., and washing in
0.1.times.SSC/0.1% SDS at 68.degree. C. (Ausubel et al., 1989,
above), and is useful in practicing the present invention. In a
more preferred embodiment, the homologous polynucleotide molecule
hybridizes under highly stringent conditions to the complement of a
polynucleotide molecule consisting of the nucleotide sequence of
SEQ ID NO:3 from nt 439 to nt 1023, and is useful in practicing the
present invention.
[0067] The present invention further provides an isolated
polynucleotide molecule comprising a nucleotide sequence that
encodes a polypeptide that is homologous to the OmpW protein of the
present invention, as "homologous polypeptide" is defined
above.
[0068] The present invention further provides a polynucleotide
molecule consisting of a nucleotide sequence that is a substantial
portion of any of the aforementioned OmpW-related polynucleotide
molecules of the present invention. As used herein, a "substantial
portion" of an OmpW-related polynucleotide molecules means a
polynucleotide molecule consisting of less than the complete
nucleotide sequence of the particular full-length OmpW-related
polynucleotide molecule, but comprising at least about 10%, and
more preferably at least about 20%, of the nucleotide sequence of
the particular full-length OmpW-related polynucleotide molecule,
and that is useful in practicing the present invention, as
"usefulness" is defined above for polynucleotide molecules. In a
non-limiting embodiment, the substantial portion of the
OmpW-related polynucleotide molecule encodes a peptide fragment of
any of the aforementioned OmpW-related proteins or polypeptides of
the present invention, as the term "peptide fragment" is defined
above.
[0069] The present invention further provides a polynucleotide
molecule comprising a nucleotide sequence, which encodes the native
OmpW signal sequence from amino acid residue 1 to amino acid
residue 21 of SEQ ID NO:4. In a preferred though non-limiting
embodiment, the OmpW signal sequence-encoding polynucleotide
molecule comprises from nt 376 to nt 438 of SEQ ID NO:3.
[0070] The present invention further provides a polynucleotide
molecule comprising a nucleotide sequence that encodes a fusion
protein comprising the OmpW protein, homologous polypeptide, or
peptide fragment, fused to a carrier or fusion partner.
5.2.3. Polynucleotide Molecules Encoding Omp27
[0071] References herein below to nucleotide sequences from SEQ ID
NO:5, and to selected and substantial portions thereof, are
intended to also refer to the corresponding Omp27-related
nucleotide sequences of plasmid pER417 which is present in host
cells of strain Pz417 (ATCC 98927), unless otherwise indicated. In
addition, references herein below to the amino acid sequences shown
in SEQ ID NO:6, and to peptide fragments thereof, are intended to
also refer to the corresponding amino acid sequences encoded by the
Omp27-related nucleotide sequence of plasmid pER417, unless
otherwise indicated.
[0072] The present invention provides an isolated polynucleotide
molecule comprising a nucleotide sequence encoding the APP protein,
Omp27, with or without signal sequence. In a preferred embodiment,
the isolated Omp27-encoding polynucleotide molecule of the present
invention comprises the nucleotide sequence of SEQ ID NO:5 from nt
238 to nt 933. In a more preferred embodiment, the isolated
Omp27-encoding polynucleotide molecule of the present invention
comprises the nucleotide sequence of SEQ ID NO:5 from nt 157 to nt
933. In a non-limiting embodiment, the isolated Omp27-encoding
polynucleotide molecule of the present invention comprises the
nucleotide sequence of SEQ ID NO:5.
[0073] The present invention further provides an isolated
polynucleotide molecule that is homologous to an APP Omp27-encoding
polynucleotide molecule of the present invention. The term
"homologous" when used to refer to an Omp27-encoding polynucleotide
molecule means a polynucleotide molecule having a nucleotide
sequence: (a) that encodes the same amino acid sequence as the
nucleotide sequence of SEQ ID NO:5 from nt 238 to nt 933, but that
includes one or more silent changes to the nucleotide sequence
according to the degeneracy of the genetic code; or (b) that
hybridizes to the complement of a polynucleotide molecule having a
nucleotide sequence that encodes amino acid residues 28 to 258 of
SEQ ID NO:6 under moderately stringent conditions, i e.,
hybridization to filter-bound DNA in 0.5 M NaHPO.sub.4, 7% SDS, 1
mM EDTA at 65.degree. C., and washing in 0.2.times.SSC/0.1% SDS at
42.degree. C. (Ausubel et al., 1989, above), and that is useful in
practicing the present invention, as "usefulness" is defined above
for polynucleotide molecules. In a preferred embodiment, the
homologous polynucleotide molecule hybridizes to the complement of
a polynucleotide molecule having a nucleotide sequence that encodes
amino acid residues 28 to 258 of SEQ ID NO:6 under highly stringent
conditions, i.e., hybridization to filter-bound DNA in 0.5 M
NaHPO.sub.4, 7% SDS, 1 mM EDTA at 65.degree. C., and washing in
0.1.times.SSC/0.1% SDS at 68.degree. C. (Ausubel et al., 1989,
above), and is useful in practicing the present invention. In a
more preferred embodiment, the homologous polynucleotide molecule
hybridizes under highly stringent conditions to the complement of a
polynucleotide molecule consisting of the nucleotide sequence of
SEQ ID NO:5 from nt 238 to nt 933, and is useful in practicing the
present invention.
[0074] The present invention further provides an isolated
polynucleotide molecule comprising a nucleotide sequence that
encodes a polypeptide that is homologous to the Omp27 protein of
the present invention, as "homologous polypeptide" is defined
above.
[0075] The present invention further provides a polynucleotide
molecule consisting of a nucleotide sequence that is a substantial
portion of any of the aforementioned Omp27-related polynucleotide
molecules of the present invention. As used herein, a "substantial
portion" of an Omp27-related polynucleotide molecules means a
polynucleotide molecule consisting of less than the complete
nucleotide sequence of the particular full-length Omp27-related
polynucleotide molecule, but comprising at least about 10%, and
more preferably at least about 20%, of the nucleotide sequence of
the particular full-length Omp27-related polynucleotide molecule,
and that is useful in practicing the present invention, as
"usefulness" is defined above for polynucleotide molecules. In a
non-limiting embodiment, the substantial portion of the
Omp27-related polynucleotide molecule encodes a peptide fragment of
any of the aforementioned Omp27-related proteins or polypeptides of
the present invention, as the term "peptide fragment" is defined
above.
[0076] The present invention further provides a polynucleotide
molecule comprising a nucleotide sequence, which encodes the native
Omp27 signal sequence from amino acid residue I to amino acid
residue 27 of SEQ ID NO:6. In a preferred though non-limiting
embodiment, the Omp27 signal sequence-encoding polynucleotide
molecule comprises from nt 157 to nt 237 of SEQ ID NO:5.
[0077] The present invention further provides a polynucleotide
molecule comprising a nucleotide sequence that encodes a fusion
protein comprising the Omp27 protein, homologous polypeptide, or
peptide fragment, fused to a carrier or fusion partner.
5.2.4. Polynucleotide Molecules Encoding OmpA1
[0078] References herein below to nucleotide sequences from SEQ ID
NO:7, and to selected and substantial portions thereof, are
intended to also refer to the corresponding OmpA1-related
nucleotide sequences of plasmid pER419 which is present in host
cells of strain Pz419 (ATCC 98929), unless otherwise indicated. In
addition, references herein below to the amino acid sequences shown
in SEQ ID NO:8, and to peptide fragments thereof, are intended to
also refer to the corresponding amino acid sequences encoded by the
OmpA1-related nucleotide sequence of plasmid pER419, unless
otherwise indicated.
[0079] The present invention provides an isolated polynucleotide
molecule comprising a nucleotide sequence encoding the APP protein,
OmpA1, with or without signal sequence. In a preferred embodiment,
the isolated OmpA1-encoding polynucleotide molecule of the present
invention comprises the nucleotide sequence of SEQ ID NO:7 from nt
671 to nt 1708. In a more preferred embodiment, the isolated
OmpA1-encoding polynucleotide molecule of the present invention
comprises the nucleotide sequence of SEQ ID NO:7 from nt 614 to nt
1708. In a non-limiting embodiment, the isolated OmpA1-encoding
polynucleotide molecule of the present invention comprises the
nucleotide sequence of SEQ ID NO:7.
[0080] The present invention further provides an isolated
polynucleotide molecule that is homologous to an APP OmpA1-encoding
polynucleotide molecule of the present invention. The term
"homologous" when used to refer to an OmpA1-encoding polynucleotide
molecule means a polynucleotide molecule having a nucleotide
sequence: (a) that encodes the same amino acid sequence as the
nucleotide sequence of SEQ ID NO:7 from nt 671 to nt 1708, but that
includes one or more silent changes to the nucleotide sequence
according to the degeneracy of the genetic code; or (b) that
hybridizes to the complement of a polynucleotide molecule having a
nucleotide sequence that encodes amino acid residues 20 to 364 of
SEQ ID NO:8 under moderately stringent conditions, i.e.,
hybridization to filter-bound DNA in 0.5 M NaHPO.sub.4, 7% SDS, 1
mM EDTA at 65.degree. C., and washing in 0.2.times.SSC/0.1% SDS at
42.degree. C. (Ausubel et al., 1989, above), and that is useful in
practicing the present invention, as "usefulness" is defined above
for polynucleotide molecules. In a preferred embodiment, the
homologous polynucleotide molecule hybridizes to the complement of
a polynucleotide molecule having a nucleotide sequence that encodes
amino acid residues 20 to 364 of SEQ ID NO:8 under highly stringent
conditions, i.e., hybridization to filter-bound DNA in 0.5 M
NaHPO.sub.4, 7% SDS, 1 mM EDTA at 65.degree. C., and washing in
0.1.times.SSC/0.1% SDS at 68.degree. C. (Ausubel et al., 1989,
above), and is useful in practicing the present invention. In a
more preferred embodiment, the homologous polynucleotide molecule
hybridizes under highly stringent conditions to the complement of a
polynucleotide molecule consisting of the nucleotide sequence of
SEQ ID NO:7 from nt 671 to nt 1708, and is useful in practicing the
present invention.
[0081] The present invention further provides an isolated
polynucleotide molecule comprising a nucleotide sequence that
encodes a polypeptide that is homologous to the OmpA1 protein of
the present invention, as "homologous polypeptide" is defined
above.
[0082] The present invention further provides a polynucleotide
molecule consisting of a nucleotide sequence that is a substantial
portion of any of the aforementioned OmpA1-related polynucleotide
molecules of the present invention. As used herein, a "substantial
portion" of an OmpA1-related polynucleotide molecules means a
polynucleotide molecule consisting of less than the complete
nucleotide sequence of the particular full-length OmpA1-related
polynucleotide molecule, but comprising at least about 10%, and
more preferably at least about 20%, of the nucleotide sequence of
the particular full-length OmpA1-related polynucleotide molecule,
and that is useful in practicing the present invention, as
"usefulness" is defined above for polynucleotide molecules. In a
non-limiting embodiment, the substantial portion of the
OmpA1-related polynucleotide molecule encodes a peptide fragment of
any of the aforementioned OmpA1-related polypeptides of the present
invention, as the term "peptide fragment" is defined above.
[0083] The present invention further provides a polynucleotide
molecule comprising a nucleotide sequence, which encodes the native
OmpA1 signal sequence from amino acid residue 1 to amino acid
residue 19 of SEQ ID NO:8. In a preferred though non-limiting
embodiment, the OmpA1 signal sequence-encoding polynucleotide
molecule comprises from nt 614 to nt 670 of SEQ ID NO:7.
[0084] The present invention further provides a polynucleotide
molecule comprising a nucleotide sequence that encodes a fusion
protein comprising the OmpA1 protein, homologous polypeptide, or
peptide fragment, fused to a carrier or fusion partner.
5.2.5. Polynucleotide Molecules Encoding OmpA2
[0085] References herein below to nucleotide sequences from SEQ ID
NO:9, and to selected and substantial portions thereof, are
intended to also refer to the corresponding OmpA2-related
nucleotide sequences of plasmid pER420 which is present in host
cells of strain Pz420 (ATCC 98930), unless otherwise indicated. In
addition, references herein below to the amino acid sequences shown
in SEQ ID NO:10, and to peptide fragments thereof, are intended to
also refer to the corresponding amino acid sequences encoded by the
OmpA2-related nucleotide sequence of plasmid pER420, unless
otherwise indicated.
[0086] The present invention provides an isolated polynucleotide
molecule comprising a nucleotide sequence encoding the APP protein,
OmpA2, with or without signal sequence. In a preferred embodiment,
the isolated OmpA2-encoding polynucleotide molecule of the present
invention comprises the nucleotide sequence of SEQ ID NO:9 from nt
254 to nt 1306. In a more preferred embodiment, the isolated
OmpA2-encoding polynucleotide molecule of the present invention
comprises the nucleotide sequence of SEQ ID NO:9 from nt 197 to nt
1306. In a non-limiting embodiment, the isolated OmpA2-encoding
polynucleotide molecule of the present invention comprises the
nucleotide sequence of SEQ ID NO:9.
[0087] The present invention further provides an isolated
polynucleotide molecule that is homologous to an APP OmpA2-encoding
polynucleotide molecule of the present invention. The term
"homologous" when used to refer to an OmpA2-encoding polynucleotide
molecule means a polynucleotide molecule having a nucleotide
sequence: (a) that encodes the same amino acid sequence as the
nucleotide sequence of SEQ ID NO:9 from nt 254 to nt 1306, but that
includes one or more silent changes to the nucleotide sequence
according to the degeneracy of the genetic code; or (b) that
hybridizes to the complement of a polynucleotide molecule having a
nucleotide sequence that encodes amino acid residues 20 to 369 of
SEQ ID NO:10 under moderately stringent conditions, i.e.,
hybridization to filter-bound DNA in 0.5 M NaHPO.sub.4, 7% SDS, 1
mM EDTA at 65.degree. C., and washing in 0.2.times.SSC/0.1% SDS at
42.degree. C. (Ausubel et al., 1989, above), and that is useful in
practicing the present invention, as "usefulness" is defined above
for polynucleotide molecules. In a preferred embodiment, the
homologous polynucleotide molecule hybridizes to the complement of
a polynucleotide molecule having a nucleotide sequence that encodes
amino acid residues 20 to 369 of SEQ ID NO:10 under highly
stringent conditions, i.e., hybridization to filter-bound DNA in
0.5 M NaHPO.sub.4, 7% SDS, 1 mM EDTA at 65.degree. C., and washing
in 0.1.times.SSC/0.1% SDS at 68.degree. C. (Ausubel et al., 1989,
above), and is useful in practicing the present invention. In a
more preferred embodiment, the homologous polynucleotide molecule
hybridizes under highly stringent conditions to the complement of a
polynucleotide molecule consisting of the nucleotide sequence of
SEQ ID NO:9 from nt 254 to nt 1306, and is useful in practicing the
present invention.
[0088] The present invention further provides an isolated
polynucleotide molecule comprising a nucleotide sequence that
encodes a polypeptide that is homologous to the OmpA2 protein of
the present invention, as "homologous polypeptide" is defined
above.
[0089] The present invention further provides a polynucleotide
molecule consisting of a nucleotide sequence that is a substantial
portion of any of the aforementioned OmpA2-related polynucleotide
molecules of the present invention. As used herein, a "substantial
portion" of an OmpA2-related polynucleotide molecules means a
polynucleotide molecule consisting of less than the complete
nucleotide sequence of the particular full-length OmpA2-related
polynucleotide molecule, but comprising at least about 10%, and
more preferably at least about 20%, of the nucleotide sequence of
the particular full-length OmpA2-related polynucleotide molecule,
and that is useful in practicing the present invention, as
"usefulness" is defined above for polynucleotide molecules. In a
non-limiting embodiment, the substantial portion of the
OmpA2-related polynucleotide molecule encodes a peptide fragment of
any of the aforementioned OmpA2-related polypeptides of the present
invention, as the term "peptide fragment" is defined above.
[0090] The present invention further provides a polynucleotide
molecule comprising a nucleotide sequence, which encodes the native
OmpA2 signal sequence from amino acid residue 1 to amino acid
residue 19 of SEQ ID NO:10. In a preferred though non-limiting
embodiment, the OmpA2 signal sequence-encoding polynucleotide
molecule comprises from nt 197 to nt 253 of SEQ ID NO:9.
[0091] The present invention further provides a polynucleotide
molecule comprising a nucleotide sequence that encodes a fusion
protein comprising the OmpA2 protein, homologous polypeptide, or
peptide fragment, fused to a carrier or fusion partner.
5.3. Oligonucleotide Molecules
[0092] The present invention further provides oligonucleotide
molecules that hybridize to any of the aforementioned
polynucleotide molecules of the present invention, or that
hybridize to a polynucleotide molecule having a nucleotide sequence
that is the complement of any of the aforementioned polynucleotide
molecules of the present invention. Such oligonucleotide molecules
are preferably at least about 10 to 15 nucleotides in length, but
can extend up to the length of any sub-sequence of SEQ ID NOS:1, 3,
5, 7 or 9, or homologous polynucleotide molecule thereof, and can
hybridize to one or more of the aforementioned polynucleotide
molecules under highly stringent conditions. For shorter
oligonucleotide molecules, an example of highly stringent
conditions includes washing in 6.times.SSC/0.5% sodium
pyrophosphate at about 37.degree. C for--14-base oligos, at about
48.degree. C. for .about.17-base oligos, at about 55.degree. C for
.about.20-base oligos, and at about 60.degree. C. for
.about.23-base oligos. For longer oligonucleotide molecules (i.e.,
greater than about 100 nts), examples of highly stringent
conditions are provided in Section 5.2 above. Other appropriate
hybridization conditions can be determined and adjusted as known in
the art, depending upon the particular oligonucleotide and
polynucleotide molecules utilized.
[0093] In a preferred embodiment, an oligonucleotide molecule of
the present invention hybridizes under highly stringent conditions
to a polynucleotide molecule consisting of a nucleotide sequence
selected from SEQ ID NOS:1, 3, 5, 7, or 9, or to a polynucleotide
molecule consisting of a nucleotide sequence that is the complement
of a nucleotide sequence selected from SEQ ID NOS:1, 3, 5, 7, or
9.
[0094] In a non-limiting embodiment, an oligonucleotide molecule of
the present invention comprises a nucleotide sequence selected from
the group consisting of SEQ ID NOS:15-47 and 49-93 and the
complements of said sequences. In a non-limiting embodiment, the
oligonucleotide molecule consists of a nucleotide sequence selected
from the group consisting of SEQ ID NOS: 15-47 and 49-93 and the
complements of said sequences.
[0095] The oligonucleotide molecules of the present invention are
useful for a variety of purposes, including as primers in
amplification of an APP protein-encoding polynucleotide molecule
for use, e.g., in differential disease diagnosis, or to encode or
act as antisense molecules useful in gene regulation. Amplification
can be used to detect the presence of a polynucleotide molecule
encoding an APP protein in a tissue or fluid sample, e.g., in
mucous or bronchial fluid, from an infected animal. The production
of a specific amplification product can help support a diagnosis of
APP bacterial infection, while lack of an amplified product can
indicate a lack of such infection. The oligonucleotide molecules
disclosed herein can also be used to isolate homologous genes from
other species or strains of Actinobacillus, or from other
bacteria.
[0096] Amplification can be carried out using suitably designed
oligonucleotide molecules in conjunction with standard techniques,
such as the polymerase chain reaction (PCR), although other
amplification techniques known in the art, e.g., the ligase chain
reaction, can be used. For example, for PCR, a mixture comprising
suitably designed primers, a template comprising the nucleotide
sequence to be amplified, and appropriate PCR enzymes and buffers
as known in the art, is prepared and processed according to
standard protocols to amplify a specific APP-related polynucleotide
sequence of the template. Methods for conducting PCR are described,
among other places, in Innis et al. (eds), 1995, above; and Erlich
(ed), 1992, above.
5.4. Recombinant Expression Systems
5.4.1. Cloning and Expression Vectors
[0097] The present invention further provides compositions for
cloning and expressing any of the polynucleotide molecules of the
present invention, including recombinant cloning vectors and
recombinant expression vectors comprising a polynucleotide molecule
of the present invention, host cells transformed with any of said
vectors, and cell lines derived therefrom. Recombinant vectors of
the present invention, particularly expression vectors, are
preferably constructed so that the coding sequence of the
polynucleotide molecule (referred to hereinafter as the "APP coding
sequence") is in operative association with one or more regulatory
elements necessary for transcription and translation of the APP
coding sequence to produce a polypeptide.
[0098] As used herein, the term "regulatory element" includes but
is not limited to nucleotide sequences that encode inducible and
non-inducible promoters, enhancers, operators and other elements
known in the art that serve to drive and/or regulate expression of
polynucleotide coding sequences. Also, as used herein, the APP
coding sequence is in "operative association" with one or more
regulatory elements where the regulatory elements effectively
regulate and allow for the transcription of the coding sequence or
the translation of its mRNA, or both.
[0099] Methods are well-known in the art for constructing
recombinant vectors containing particular coding sequences in
operative association with appropriate regulatory elements,
including in vitro recombinant techniques, synthetic techniques,
and in vivo genetic recombination. See, e.g., the techniques
described in Maniatis et al., 1989, above; Ausubel et al., 1989,
above; Sambrook et al., 1989, above; Innis et al., 1995, above; and
Erlich, 1992, above.
[0100] A variety of expression vectors are known in the art that
can be utilized to express any of the APP coding sequences of the
present invention, including recombinant bacteriophage DNA, plasmid
DNA, and cosmid DNA expression vectors containing an APP coding
sequence. Typical prokaryotic expression vector plasmids that can
be engineered to contain an APP coding sequence of the present
invention include pUC8, pUC9, pBR322 and pBR329 (Biorad
Laboratories, Richmond, Calif.), pPL and pKK223 (Pharmacia,
Piscataway, N.J.), pQE50 (Qiagen, Chatsworth, Calif.), and pGEX
series plasmids (Pharmacia), among many others. Typical eukaryotic
expression vectors that can be engineered to contain an APP coding
sequence of the present invention include an ecdysone-inducible
mammalian expression system (Invitrogen, Carlsbad, Calif.),
cytomegalovirus promoter-enhancer-based systems (Promega, Madison,
Wis.; Stratagene, La Jolla, Calif.; Invitrogen), baculovirus-based
expression systems (Promega), and plant-based expression systems,
among others.
[0101] The regulatory elements of these and other vectors can vary
in their strength and specificities. Depending on the host/vector
system utilized, any of a number of suitable transcription and
translation elements can be used. For instance, when cloning in
mammalian cell systems, promoters isolated from the genome of
mammalian cells, e.g., mouse metallothionein promoter, or from
viruses that grow in these cells, e.g., vaccinia virus 7.5K
promoter or Moloney murine sarcoma virus long terminal repeat, can
be used. Promoters obtained by recombinant DNA or synthetic
techniques can also be used to provide for transcription of the
inserted coding sequence. In addition, expression from certain
promoters can be elevated in the presence of particular inducers,
e.g., zinc and cadmium ions for metallothionein promoters.
Non-limiting examples of transcriptional regulatory regions or
promoters include for bacteria, the .beta.-gal promoter, the T7
promoter, the TAC promoter, .lambda. left and right promoters, trp
and lac promoters, trp-lac fusion promoters, etc.; for yeast,
glycolytic enzyme promoters, such as ADH-I and II promoters, GPK
promoter, PGI promoter, TRP promoter, etc.; and for mammalian
cells, SV40 early and late promoters, adenovirus major late
promoters, among others.
[0102] Specific initiation signals are also required for sufficient
translation of inserted coding sequences. These signals typically
include an ATG initiation codon and adjacent sequences. In cases
where the APP coding sequence of the present invention including
its own initiation codon and adjacent sequences are inserted into
the appropriate expression vector, no additional translation
control signals are needed. However, in cases where only a portion
of an APP coding sequence is inserted, exogenous translational
control signals, including the ATG initiation codon, can be
required. These exogenous translational control signals and
initiation codons can be obtained from a variety of sources, both
natural and synthetic. Furthermore, the initiation codon must be in
phase with the reading frame of the coding regions to ensure
in-frame translation of the entire insert.
[0103] Expression vectors can also be constructed that will express
a fusion protein comprising any of the APP-related polypeptides of
the present invention fused to a carrier or fusion partner. Such
fusion proteins can be used for a variety of purposes, such as to
increase the stability of a recombinantly-expressed APP protein, to
raise antisera against an APP protein, to study the biochemical
properties of an APP protein, to engineer an APP protein exhibiting
altered immunological properties, or to aid in the identification
or purification of a recombinantly-expressed APP protein. Possible
fusion protein expression vectors include but are not limited to
vectors incorporating sequences that encode a protective peptide,
such as that described below in Section 8.2, as well as
.beta.-galactosidase and trpE fusions, maltose-binding protein
fusions, glutathione-S-transferase (GST) fusions and polyhistidine
fusions (carrier regions). Methods are well-known in the art that
can be used to construct expression vectors encoding these and
other fusion proteins.
[0104] Fusion proteins can be useful to aid in purification of the
expressed protein. In non-limiting embodiments, e.g., an APP
protein-maltose-binding fusion protein can be purified using
amylose resin; an APP protein-GST fusion protein can be purified
using glutathione-agarose beads; and an APP protein-polyhistidine
fusion protein can be purified using divalent nickel resin.
Alternatively, antibodies against a carrier protein or peptide can
be used for affinity chromatography purification of the fusion
protein. For example, a nucleotide sequence coding for the target
epitope of a monoclonal antibody can be engineered into the
expression vector in operative association with the regulatory
elements and situated so that the expressed epitope is fused to an
APP protein of the present invention. In a non-limiting embodiment,
a nucleotide sequence coding for the FLAG.TM. epitope tag
(International Biotechnologies Inc.), which is a hydrophilic marker
peptide, can be inserted by standard techniques into the expression
vector at a point corresponding to the amino or carboxyl terminus
of the APP protein. The expressed polypeptide-FLAG.TM. epitope
fusion product can then be detected and affinity-purified using
commercially available anti-FLAG.TM. antibodies.
[0105] The expression vector of the present invention can also be
engineered to contain polylinker sequences that encode specific
protease cleavage sites so that the expressed APP protein can be
released from the carrier region or fusion partner by treatment
with a specific protease. For example, the fusion protein vector
can include a nucleotide sequence encoding a thrombin or factor Xa
cleavage site, among others.
[0106] A signal sequence upstream from and in reading frame with
the APP coding sequence can be engineered into the expression
vector by known methods to direct the trafficking and secretion of
the expressed APP polypeptide. Non-limiting examples of signal
sequences include those which are native to the APP proteins of the
present invention as disclosed herein, as well as signal sequences
from .alpha.-factor, immunoglobulins, outer membrane proteins,
penicillinase, and T-cell receptors, among others.
[0107] To aid in the selection of host cells transformed or
transfected with a recombinant vector of the present invention, the
vector can be engineered to further comprise a coding sequence for
a reporter gene product or other selectable marker. Such a coding
sequence is preferably in operative association with the regulatory
elements, as described above. Reporter genes that are useful in
practicing the invention are well-known in the art and include
those encoding chloramphenicol acetyltransferase (CAT), green
fluorescent protein, firefly luciferase, and human growth hormone,
among others. Nucleotide sequences encoding selectable markers are
well-known in the art, and include those that encode gene products
conferring resistance to antibiotics or anti-metabolites, or that
supply an auxotrophic requirement. Examples of such sequences
include those that encode thymidine kinase activity, or resistance
to methotrexate, ampicillin, kanamycin, chloramphenicol, zeocin,
tetracycline, and carbenicillin, among many others.
[0108] In specific though non-limiting embodiments, the present
invention provides the following plasmid cloning vectors,
constructed as described below in Section 11, which are present in
host cells that have been deposited with the American Type Culture
Collection (ATCC): plasmid pER416, which is present in host cells
of strain Pz416 (ATCC 98926), and which plasmid comprises the ORF
of omp20; plasmid pER418, which is present in host cells of strain
Pz418 (ATCC 98928), and which plasmid comprises the ORF of ompW,
plasmid pER417, which is present in host cells of strain Pz417
(ATCC 98927), and which plasmid comprises the ORF of omp27; plasmid
pER419, which is present in host cells of strain Pz419 (ATCC
98929), and which plasmid comprises the ORF of ompA1; and plasmid
pER420, which is present in host cells of strain Pz420 (ATCC
98930), and which plasmid comprises the ORF of ompA2.
5.4.2. Transformation of Host Cells
[0109] The present invention further provides host cells
transformed with a polynucleotide molecule or recombinant vector of
the invention, and cell lines derived therefrom. Host cells useful
in practicing the present invention can either be prokaryotic or
eukaryotic. Such transformed host cells include but are not limited
to microorganisms, such as bacterial cells transformed with
recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression
vectors; or yeast cells transformed with recombinant expression
vectors; or animal cells, such as insect cells infected with
recombinant virus expression vectors, e.g., baculovirus, or
mammalian cells infected with recombinant virus expression vectors,
e.g., adenovirus or vaccinia virus, among others.
[0110] Bacterial cells are generally preferred as host cells. A
strain of E. coli can typically be used, such as, e.g., strain
DH5.alpha., available from Gibco BRL, Life Technologies
(Gaithersburg, Md.), or E. coli strain LW14, as described below.
Eukaryotic host cells, including yeast cells, and mammalian cells,
such as from a mouse, hamster, pig, c6w, monkey, or human cell
line, can also be used effectively. Examples of eukaryotic host
cells that can be used to express the recombinant protein of the
invention include Chinese hamster ovary (CHO) cells (e.g., ATCC
Accession No. CCL61) and NIH Swiss mouse embryo cells NIH/3T3
(e.g., ATCC Accession No. CRL 1658).
[0111] A recombinant vector of the invention is preferably
transformed or transfected into one or more host cells of a
substantially homogeneous culture of cells. The vector is generally
introduced into host cells in accordance with known techniques such
as, e.g., by calcium phosphate precipitation, calcium chloride
treatment, microinjection, electroporation, transfection by contact
with a recombined virus, liposome-mediated transfection,
DEAE-dextran transfection, transduction, conjugation, or
microprojectile bombardment. Selection of transformants can be
conducted by standard procedures, such as by selecting for cells
expressing a selectable marker, e.g., antibiotic resistance,
associated with the recombinant vector.
[0112] Once the vector is introduced into the host cell, the
integration and maintenance of the APP coding sequence either in
the host cell genome or episomally can be confirmed by standard
techniques, e.g., by Southern hybridization analysis, restriction
enzyme analysis, PCR analysis, including reverse transcriptase PCR
(rt-PCR), or by immunological assay to detect the expected protein
product. Host cells containing and/or expressing the APP coding
sequence can be identified by any of at least four general
approaches, which are well-known in the art, including: (i)
DNA-DNA, DNA-RNA, or RNA-antisense RNA hybridization; (ii)
detecting the presence of "marker" gene functions; (iii) assessing
the level of transcription as measured by the expression of
APP-specific mRNA transcripts in the host cell; and (iv) detecting
the presence of mature APP protein product as measured, e.g., by
immunoassay.
5.4.3. Expression of Recombinant Polypeptides
[0113] Once the APP coding sequence has been stably introduced into
an appropriate host cell, the transformed host cell is clonally
propagated, and the resulting cells are grown under conditions
conducive to the maximum production of the APP protein. Such
conditions typically include growing such cells to high density.
Where the expression vector comprises an inducible promoter,
appropriate induction conditions such as, e.g., temperature shift,
exhaustion of nutrients, addition of gratuitous inducers (e.g.,
analogs of carbohydrates, such as isopropyl-P-D-thiogalac-
topyranoside (IPTG)), accumulation of excess metabolic by-products,
or the like, are employed as needed to induce expression.
[0114] Where the recombinantly-expressed APP protein is retained
inside the host cells, the cells are harvested and lysed, and the
APP protein is substantially purified or isolated from the lysate
under extraction conditions known in the art to minimize protein
degradation such as, e.g., at 4.degree. C., or in the presence of
protease inhibitors, or both. Where the recombinantly-expressed APP
protein is secreted from the host cells, the exhausted nutrient
medium can simply be collected and the APP polypeptide
substantially purified or isolated therefrom.
[0115] The recombinantly-expressed APP protein can be partially or
substantially purified or isolated from cell lysates or culture
medium, as appropriate, using standard methods, including but not
limited to any combination of the following methods: ammonium
sulfate precipitation, size fractionation, ion exchange
chromatography, HPLC, density centrifugation, and affinity
chromatography. Increasing purity of the APP polypeptide of the
present invention can be determined as based, e.g., on size, or
reactivity with an antibody specific to the APP polypeptide, or by
the presence of a fusion tag. For use in practicing the present
invention, e.g., in a vaccine composition, the APP protein can be
used in an unpurified state as secreted into the culture fluid, or
as present in host cells or in a cell lysate, or in substantially
purified or isolated form. As used herein, an APP protein is
"substantially purified" where the protein constitutes more than
about 20 wt % of the protein in a particular preparation. Also, as
used herein, an APP protein is "isolated" where the protein
constitutes at least about 80 wt % of the protein in a particular
preparation.
[0116] The present invention thus provides a method for preparing
an APP protein, homologous polypeptide, peptide fragment or fusion
protein of the present invention, comprising culturing a host cell
transformed with a recombinant expression vector, said recombinant
expression vector comprising a polynucleotide molecule comprising a
nucleotide sequence encoding: (a) an APP protein comprising the
amino acid sequence of SEQ ID NO:2, 4, 6, 8, or 10, either with or
without its native signal sequence; or (b) a polypeptide that is
homologous to the APP protein of (a); or (c) a peptide fragment of
the APP protein of (a) or homologous polypeptide of (b); or (d) a
fusion protein comprising the APP protein of (a), homologous
polypeptide of (b), or peptide fragment of (c), fused to a fusion
partner; which polynucleotide molecule is in operative association
with one or more regulatory elements that control expression of the
polynucleotide molecule in the host cell, under conditions
conducive to the production of the APP protein, homologous
polypeptide, peptide fragment or fusion protein, and recovering the
APP protein, homologous polypeptide, peptide fragment or fusion
protein from the cell culture.
[0117] Once an APP protein of the present invention has been
obtained in sufficient purity, it can be characterized by standard
methods, including by SDS-PAGE, size exclusion chromatography,
amino acid sequence analysis, serological reactivity, etc. The
amino acid sequence of the APP protein can be determined using
standard peptide sequencing techniques. The APP protein can be
further characterized using hydrophilicity analysis (see, e.g.,
Hopp and Woods, 1981, Proc. Natl. Acad. Sci. USA 78:3824), or
analogous software algorithms, to identify hydrophobic and
hydrophilic regions. Structural analysis can be carried out to
identify regions of the APP protein that assume specific secondary
structures. Biophysical methods such as X-ray crystallography
(Engstrom, 1974, Biochem. Exp. Biol. 11: 7-13), computer modelling
(Fletterick and Zoller (eds), 1986, in: Current Communications in
Molecular Biology, Cold Spring Harbor Laboratory, Cold Spring
Harbor, N.Y.), nuclear magnetic resonance (NMR), and mass
spectrometry can also be used to characterize the protein.
Information obtained from these studies can be used, e.g., to
design more effective vaccine compositions, or to select vaccines
comprising only specific portions of the APP protein.
[0118] An APP protein that is useful in practicing the present
invention is a polypeptide that: (a) is immunogenic, i.e., capable
of inducing by itself, or capable of contributing in combination
with other APP proteins or other APP-related antigens to the
induction of, a protective response against APP when administered
to swine; or (b) is capable of inducing the production of anti-APP
antibodies when administered to a member of a mammalian species; or
(c) can be used as a diagnostic reagent to detect the presence of
anti-APP antibodies in a blood or serum sample from a pig resulting
from infection with APP or from vaccination with a vaccine of the
present invention. Such a protein, once prepared, can be identified
using routine screening procedures known in the art. For example,
the ability to induce, or contribute to the induction of, a
protective immune response against APP can be identified by
administering the APP protein, either alone or in combination with
other APP proteins or other APP-related antigens, respectively, to
a pig, and testing for the resulting induction of APP-neutralizing
antibodies, or for the resulting ability of the vaccinated animal
to resist subsequent challenge with APP compared to an unvaccinated
control. The ability to induce the production of APP-specific
antibodies can be identified by administering the APP protein to a
model animal, such as a mouse, pig, sheep, goat, horse, cow, etc.,
and testing the animal's serum for the presence of APP-specific
antibodies using standard techniques. The ability to use the APP
protein as a diagnostic reagent can be determined by exposing the
APP protein to a blood or serum sample of an animal previously or
currently infected with APP, or previously vaccinated with a
vaccine of the present invention, and detecting the binding to the
APP protein of APP-specific antibodies from the sample using
standard techniques, such as with an ELISA assay.
5.5. APP Vaccines
[0119] The present invention further provides a vaccine for
protecting swine against APP, comprising an immunologically
effective amount of one or more of the following: (a) an APP
protein comprising an amino acid sequence selected from the group
consisting of SEQ ID NOS:2, 4, 6, 8 or 10, either with or without
its native signal sequence; (b) a polypeptide that is homologous to
the APP protein of (a); (c) a peptide fragment consisting of a
sub-sequence of the APP protein of (a) or the homologous
polypeptide of (b); (d) a fusion protein comprising the APP protein
of (a), homologous polypeptide of (b), or peptide fragment of (c),
fused to a fusion partner; (e) an analog or derivative of the APP
protein of (a), homologous polypeptide of (b), peptide fragment of
(c), or fusion protein of (d); or (f) a polynucleotide molecule
comprising a nucleotide sequence encoding the APP protein of (a),
homologous polypeptide of (b), peptide fragment of (c), fusion
protein of (d), or analog or derivative of (e); which APP protein,
homologous polypeptide, peptide fragment, fusion protein, analog,
derivative or polynucleotide molecule, can induce by itself, or in
combination with one or more other such antigens contribute to the
induction of, a protective response against APP in swine; and a
veterinarily acceptable carrier. As used herein, the term
"immunologically effective amount" refers to that amount of antigen
that is capable of inducing, or contributing to the induction of, a
protective immune response in swine against one or more serotypes
of APP after either a single administration or after administration
of divided doses.
[0120] The phrase "capable of inducing a protective immune
response" is used broadly herein to include the induction of, or
increase in, any immune-based response in the pig in response to
vaccination, including either an antibody or cell-mediated immune
response, or both, that serves to protect the vaccinated animal
against APP. The terms "protective immune response", "protect", and
the like, as used herein, are not limited to absolute prevention of
APP-associated swine pneumonia or absolute prevention of infection
of pigs by APP, but are intended to also refer to any reduction in
the degree or rate of infection by the pathogen, or any reduction
in the severity of the disease or in any symptom or condition
resulting from infection with the pathogen, including, e.g., any
detectable decrease in lung pathology, as compared to that
occurring in an unvaccinated, infected control animal.
[0121] Vaccine compositions of the present invention can be
formulated following accepted convention using standard buffers,
carriers, stabilizers, diluents, preservatives, and solubilizers,
and can also be formulated to facilitate sustained release.
Diluents can include water, saline, dextrose, ethanol, glycerol,
and the like. Additives for isotonicity can include sodium
chloride, dextrose, mannitol, sorbitol, and lactose, among others.
Stabilizers include albumin, among others.
[0122] Adjuvants can optionally be employed in the vaccine.
Non-limiting examples of adjuvants include the RIBI adjuvant system
(Ribi Inc.), alum, aluminum hydroxide gel, oil-in-water emulsions,
water-in-oil emulsions such as, e.g., Freund's complete and
incomplete adjuvants, Block co polymer (CytRx, Atlanta Ga.), SAF-M
(Chiron, Emeryville Calif.), AMPHIGEN.RTM. adjuvant, saponin, Quil
A, QS-21 (Cambridge Biotech Inc., Cambridge Mass.), or other
saponin fractions, SEAM-1, monophosphoryl lipid A, Avridine
lipid-amine adjuvant, heat-labile enterotoxin from E. coli
(recombinant or otherwise), cholera toxin, or muramyl dipeptide,
among many others. The vaccine can further comprise one or more
other immunomodulatory agents such as, e.g., interleukins,
interferons, or other cytokines.
[0123] Suitable veterinarily acceptable vaccine vehicles, carriers,
and additives are known, or will be apparent to those skilled in
the art; see, e.g., Remington's Pharmaceutical Science, 18th Ed.,
1990, Mack Publishing, which is incorporated herein by reference.
The vaccine can be stored in solution, or alternatively in
lyophilized form to be reconstituted with a sterile diluent
solution prior to administration.
[0124] The present invention further provides vaccine formulations
for the sustained release of the antigen. Examples of such
sustained release formulations include the antigen in combination
with composites of biocompatible polymers, such as, e.g.,
poly(lactic acid), poly(lactic-cd-glycolic acid), methylcellulose,
hyaluronic acid, collagen and the like. The structure, selection
and use of degradable polymers in drug delivery vehicles have been
reviewed in several publications, including A. Domb et al., 1992,
Polymers for Advanced Technologies 3: 279-292, which is
incorporated herein by reference. Additional guidance in selecting
and using polymers in pharmaceutical formulations can be found in
the text by M. Chasin and R. Langer (eds), 1990, "Biodegradable
Polymers as Drug Delivery Systems" in: Drugs and the Pharmaceutical
Sciences, Vol. 45, M. Dekker, NY, which is also incorporated herein
by reference. Alternatively, or additionally, the antigen can be
microencapsulated to improve administration and efficacy. Methods
for microencapsulating antigens are well-known in the art, and
include techniques described, among other places, in U.S. Pat. No.
3,137,631; U.S. Pat. No. 3,959,457; U.S. Pat. No. 4,205,060; U.S.
Pat. No. 4,606,940; U.S. Pat. No. 4,744,933; U.S. Pat. No.
5,132,117; and International Patent Publication WO 95/28227, all of
which are incorporated herein by reference.
[0125] Liposomes and liposome derivatives (e.g., cochleates,
vesicles) can also be used to provide for the sustained release of
the antigen. Details regarding how to make and use liposomal
formulations can be found, among other places, in U.S. Pat. No.
4,016,100; U.S. Pat. No. 4,452,747; U.S. Pat. No. 4,921,706; U.S.
Pat. No. 4,927,637; U.S. Pat. No. 4,944,948; U.S. Pat. No.
5,008,050; and U.S. Pat. No. 5,009,956, all of which are
incorporated herein by reference.
[0126] In a non-limiting embodiment, the vaccine of the present
invention can be a combination vaccine for protecting swine against
APP and, optionally, one or more other diseases or pathological
conditions that can afflict swine, which combination vaccine
comprises a first component comprising an immunologically effective
amount of an antigen of the present invention selected from the
group consisting of an APP protein, homologous polypeptide, peptide
fragment, fusion protein, analog, derivative, or polynucleotide
molecule of the present invention which is capable of inducing, or
contributing to the induction of, a protective response against APP
in swine; a second component comprising an immunologically
effective amount of an antigen that is different from the antigen
in the first component, and which is capable of inducing, or
contributing to the induction of, a protective response against a
disease or pathological condition that can afflict swine; and a
veterinarily acceptable carrier or diluent.
[0127] The second component of the combination vaccine is selected
based on its ability to induce, or to contribute to the induction
of, a protective response against either APP or another pathogen,
disease, or pathological condition which afflicts swine, as known
in the art. Any immunogenic composition now known or to be
determined in the future to be useful in a vaccine composition for
swine can be used in the second component of the combination
vaccine. Such immunogenic compositions include but are not limited
to those that provide protection against Actinobacillus suis,
Pasteurella multocida, Salmonella cholerasuis, Streptococcus suis,
Erysipelothrix rhusiopathiae, Leptospira sp., Staphylococcus
hyicus, Haemophilus parasuis, Bordetella bronchiseptica, Mycoplasma
hyopneumoniae, Lawsonia intracellularis, Escherichia coli, porcine
reproductive and respiratory syndrome virus, swine influenza virus,
transmissible gastroenteritis virus, porcine parvovirus,
encephalomyocarditis virus, coronavirus, pseudorabies virus, and
circovirus. In a non-limiting embodiment, the combination vaccine
comprises a combination of components including one or more APP
proteins of the present invention, and one or more other APP
bacterial components such as APXI, ApxII, and OmIA.
[0128] The antigen comprising the second component can optionally
be covalently linked to the antigen of the first component to
produce a chimeric molecule. In a non-limiting embodiment, the
antigen of the second component comprises a hapten, the
immunogenicity of which is detectably increased by conjugation to
the antigen of the first component. Chimeric molecules comprising
covalently-linked antigens of the first and second components of
the combination vaccine can be synthesized using one or more
techniques known in the art. For example, a chimeric molecule can
be produced synthetically using a commercially available peptide
synthesizer utilizing standard chemical synthetic processes (see,
e.g., Merrifield, 1985, Science 232:341-347). Alternatively, the
separate antigens can be separately synthesized and then linked
together by the use of chemical linking groups, as known in the
art. Alternatively, a chimeric molecule can be produced using
recombinant DNA technology whereby, e.g., separate polynucleotide
molecules having sequences encoding the different antigens of the
chimeric molecule are spliced together in-frame and expressed in a
suitable transformed host cell for subsequent isolation of the
chimeric fusion polypeptide. Where the vaccine of the invention
comprises a polynucleotide molecule rather than a polypeptide, the
spliced polynucleotide molecule can itself be used in the vaccine
composition. Ample guidance for carrying out such recombinant
techniques is provided, among other places, in Maniatis et al.,
1989, above; Ausubel et al., 1989, above; Sambrook et al., 1989,
above; Innis et al., 1995, above; and Erlich, 1992, above.
[0129] The present invention further provides a method of preparing
a vaccine for protecting swine against APP, comprising combining an
immunologically effective amount of one or more antigens of the
present invention selected from the group consisting of an APP
protein, homologous polypeptide, peptide fragment, fusion protein,
analog, derivative, or polynucleotide molecule of the present
invention which is capable of inducing, or contributing to the
induction of, a protective response against APP in swine, with a
veterinarily acceptable carrier or diluent, in a form suitable for
administration to swine.
[0130] The present invention further provides a method of
vaccinating swine against APP, comprising administering a vaccine
of the present invention to a pig. The amount of antigen
administered depends upon such factors as the age, weight, health
and general physical characteristics of the animal being
vaccinated, as well as the particular vaccine composition to be
administered. Determination of the optimum dosage for each
parameter can be made using routine methods in view, e.g., of
empirical studies. The amount of APP protein administered will
preferably range from about 0.1 .mu.g to about 10 mg of
polypeptide, more preferably from about 10 .mu.g to about 1 mg, and
most preferably from about 25 .mu.g to about 0.1 mg. For a DNA
vaccine, the amount of a polynucleotide molecule will preferably
range from about 0.05 .mu.g to about 500 mg, more preferably from
about 0.5 .mu.g to about 50 mg. In addition, the typical dose
volume of the vaccine will range from about 0.5 ml to about 5 ml
per dose per animal.
[0131] Animals can be vaccinated at any appropriate time, including
within 1 Week after birth, or at weaning age, or just prior to or
at the time of breeding, or at the time that APP infection first
begins to appear in one or more members of an animal population.
Supplemental administrations, or boosters, may be required to
achieve full protection. Methods for determining whether adequate
immune protection has been achieved in an animal are well-known in
the art, and include, e.g., determining seroconversion.
[0132] The vaccine can be administered by any appropriate route
such as, e.g., by oral, intranasal, intramuscular, intra-lymph
node, intradermal, intraperitoneal, subcutaneous, rectal or vaginal
administration, or by a combination of routes. The skilled artisan
will readily be able to formulate the vaccine composition according
to the route chosen.
[0133] The present invention further provides a vaccine kit for
vaccinating swine against infection or disease caused by APP,
comprising a first container comprising an immunologically
effective amount of one or more antigens of the present invention
selected from the group consisting of an APP protein, homologous
polypeptide, peptide fragment, fusion protein, analog, derivative,
or polynucleotide molecule of the present invention which is
capable of inducing, or contributing to the induction of, a
protective response against APP in swine. The kit can optionally
further comprise a second container comprising a veterinarily
acceptable carrier or diluent. The vaccine composition can be
stored in the first container either in solution or in lyophilized
form to be reconstituted using the carrier or diluent of the second
container.
5.6. Anti-APP Antibodies
[0134] The present invention further provides isolated antibodies
that bind to an APP protein of the present invention. Such
antibodies are useful for a variety of purposes including, e.g., as
affinity reagents to purify the APP protein, or to detect the
presence of the APP protein in a cell, tissue or fluid sample
collected from an APP-infected animal, e.g.; by use of an ELISA or
Western blot assay, or as a therapeutic agent to prevent, control
or treat APP infection.
[0135] Antibodies against an APP protein of the, present invention
can be raised according to known methods by administering an
appropriate antigen of the present invention to a host animal
selected from pigs, cows, horses, rabbits, goats, sheep, and mice,
among others. Various adjuvants, such as those described above, can
be used to enhance antibody production. Antibodies of the present
invention can either be polyclonal or monoclonal. Polyclonal
antibodies can be prepared and isolated from the serum of immunized
animals and tested for anti-APP protein specificity using standard
techniques. Alternatively, monoclonal antibodies against an APP
protein can be prepared and isolated using any technique that
provides for the production of antibody molecules by continuous
cell lines in culture. These include but are not limited to the
hybridoma technique originally described by Kohler and Milstein
(Nature, 1975, 256: 495-497); the human B-cell hybridoma technique
(Kosbor et al., 1983, Immunology Today 4:72; Cote et al., 1983,
Proc. Natl. Acad. Sci. USA 80: 2026-2030); and the EBV-hybridoma
technique (Cole et al., 1985, Monoclonal Antibodies and Cancer
Therapy, Alan R. Liss, Inc., pp. 77-96). Alternatively, techniques
described for the production of single chain antibodies (see, e.g.,
U.S. Patent 4,946,778) can be adapted to produce APP
protein-specific single chain antibodies.
[0136] Also encompassed within the scope of the present invention
are antibody fragments that contain specific binding sites for an
APP protein of the present invention. Such fragments include but
are not limited to F(ab').sub.2 fragments, which can be generated
by pepsin digestion of an intact antibody molecule, and Fab
fragments, which can be, generated by reducing the disulfide
bridges of the F(ab').sub.2 fragments. Alternatively, Fab and/or
scFv expression libraries can be constructed (see, e.g., Huse et
al., 1989, Science 246: 1275-1281) to allow rapid identification of
fragments having the desired specificity to an APP protein of the
present invention.
[0137] Techniques for the production and isolation of monoclonal
antibodies and antibody fragments are well-known in the art, and
are additionally described, among other places, in Harlow and Lane,
1988, Antibodies: A Laboratory Manual, Cold Spring Harbor
Laboratory, and in J. W. Goding, 1986, Monoclonal Antibodies:
Principles and Practice, Academic Press, London. All of the
above-cited publications are incorporated herein by reference.
5.7. Diagnostic Kits
[0138] The present invention further provides diagnostic kits. In a
non-limiting embodiment, the diagnostic kit of the present
invention comprises a first container comprising an APP protein,
homologous polypeptide, peptide fragment, fusion protein, analog,
or derivative of the present invention that can specifically bind
to antibodies directed against the APP protein; and a second
container comprising a secondary antibody directed against porcine
antibodies. The secondary antibody preferably comprises a
detectable label. Such a diagnostic kit is useful to detect pigs
that currently are, or have previously been, infected with APP, or
that have seroconverted as a result of vaccination with a vaccine
of the present invention.
[0139] In an alternative embodiment, the present invention provides
a diagnostic kit comprising a first container comprising a primary
antibody that binds to an APP protein; and a second container
comprising a secondary antibody that binds to a different epitope
on the APP protein, or that is directed against the primary
antibody. The secondary antibody preferably comprises a detectable
label. In an alternative embodiment, the diagnostic kit comprises a
container comprising a polynucleotide molecule or oligonucleotide
molecule of the present invention that can specifically hybridize
to, or amplify, an APP-specific polynucleotide molecule. These
latter two diagnostic kits are useful to detect pigs that are
currently infected with APP.
5.8. Anti-Sense Oligonucleotides and Ribozymes
[0140] The present invention further provides oligonucleotide
molecules that include anti-sense oligonucleotides,
phosphorothioates and ribozymes that function to bind to, degrade
and/or inhibit the translation of an APP protein-encoding mRNA.
[0141] Anti-sense oligonucleotides, including anti-sense RNA
molecules and anti-sense DNA molecules, act to directly block the
translation of mRNA by binding to targeted mRNA and thereby
preventing protein translation. For example, antisense
oligonucleotides of at least about 15 bases and complementary to
unique regions of the mRNA transcript sequence encoding an APP
protein can be synthesized, e.g., by conventional phosphodiester
techniques.
[0142] Ribozymes are enzymatic RNA molecules capable of catalyzing
the specific cleavage of RNA. The mechanism of ribozyme action
involves sequence specific hybridization of the ribozyme molecule
to complementary target RNA, followed by endonucleolytic cleavage.
Engineered hammerhead motif ribozyme molecules that specifically
and efficiently catalyze endonucleolytic cleavage of APP protein
mRNA sequences are also within the scope of the invention.
[0143] Specific ribozyme cleavage sites within any potential RNA
target are initially identified by scanning the target molecule for
ribozyme cleavage sites which include the following sequences, GUA,
GUU, and GUC. Once identified, short RNA sequences of between about
15 and 20 ribonucleotides corresponding to the region of the target
gene containing the cleavage site can be evaluated for predicted
structural features, such as secondary structure, that can render
the oligonucleotide sequence unsuitable. The suitability of
candidate targets can also be evaluated by testing their
accessibility to hybridization with complementary oligonucleotides,
using, e.g., ribonuclease protection assays.
[0144] Both the anti-sense oligonucleotides and ribozymes of the
invention can be prepared by known methods. These include
techniques for chemical synthesis such as, e.g., by solid phase
phosphoramadite chemical synthesis. Alternatively, anti-sense RNA
molecules can be generated by in vitro or in vivo transcription of
DNA sequences encoding the RNA molecule. Such DNA sequences can be
incorporated into a wide variety of vectors which incorporate
suitable RNA polymerase promoters such as the T7 or SP6 polymerase
promoters.
[0145] Various modifications to the oligonucleotides of the
invention can be introduced as a means of increasing intracellular
stability and half-life. Possible modifications include but are not
limited to the addition of flanking sequences of ribonucleotides or
deoxyribonucleotides to the 5' and/or 3' ends of the molecule, or
the use of phosphorothioate or 240 -O-methyl rather than
phosphodiesterase linkages within the oligonucleotide backbone.
[0146] The following examples are illustrative only, and are not
intended to limit the scope of the present invention.
6. EXAMPLE
Identification of Novel APP Proteins
[0147] Results of the following experiment demonstrate the
specificities of local antibody responses induced when pigs that
were previously challenged with APP serotype-5 were heterologously
rechallenged with APP serotype-7. The antibody specificities were
used to identify previously unrecognized APP proteins, three of
which (Omp20, OmpW, Omp27) were shown by Western blot analysis to
be present in all twelve APP serotypes. The two additional novel
proteins (OmpA1, OmpA2) were identified following protein fraction
isolation and concentration (see Section 6.1.6, below).
6.1. Materials and Methods
6.1.1. Bacterial Challenge
[0148] The APP serotype-5 culture (strain K-17) used to prepare
porcine challenge material was obtained from Dr. R. A. Schultz,
Avoca, Iowa, USA. The APP serotype-7 culture (strain WF-83) used to
prepare porcine challenge material was obtained from Dr. E. Jones,
Swedeland, Pa., USA.
[0149] Clinically healthy 7-8 week old cross-bred pigs were
obtained from a herd in Nebraska with no previous history of APP
infection, and were housed in isolation facilities at Pfizer Animal
Health, Lincoln, Nebr., according to IACUC guidelines. Animals were
examined by a veterinarian to determine their health status prior
to initiation of the study. Following a 2-week acclimatization
period, pigs were anesthetized using a combination of 100 mg/ml of
Telazol, 50 mg/ml of Xylazine, and 50 mg/ml of Ketamine
administered at a rate of approximately 1 ml/50 lb of body weight,
and intranasally inoculated with 2.6.times.10.sup.6 cfu of APP
serotype-5. Seventy-eight days after primary challenge, six of the
surviving pigs, which had demonstrated clinical APP disease but had
recovered, were again anesthetized as described above. The first
and second of these 6 pigs were intranasally rechallenged with
1.times.10.sup.7 cfu of APP serotype-5 (homologous rechallenge);
the third and fourth of these pigs were intranasally rechallenged
with 1.times.10.sup.8 cfu of APP serotype-7 (heterologous
rechallenge); and the fifth and sixth pigs were intranasally
inoculated with bacterial growth medium only (control). All
challenge inoculums were administered in 1 ml volumes (0.5
ml/nostril).
[0150] All pigs were sacrificed 48 hrs post-rechallenge, their
serum and organs were collected, and tissue pieces from the organs
were cultured in vitro for 24 or 48 hr, as described in Sections
6.1.2 and 6.1.3 below. Antibody-containing supernatants from the
tissue explant cultures were used to compare the memory antibody
profile elicited by heterologous rechallenge to that elicited by
homologous rechallenge or single challenge (control). The
specificity of antibodies produced by the tissue explants in vitro
was determined by Western blot analysis against a panel of whole
bacterial cell preparations representing all 12 APP serotypes. This
analysis established the presence of an antibody profile following
heterologous rechallenge that was distinct from the antibody
profile following homologous rechallenge or single challenge
(control).
[0151] APP serotype-1 (strain 4074), serotype-2 (strain 4226),
serotype-3 (strain 1421), serotype-4 (strain M62), serotype-5
(strain K-17), serotype-6 (strain Femo FE 171D), serotype-7 (strain
WF-83), serotype-8 (strain 405), serotype-9 (strain CVJ-13261),
serotype-10 (strain 13039), serotype-11 (strain 56153), and
serotype-12 (strain 8329) reference cultures used to prepare
material for Western blots were obtained from Dr. B. Fenwick,
Kansas State University, Manhattan, Kans., USA.
6.1.2. Tissue Collection
[0152] Serum samples were obtained from pigs prior to rechallenge,
and again prior to necropsy (48 hr post-rechallenge). Pigs were
euthanized 48 hr post-rechallenge with an overdose of intravenous
pentobarbital. Lungs were removed and examined for characteristic
gross lesions attributable to APP infection, and a complete gross
examination was done of all major organs. Tissue samples of lung,
lymph nodes (mesenteric, popliteal, and bronchial), Peyers patches,
and tonsils were collected, washed with 70% ethanol, and rinsed
3.times. in RPMI transport media (RPMI medium (Gibco/BRL, Grand
Island, N.Y.) supplemented with 10 mM HEPES, 5% FBS, 50 U/ml of
penicillin and 50 .mu.g/ml of streptomycin). After washing, tissues
were placed in 50 ml centrifuge tubes containing 10 ml of transport
media and placed on ice until processed in the laboratory (within 3
hr of collection). Additional tissues samples were frozen in liquid
nitrogen for future mRNA isolation and immunohistochemistry, or
fixed in formalin for histopathology. A sample of lung tissue was
also submitted to the University of Nebraska-Lincoln diagnostic
laboratory for bacterial identification.
6.1.3. Tissue Explant Cultures
[0153] Tissues were placed in individual Petri dishes containing
approximately 5 ml of transport media. Small tissue pieces of
approximately 2.times.2 mm were cut off from the original sample
with a scalpel blade and/or scissors and placed in individual wells
of 12- or 24-well plates (Costar, Cambridge, Mass.) containing 2 ml
of wash media (Ca.sup.2+ and Mg.sup.2+ free Hank's balanced salt
solution (HBSS) supplemented with 10 mM HEPES and 50 .mu.g/ml of
gentamicin). The tissue pieces were rinsed in the wash media and
transferred to another well containing wash media. This
washing/rinsing operation was repeated four times, and the tissue
pieces were then transferred to wells containing RPMI media
supplemented with 10% FBS, 10 mM HEPES, 2 mM glutamine, 50 .mu.g/ml
gentamicin, 62 .mu.g/ml amphotericin B, 40 .mu.g/ml sodium
desoxycholate, 50 U/ml penicillin and 50 .mu.g/ml of streptomycin.
Plates were incubated at 38.5.degree. C. for 24 or 48 hr in a
humidified chamber having 5% CO.sub.2. After incubation,
supernatant fluids were removed and frozen at -70.degree. C.
6.1.4. Western Blot Analysis
[0154] The specificity of recovered antibodies in the tissue
explant supernatants was examined by Western blot analysis as
follows. Representative isolates of each of the 12 APP serotypes
were each grown separately to generate whole bacterial cell antigen
to test the supernatants. Each strain was cultured (1% seed) in
minimal medium-3 (MM3) (1.8% Bacterin HP medium, 1.7% lactic acid,
0.3% glycerol, 0.05M HEPES, 0.011 M L-glutamic acid (monosodium
salt), 5.times.10.sup.-5 M nicotinamide, and 0.2% casamino acids)
supplemented with 10 .mu.g/ml of p-nicotinamide adenine
dinucleotide (.beta.-NAD), for 5-6 hr at 37.degree. C. and 180 rpm
until OD.sub.560 of .about.0.5-0.6. The cells were pelleted by
centrifugation at 12,000.times.g for 10 min, the medium was
reserved for analysis, and the pellet was resuspended in 5 ml
Dulbecco's phosphate buffered saline (DPBS). Prior to the protein
assay, the resuspended pellet was frozen at -20.degree. C. and then
thawed in order to lyse any intact bacterial cells. The protein
concentration of each preparation was determined using a BCA
Protein Assay Reagent Kit (Pierce, Rockford, Ill.). APP antigen
preparations (5 .mu.g/lane) were loaded onto a 4-20% Tris-glycine
gel (Novex, San Diego, Calif.), and proteins were separated by
electrophoresis at rm temp with a constant current of 20 mA.
[0155] Separated proteins were transferred to ProBlot.TM. membranes
(Applied Biosystems, Foster City, Calif.) using a semi-dry graphite
electroblotter (Milliblot, Millipore, Seattle, Wash.). Transfer was
performed at rm temp for 30 min at a constant current of 200 mA.
After transfer was complete, membranes were blocked by incubating
overnight at rm temp with Buffer A (50 mM Tris HCl, 150 mM NaCl, pH
7.4 and 5% (w/v) nonfat dried milk). The blocking buffer was then
decanted and replaced with either serum (1:100 dilution) or tissue
explant culture supernatants (1:3 dilution) in Buffer A, and the
membranes were incubated for 1 hr at rm temp, followed by a 10 min
wash in Buffer B (Buffer A containing 0.2% (v/v) Triton X-100) and
two 10 min washes in Buffer A. After washing was complete, the
membranes were incubated for 1 hr at rm temp with
phosphatase-conjugated goat anti-swine antibodies (Kirkegaard &
Perry Laboratories, Gaithersburg, Md.) diluted 1:1000 in Buffer A.
The membranes were then washed in Buffer A for 10 min, and
incubated for 15 min with
5-bromo4-chloro-3-indolyl-phosphate/nitroblue tetrazolium
(BCIP/NBT) substrate system (Kirkegaard & Perry
Laboratories).
6.1.5. Preparation of APP Serotype-7 Membranes
[0156] An aliquot of APP serotype-7 was seeded (1%) into MM3
supplemented with 10 .mu.g/ml .beta.-NAD and cultured overnight at
37.degree. C. (180 rpm). A portion of the overnight culture was
inoculated into fresh medium (bacterial inoculum was 3% of total
volume) and incubated for 5-6 hr or to a culture density of 274
Klett units. The cells were pelleted by centrifugation at 4,500 rpm
for 40 min at 10.degree. C., the supernatant was removed, and the
pellet was resuspended in 5 ml of 50 mM Tris-HCl, pH 8.0, with
sufficient PMSF (phenyl methylsulfonyl fluoride) to result in a
final concentration of 1 mM PMSF. Bacterial cells were lysed using
a French Press under 16,000 lb/in.sup.2 in a 40K PSI pressure cell
(Sim Aminco., Rochester, N.Y.). The broken cells were centrifuged
at 1,000.times.g for 15 min to remove large bacterial debris. Crude
total membranes were collected by centrifugation at 45,000 rpm for
60 min at 18.degree. C. The supernatant was discarded, the pellet
was resuspended in 50 mM Tris-HCl, pH 8.0, and protein was
determined using the Bradford Standard Protein Assay.
[0157] To 15 mg of crude membrane in a 3 ml volume was added 30
.mu.l of 100 mM PMSF and 750 .mu.l of 2.5% sarkosyl, and the entire
volume was mixed thoroughly. After a 30 min incubation on ice, the
membranes were pelleted by centrifugation at 200,000.times.g for 15
min at 10.degree. C. The supernatant was removed from the pelleted
membrane fraction and the pellet was resuspended in 3 ml of 50 mM
Tris-HCl/100 mM NaCl, pH 8.0. This membrane preparation, which
represented the APP serotype-7 membrane antigen, was then stored at
-20.degree. C.
6.1.6. Membrane Protein Fractionation and Purification
[0158] Purification of APP proteins for N-terminal sequencing was
achieved through continuous-elution SDS-PAGE using a BioRad Model
491 Prep Cell (BioRad, Richmond, Calif.). A 10 ml volume (4.5 mg
total protein) of APP serotype-7 membrane protein fraction was
mixed with an equal volume of non-reducing sample buffer (125 mM
Tris-HCL, pH 6.8, 4% SDS, 20% glycerol, and 0.1% bromphenol blue).
The protein-buffer mixture was boiled for 5 min and applied to a 3%
stacking/15% separation SDS-polyacrylamide gel. Samples were
electrophoresed at 20 mA constant current (initial voltage 175-250
V, final voltage 200-300 V) for 72 hr. Approximately 800.times.5 ml
fractions were collected at a flow rate of 1 ml/min throughout the
run, and analyzed for protein content by spectrophotometry at
A.sub.280. Every 10th fraction was analyzed by SDS-PAGE and silver
staining (Bio-Rad, Richmond, Calif.). Fractions which putatively
contained the same protein, as determined by molecular weight, were
pooled and stored at 4.degree. C. Pooled samples were desalted and
concentrated in preparation for N-terminal sequencing. Desalting
was performed by applying aliquots of pooled sample to a Presto.TM.
desalting column (Pierce, Rockford, Ill.) with a 10 ml bed volume.
Three ml aliquots of each protein pool were applied to separate
columns and eluted in ddH.sub.2O in 10.times.2 ml fractions. This
was repeated 10 times for each protein pool until 30 ml had been
desalted. As determined by Western blot analysis, the majority of
the desalted protein was found in fraction No. 2. Therefore, the
second fraction from each of the 10 elutions were pooled for each
individual protein. The resulting 20 ml samples were lyophilized
and resuspended in 0.5 ml ddH.sub.2O for N-terminal sequencing.
N-terminal sequences were obtained at the Pfizer Central Research
Molecular Sciences Sequence Facility.
6.2. Results
6.2.1. Clinical Signs and Pathological Findings After
Rechallenge
[0159] Pigs did not show any signs of clinical disease after either
homologous (serotype-5) or heterologous (serotype-7) rechallenge.
Pathological examination confirmed that the animals had not
developed lung lesions that would be consistent with acute APP
infection following rechallenge. However, bronchial lymph nodes of
the animals rechallenged with serotype-7 were hemorrhagic and
enlarged compared to those from animals homologously rechallenged
with serotype-5 or from control animals.
6.2.2. Specificity of Antibodies Elicited by Rechallenge
[0160] The specificity of antibodies present in serum and tissue
explant supernatants was assessed by Western blot analysis as
described above. All tissue-derived supernatants collected after 24
or 48 hr incubation contained antibodies that specifically
recognized APP proteins. In general, the reactivity against
serotype-5 antigens was greater than the reactivity against
antigens of serotype-7 or serotype-1. However, the reactivity of
most tissue-derived supernatants was less intense and narrower in
spectrum than the reactivity of serum (FIG. 1a). In general, the
reactivity of tissue explant supernatants had no particular
pattern. Nevertheless, the pattern of Western blot reactivity of
tissue explant supernatants from one specific animal (No. 803,
heterologously rechallenged with serotype-7) was strong, and
highlighted several low molecular weight proteins present in APP
serotypes-1, -5, and -7 (FIG. 1b). This supernatant was used as the
antibody source to further characterize the degree of
cross-reactivity of the secondary (memory) antibody response
elicited by heterologous rechallenge with APP serotype-7 in pigs
that had been challenged initially with APP serotype-5.
[0161] The degree of cross-reactivity of the antibodies in the BLN
tissue explant supernatants from pig No. 803 was assessed by
Western blot analysis using whole bacterial cell antigens prepared
from each of the twelve different APP serotypes. This analysis
showed that three of the low molecular weight proteins recognized
by the antibodies were present in all twelve serotypes (FIG. 2).
Antibodies present in this BLN tissue explant supernatant also
recognized other protein bands. A high molecular weight band
present in serotypes-1, 2, 4, 5, 6, 7, 8, and 9, which may
correspond to the exotoxin, Apx II (see Nakai, 1983, above), and
another protein band present in serotypes 2, 5, 8 and 10,
represented the second most cross-reactive patterns. Western blot
analysis of APP cell pellets and supernatants revealed that
reactivity of antibodies in the BLN tissue explant supernatants to
the low molecular weight proteins was restricted to proteins
present in the cell pellets (FIG. 3), indicating that the proteins
are associated with the bacterial cell and are not secreted.
6.2.3. Proteins Recognized by Cross-Reactive Antibodies
[0162] The low molecular weight proteins were purified as described
above, yielding partially purified preparations containing the
protein of interest as identified by Western blot analysis using:
(a) BLN tissue explant supernatant fluids from pig No. 803; or (b)
serum from pig No. 803 (FIG. 4). Upon fractionation of membrane
proteins, four protein bands with molecular weights of about 19-20,
about 23, about 27, and about 29 kDa, respectively, were identified
using this procedure.
[0163] N-terminal sequence analysis of the proteins in the four
bands yielded a primary sequence and tentative residues (in
parentheses) as shown in TABLE 1, below, and designated therein as
"Pep-1" (SEQ ID NO:11), "Pep-2" (SEQ ID NO:12), "Pep-3" (SEQ ID
NO:13), and "Pep-4" (SEQ ID NO:14). Occasional secondary signals
were observed and were probably due to the presence of minor
contaminants (data not shown). The partial N-terminal sequences
shown in TABLE 1 were used to design probes and primers to obtain
the primary DNA sequences encoding the cross-reactive APP proteins.
Sequence homology comparisons suggested that the four proteins
recognized by the dominant local antibody response elicited after
heterologous rechallenge have not previously been described for
APP.
1TABLE 1 N-Terminal Amino acid Sequences of Low Molecular Weight
APP Proteins Approx. Peptide Mol. Wt Protein Sequence.sup.1 (SEQ ID
NO) (kDa) Name (NH.sub.2 to COOH) Pep-1 (11) 19-20 Omp20
Ala-Pro-Val-Gly-Asn-Thr-Phe-T- hr-Gly-Val-(Lys)-
Val-(Tyr)-Val-Asp-Leu-Thr-Xaa-Val-Al- a Pep-2 (12) 23 OmpW
His-Gln-Ala-Gly-Asp-Val-Ile-Phe-Arg-A- la-Gly-Ala-
Ile-Gly-Val-Ile-Ala-Asn-Ser-Ser-Ser-Asp-Tyr- -(Gln)-
Thr-(Gln)-Ala-Asp-Val-(Asn/Val)-Leu-Asp-Val-Asn- - Asn Pep-3 (13)
27 Omp27 Ala-Glu-Ile-Gly-Leu-Gly-(Gly)-Ala-Arg-Glu-(Ser)-
(Ser)-Ile-Tyr-Tyr-(Ser)-Lys-His-Lys-Val-Ala-Thr-
Asn-Pro-Phe-Leu-Ala-Leu-Asp-Leu Pep-4 (14) 29 OmpA
Ala-(Asp/Glu)-Pro-Glu-Asn-Thr-Phe-Tyr-Pro-Gly-
Ala-Lys-Val-Xaa-Xaa-(Ser)-Xaa-(Phe)-(His) .sup.1Xaa indicates that
the amino acid residue at the particular position could not be
determined.
7. EXAMPLE
Molecular Cloning of DNA Encoding the APP Proteins
7.1. Isolation of Chromosomal DNA And Construction Of Genomic
Libraries
[0164] Genomic DNA from each of the twelve APP serotypes was
separately isolated by either the hexadecyltrimethyl ammonium
bromide (CTAB)-proteinase K method (Ausubel et al., 1988, Curr.
Protocols Mol. Biol. Wiley Interscience, NY), or the DNA Isolator
Genomic DNA Isolation Reagent (Genosys Biotechnologies, Inc., The
Woodlands, Texas). The APP DNA was dissolved in TE buffer (10 mM
Tris-HCl, pH 8.0, 1 mM EDTA) at <1 .mu.g/ml and quantitated by
UV spectrophotometry.
[0165] To facilitate cloning of the APP gene sequences encoding
Omp20, OmpW, Omp27, and OmpA, several genomic libraries were
constructed. These libraries were specifically modified by ligation
of a known sequence (Vectorette II.TM., Genosys Biotechnologies,
Inc., The Woodlands, Texas) to the 5' and 3' ends of restricted DNA
fragments essentially as recommended by the supplier. Thus,
Vectorette libraries were constructed by separately digesting two
pg chromosomal DNA FROM APP 7-1 (serotype 7, passage 1) with
restriction endonuclease BamHI, Bg/II, HindIII, EcoRI, DraI or HpaI
at 37.degree. C. overnight. The reaction was then spiked with
additional fresh restriction enzyme and adjusted to 2 mM ATP, 2 mM
DTT final concentration. Vectorette tailing was carried out by
addition of T4 DNA Ligase (400 U) plus 3 pMol of the appropriate
compatible Vectorette linker (BamHI Vectorette: BamHI, Bg/II;
HindIII Vectorette: HindIII; EcoRI Vectorette: EcoRI; Blunt
Vectorette: DraI, HpaI). The mixture was incubated for three cycles
at 20.degree. C., 60 min; 37.degree. C., 30 min to complete the
tailing reaction, and then adjusted to 200 .mu.l with dH.sub.2O and
stored at -20.degree. C.
7.2. Molecular Cloning of omp20
[0166] Screening of the Vectorette libraries was carried out to
obtain DNA fragments encoding Omp20 and flanking regions.
Degenerate oligonucleotide ER49 (SEQ ID NO: 39) was designed based
on the N-terminal amino acid sequence of this protein (TABLE 1,
Pep-1 (SEQ ID NO:11), aa 1-9).
[0167] For PCR amplification of a fragment of the omp20 gene,
oligonucleotide ER49 (SEQ ID NO: 39) was used in combination with a
Vectorette specific primer, ER70 (SEQ ID NO: 48) in 50 .mu.l
reactions containing 1.times.PCR Buffer II (Perkin Elmer), 1.5 mM
MgCl.sub.2, 200 lM each deoxy-NTP, 100 pMol each primer, and 2.5 U
AmpIITaq Gold (Perkin Elmer) thermostable polymerase. Multiple
single reactions were performed with 5 .mu.l of the Vectorette
libraries as DNA template. Amplification was carried out as
follows: denaturation (95.degree. C., 9 min); 35 cycles of
denaturation (95.degree. C., 30 sec), annealing (55.degree. C., 1
min), and polymerization (72.degree. C., 3 min); followed by a
final extension (72.degree. C., 7 min).
[0168] The amplified products were visualized by separation on a
1.2% agarose gel (Sigma). A 433-bp product resulted from
amplification of the EcoRI Vectorette library. The fragment was
cloned into pGEM.RTM.-T Easy PCR cloning vector (Promega, Madison,
Wis.) and sequenced. Analysis of the sequence confirmed the
identity of the fragment as partially encoding Omp20 based on the
N-terminal amino acid sequence (Pep-1).
[0169] Based on the newly identified sequence of this partial gene,
specific primers ER67 (SEQ ID NO: 46) and ER68 (SEQ ID NO: 47) were
designed to obtain additional 5' and 3' flanking sequences by a
second round of screening the Vectorette libraries by PCR
amplification as described above. Screening of EcoRI, HindIII,
DraI, and HpaI Vectorette libraries by PCR, employing ER68 (SEQ ID
NO: 47) plus ER70 (SEQ ID NO: 48), resulted in successful
amplification of an approximately 600-bp fragment from the DraI
Vectorette library. This fragment was sequenced to determine the 3'
end of the omp20 gene. Since no unique products were observed
during screening of these libraries using ER67 (SEQ ID NO: 46) plus
ER70 (SEQ ID NO: 48), additional specific primers ER71 (SEQ ID NO:
49), ER72 (SEQ ID NO: 50), ER76 (SEQ ID NO:52), and ER77 (SEQ ID
NO:53) were designed to, obtain DNA fragments located 5' of the
ER49 binding site. Such "genome walking" by amplification from
numerous Vectorette DNA libraries was reiterated until the outer
boundaries of the ORF, i.e., translational start and stop codons as
well as flanking nucleotide sequences, were characterized.
Generally, PCR products were sequenced directly or cloned into
pGEM.RTM.-T Easy PCR cloning vector prior to sequence analysis.
7.3. Molecular Cloning of omp27
[0170] Screening of the Vectorette libraries for omp27 was carried
out essentially as described above for omp20, except that
degenerate oligonucleotide ER50 (SEQ ID NO:40), which was designed
based on the N-terminal amino acid sequence of the purified Omp27
protein (TABLE 1, Pep-3 (SEQ ID NO:13), aa 13-24), was used.
Following PCR amplification of Vectorette libraries as described
above, a 152-bp fragment was confirmed to encode a portion of Omp27
based on the N-terminal amino acid sequence of Pep-3 (SEQ ID
NO:13). A second round of genome walking using BamHI, Bg/II,
HindIII, EcoRI, HpaI, and DraI Vectorette libraries and specific
primers ER88 (SEQ ID NO:64), and ER89 (SEQ ID NO:65) resulted in
PCR amplification of an approximately 2 kb fragment from the DraI
library, and an approximately 1.5 kb fragment from the HindIII
library. These DNA fragments were cloned into pGEM.RTM.-T Easy PCR
cloning vector, which allowed for derivation of the nucleotide
sequence 5' and 3' of the omp27 gene, respectively.
7.4. Molecular Cloning of ompA1 And ompA2
[0171] The N-terminal amino acid sequence obtained from the
purified 29 kDa protein (TABLE 1, Pep-4 (SEQ ID NO:14)) was used to
design degenerate N-terminal primers RA22 (SEQ ID NO: 73) and RA34
(SEQ ID NO:77) for use in direct PCR amplification of the gene
encoding the 29 kDa protein from APP chromosomal DNA. The sequence
of Pep-4 was analyzed using a BlastP computer program comparison
(National Center for Biotechnology Information) against the GenBank
protein database (Altschul et al., 1990, J. Mol. Biol.
215:403-10.), and was found to exhibit homology with OmpA proteins
of several different eubacteria, such as Pasteurella multocida, P.
haemolytica, Haemophilus ducreyi, H. somnus, H. influenzae,
Actinobacillus actinomycetemcomitans, E. coli, Shigella, and
Salmonella. N-terminal sequences of the OmpA-related proteins from
the Pasteurellaceae were aligned to produce the consensus sequence
Ala-Pro-Gln-Ala/Glu-Asn-Thr-Phe-Tyr-AlaNal-Gly-Ala-Lys-Ala (SEQ ID
NO:94). These alignments were analyzed and used to design several
additional N-terminal degenerate primers. Oligonucleotide primers
RA53 (SEQ ID NO:81), RA54 (SEQ ID NO:82), RA55 (SEQ ID NO:83), RA56
(SEQ ID NO:84), and RA57 (SEQ ID NO:85) overlap each other, and
were designed to bind to the region encoding aa 4-11, aa 5-12, aa
3-10, aa 1-8, and aa 1-7, respectively, of this consensus
peptide.
[0172] Other degenerate oligonucleotide primers were designed
following alignment of the C-terminal regions of the OmpA-related
proteins. This alignment indicated a highly conserved region near
the C-terminus which included the amino acid sequence
Cys-Leu-Ala-Pro-Asp-Arg-Arg-Val-Glu-Ile (SEQ ID NO:95). Both RA49
(SEQ ID NO:78) and RA50 (SEQ ID NO:79) reverse primers were
designed to bind to the negative DNA strand in this region of OmpA.
These reverse primers were applied in a two-dimensional matrix in
which RA49 (SEQ ID NO:78) and RA50 (SEQ ID NO:79) were each
combined pairwise with RA22 (SEQ ID NO:73), RA34 (SEQ ID NO:77),
RA53 (SEQ ID NO:81), RA54 (SEQ ID NO:82), RA55 (SEQ ID NO:83), RA56
(SEQ ID NO:84), and RA57 (SEQ ID NO:85) in HotStart 50.TM. tubes
(Molecular Bioproducts Inc., San Diego, Calif.) with combined Klen
Taq1 and Pfu polymerases. The following references describe "hot
start" methods: D'Aquila et al., 1991, Nucl. Acids Res. 19:3749;
and Horton et al., 1994, Biotechniques 16:42-43. The cycling
program for PCR was a variant of "touchdown PCR" protocols and was
carried out as follows: denaturation (94.degree. C., 5 min); 30
cycles of denaturation (94.degree. C., 30 sec), annealing
(59.degree. C., 30 sec initial cycle, then -0.1.degree. C. per
additional cycle), and polymerization (72.degree. C., 1 min);
followed by a final extension (72.degree. C., 15 min). The
following references describe "touchdown PCR" protocols: Roux,
1994, BioTechniques, 16:812-814; and Hecker and Roux, 1996,
BioTechniques 20:478-485.
[0173] Among the PCR products generated, an approximately 950-bp
band was produced from reactions with either RA49 (SEQ ID NO:78) or
RA50 (SEQ ID NO:79), when each was combined pairwise with the
forward primers RA34 (SEQ ID NO:77), RA53 (SEQ ID NO:81), RA56 (SEQ
ID NO:84), or RA57 (SEQ ID NO:85). These DNA fragments were cloned
into pGEM.RTM.-T Easy PCR cloning vector and sequenced. Analysis of
these sequenced fragments indicated the existence of two different
variant sequences. Products derived from RA49 (SEQ ID NO:78)/RA57
(SEQ ID NO:85), and RA50 (SEQ ID NO:79)/RA56 (SEQ ID NO:84),
represented variant A1, and the partially encoded protein was
designated as "OmpA1." Products derived from RA50 (SEQ ID
NO:79)/RA34 (SEQ ID NO:77), and RA50 (SEQ ID NO:79)/RA53 (SEQ ID
NO:81) represented variant A2, and the partially encoded protein
was designated as "OmpA2."
[0174] The two similar yet distinct ompA partial DNA sequences were
expanded to include the entire ORFs and flanking 5' and 3'
sequences through genome walking by application of the previously
described Vectorette libraries. Alignment of the partial ompA1 and
ompA2 DNA sequences allowed the design of specific oligonucleotide
primers capable of differentiating these closely related gene
sequences. Outward facing, differentiating primers specific for the
5' or 3' regions of ompA1, i.e., ER55 (SEQ ID NO:41), ER58 (SEQ ID
NO:42), and for ompA2, i.e., ER59 (SEQ ID NO:43), ER62 (SEQ ID
NO:44), respectively, were used to probe Vectorette libraries as
described above. Unique fragments of approximately 1100, 400, 450,
and 280-bp were obtained from an EcoRI Vectorette library when
probed with ER70 (SEQ ID NO:48) plus either ER55 (SEQ ID NO:41),
ER58 (SEQ ID NO:42), ER59 (SEQ ID NO:43), or ER62 (SEQ ID NO:44),
respectively. Sequence analysis of the resulting fragments allowed
determination of the endpoints and flanking regions of both ompA1
and ompA2 ORFs.
7.5. Molecular Cloning of ompW
[0175] The N-terminal amino acid sequence obtained from the
purified 23 kDa protein (TABLE -1, Pep-2 (SEQ ID NO:12)) was used
to design RA20 (SEQ ID NO:72), a degenerate oligonucleotide primer
corresponding to amino acids 1-8 of Pep-2.
[0176] Purified APP DNA was used as a template in a variant of
"gene walking PCR" methods. The single PCR primer RA20 (SEQ ID
NO:72) was used in `hot start` reactions with combined KlenTaq1 (Ab
Peptides, Inc, St. Louis, Mo.) and Pfu (Stratagene, Inc., La Jolla,
Calif.) polymerases. The cycling program for PCR was a variant of
"touchdown PCR" protocols and was carried out as follows:
denaturation (94.degree. C., 5 min); 40 cycles of denaturation
(94.degree. C., 1 min), annealing (63.degree. C., 2 min initial
cycle, then -0.2.degree. C. and -2 sec per additional cycle), and
polymerization (72.degree. C., 1.5 min); followed by a final
extension (72.degree. C., 10 min).
[0177] Among the numerous PCR products generated, an approximately
220-bp product was obtained and cloned into pGEM.RTM.-T Easy PCR
cloning vector. Sequence analysis of this plasmid insert confirmed
that the cloned PCR product encoded amino acids corresponding to a
portion of the 23 kDa protein based on the N-terminal amino acid
sequence of Pep-2 (SEQ ID NO: 12).
[0178] Based on this newly identified sequence, a specific primer,
RA23 (SEQ ID NO:74), was generated for the amplification of
downstream sequences. Genomic, mini-libraries of APP serotype-7 DNA
were constructed by limited digestions with Taq.sup..alpha.I or
HinP1 I, which both create 5'-CG overhangs. This DNA was ligated
into a BspDI-cut pUC21 or pUC128 vector and transformed into E.
coli DH5.alpha. to yield the genomic libraries.
[0179] Mini-preps of these plasmid-borne genomic libraries were
used as templates in "gene walking PCR" using "hot start" methods.
The specific primer RA23 (SEQ ID NO:74), along with vector-specific
M13 Forward and M13 Reverse sequencing primers, was used with
combined KlenTaq and Pfu polymerases. The cycling program for PCR
was carried out as follows: denaturation (94.degree. C., 5 min); 32
cycles of denaturation (94.degree. C., 30 sec), annealing
(63.degree. C., 30 sec initial cycle, then -0.2.degree. C. per
cycle), and polymerization (72.degree. C., 30 sec); followed by a
final extension (72.degree. C., 7 min).
[0180] Among the numerous PCR products generated, a 0.8 kb product
and a 1.4 kb product were cloned into pGEM.RTM.-T Easy PCR cloning
vector and sequenced. Analysis and alignments of the resulting
sequences along with that obtained above yielded the sequence of
the mature protein. In order to obtain the sequence of the 5'
flanking region of the ompW gene, specific primers RA24 (SEQ ID
NO:75) and RA26 (SEQ ID NO:76) were used to probe numerous
Vectorette libraries, as described above. Amplification was carried
out as follows: denaturation (95.degree. C., 9 min); 40 cycles of
denaturation (95.degree. C., 30 sec), annealing (60.degree. C., 1
min), polymerization (72.degree. C., 3 min); followed by a final
extension (72.degree. C., 7 min). Specific 600 and 700-bp products
resulted from probing the HpaI and DraI Vectorette libraries,
respectively. The 700-bp product was directly sequenced to obtain
the nucleotide sequence encompassing the 5'-flanking region, and
encoded the N-terminus of the 23 kDa protein. Due to partial
similarity between the predicted APP 23 kDa protein and the
predicted Vibrio cholerae OmpW protein, the APP gene fragment,was
designated as "ompW."
7.6. Molecular Analysis of Genes Encoding APP Proteins
7.6.1. Specific PCR Amplification of DNA
[0181] Results from the cloning and preliminary sequencing of the
novel APP proteins, as described above, were used to design
oligonucleotide primers for the specific amplification of the
intact omp20, omp27, ompA1, ompA2 and ompW genes directly from APP
serotype-7 chromosomal DNA. This approach was preferred based on
the desire to eliminate the introduction of sequencing artifacts
due to possible mutations arising during the cloning of gene
fragments in E. coli. Accordingly, oligonucleotides which flank the
above intact APP genes were used to specifically amplify those
regions from chromosomal DNA. The 5' and 3' primer pairs utilized
for each gene amplification were as follows: for omp20, the primers
were ER80 (SEQ ID NO:56) and ER81 (SEQ ID NO:57); for omp27, the
primers were ER95 (SEQ ID NO:69) and ER96 (SEQ ID NO:70); for
ompA1, the primers were ER84 (SEQ ID NO:60) and ER86 (SEQ ID
NO:62); for ompA2, the primers were ER87 (SEQ ID NO:63) and ER66
(SEQ ID NO:45); and for ompW, the primers were ER82 (SEQ ID
NO:58).and ER83 (SEQ ID NO:59). PCR reactions were carried out in
triplicate and contained 260 ng purified chromosomal DNA,
1.times.PC2 buffer (Ab Peptides, Inc.), 200 .mu.M each dNTP, 100
pMol each primer, 7.5 U KlenTaq1 and 0.15 U cloned Pfu thermostable
polymerases in a 100 .mu.l final sample volume. Conditions for
amplification consisted of denaturation (94.degree. C., 5 min)
followed by 30 cycles of denaturation (95.degree. C., 30 sec),
annealing (65.degree. C., 30 sec), and polymerization (72.degree.
C., 2 min). A final extension (72.degree. C., 7 min) completed the
amplification of the target intact gene region. Following
amplification, each of the triplicate samples were pooled and the
specific product was purified by agarose gel electrophoresis and
extraction with spin chromatography (QlAquick.TM., QIAGEN Inc.,
Santa Clarita, Calif.) prior to direct sequence analysis using
DyeDeoxy termination reactions on an ABI automated DNA sequencer
(Applied Biosystems, Foster City, Calif.).
[0182] Synthetic oligonucleotide primers were used to sequence both
DNA strands of the amplified products from APP serotype-7. The
primers employed for sequencing the APP protein genes are presented
below in TABLE 2.
[0183] The nucleotide sequence of the ORF of omp20 is presented in
SEQ ID NO:1 from nt 272 to 790. The nucleotide sequence of the ORF
of ompW is presented in SEQ. ID NO:3 from nt 376 to 1023. The
nucleotide sequence of the ORF of omp27 is presented in SEQ ID NO:5
from nt 157 to 933. The nucleotide sequence of the ORF of ompA1 is
presented in SEQ ID NO:7 from nt 614 to 1708. The nucleotide
sequence of the ORF of ompA2 is presented in SEQ ID NO:9 from nt
197 to 1306.
2 TABLE 2 APP Gene Primer (SEQ ID NO) omp20 ER79 (55) ER80 (56)
ER81 (57) AP19.1 (15) AP19.2 (16) AP19.3 (17) AP19.4 (18) AP19.5
(19) omp27 ER88 (64) ER89 (65) ER91 (66) ER94 (68) ER95 (69) ER96
(70) ompA1 ER84 (60) ER85 (61) ER86 (62) AP21.1 (24) AP21.2 (25)
AP21.3 (26) AP21.4 (27) AP21.5 (28) AP21.6 (29) AP21.7 (30) AP21.8
(31) AP21.9 (32) ompA2 ER66 (45) ER87 (63) AP22.1 (33) AP22.2 (34)
AP22.3 (35) AP22.4 (36) AP22.5 (37) AP22.6 (38) ompW ER82 (58) ER83
(59) AP20.1 (20) AP20.2 (21) AP20.3 (22) AP20.4 (23)
7.6.2. Similarity of APP Serotype-7 OmpA1 and OmpA2 Proteins
[0184] The amino acid sequences of the OmpA1 protein (SEQ ID NO:8)
and the OmpA2 protein (SEQ ID NO:10) were deduced from SEQ ID NOS:7
and 9, respectively, and were aligned to compare their similarity.
The deduced OmpA1 protein is 364 amino acids in length, which is 5
amino acids shorter than the deduced OmpA2 protein. The alignment
of the APP proteins shown in FIG. 5 indicates that the two proteins
share 73.1% (270/369) amino acid identity.
7.6.3. Comparison of APP Serotype-7 OmpW Protein to Vibrio Cholerae
OmpW
[0185] The amino acid sequence (SEQ ID NO:4) deduced from the
nucleotide sequence (SEQ ID NO:3) of the ORF encoding the 23 kDa
APP OmpW protein was determined to be most similar to the OmpW
protein described for Vibrio cholerae (Jalajakumari, M. B., et al.,
1990, Nucleic Acids Res. 18(8):2180). The amino acid sequences of
these two proteins were aligned using the Clustal W (ver 1.4)
multiple sequence alignment algorithm (Thompson, J. D., et al.,
1994, Nucleic Acids Res., 22:4673-4680). This comparison indicated
that the APP OmpW and V. cholerae OmpW proteins, which are 215 and
216 residues in length, respectively, share 44.9% (97/216) amino
acid identity. The aligned proteins are shown in FIG. 6.
7.7. Southern Blot Hybridizations
[0186] The conservation of DNA sequences encoding the Omp20, OmpW,
Omp27, OmpA1 and OmpA2 proteins among different APP serotypes was
determined by performing Southern blot hybridization analysis using
each of the 5 different coding sequences as probes against APP DNA
from the different serotypes. Probes were generated with a PCR
DIG.TM. Probe Synthesis Kit (Boehringer Mannheim, Inc.,
Indianapolis, Ind.) according to manufacturer's instructions. For
example, the ompW probe was constructed in the following manner. A
PCR product encompassing the ompW coding sequence was generated
using ompW specific primers and APP serotype-7 DNA. APP serotype-7
genomic DNA (0.2 .mu.g), 1 .mu.M MW3 primer (SEQ ID NO:71), 1 .mu.M
primer RA52 (SEQ ID NO:80), 7.5 U KlenTaq1 polymerase (Ab Peptides,
Inc.), 0.075 U Pfu polymerase (Stratagene), 1.times. KlenTaq1
buffer, and 0.2 mM dNTPs were combined in a 50 .mu.l volume. PCR
was carried out as follows: denaturation (94.degree. C., 5 min); 35
cycles of denaturation (94.degree. C., 30 sec), annealing
(58.degree. C, 30 sec), and polymerization (72.degree. C. 1 min);
and final extension (72.degree. C. for 7 min). The .about.650-bp
ompW PCR product was purified following agarose gel electrophoresis
using a JETsorb.TM. kit (GENOMED, Inc., Research Triangle Park,
N.C.). The purified DNA was quantitated using a Low-Mass DNA
Ladder.TM. mass standard (GIBCO/BRL, Gaithersburg, Md.). A
digoxigenin-labeled probe was generated by PCR amplification of 24
pg of the ompW PCR product, produced as described above, using a
PCR DIG.TM. Probe Synthesis Kit according to manufacturer's
instructions, and the PCR-generated probe was stored unpurified at
-20.degree. C.
[0187] EcoRI-digested APP genomic DNA (1.5 .mu.g) obtained from
each of APP serotypes 1, 2, 5, 7, 8 and 9 was separated by agarose
gel electrophoresis. The DNA profiles were transferred to Hybond-N
0.45 .mu.m nylon membrane (Amersham, Inc., Cleveland, Ohio) using
alkaline transfer with a Turboblotter.TM. Kit (Schleicher &
Schuell, Inc., Keene, N.H.), according to manufacturer's
instructions. The DNA was covalently bound to the membrane by UV
irradiation using a Stratalinker.TM. UV Cross-Linker (Stratagene)
at the auto-crosslink setting (120 MJ/cm.sup.2). The blots were
allowed to dry and were stored at rm temp.
[0188] Nylon DNA blots were incubated in the presence of probe to
allow hybridization for detection of probe sequences in multiple
APP serotypes. Blots were pre-hybridized for 2.5 hr at 68.degree.
C. using an excess (0.2 ml/cm.sup.2) of 1.times. Prehybridization
Solution (GIBCO/BRL). Probe-hybridization solution was prepared by
adding 5.4 .mu.l of the unpurified digoxigenin-labeled probe to 500
.mu.l of 1.times. Hybridization Solution (GIBCO/BRL) and boiling at
100.degree. C. for 10 min. The probe was cooled to 0.degree. C. for
1 min, and then added to sufficient 1.times. Hybridization Solution
to give a total of 0.025 ml/cm.sup.2 of blot. Blots were hybridized
in this probe-hybridization solution mixture at 68.degree. C. for
16 hours. Stringency washes were carried out on blots as follows:
(i) 2 washes with an excess (0.2 ml/cm.sup.2) of 2.times.SSC/0.1%
SDS at 25.degree. C. for 5 min; (ii) 2 washes with an excess (0.2
ml/cm.sup.2) of 0.1.times.SSC/0.1% SDS at 68.degree. C. for 15 min.
Blots were then developed using a chemiluminescence method with a
DIG.TM. High Prime DNA Labeling and Detection Starter Kit II and a
DIG.TM. Wash and Block Buffer Set (Boehringer Mannheim, Inc.,
Indianapolis, Ind.), according to manufacturer's instructions.
Developed blots were exposed to X-ray film for varying lengths of
time to detect hybridizing bands.
[0189] Probes generated as above against omp20, ompW, omp27, ompA1
and ompA2 sequences hybridized with DNA in all the APP serotypes
tested (serotypes 1, 2, 5, 7, 8 and 9). The sizes of the EcoRI
bands detected were identical across all serotypes for ompA1 and
ompA2, but were not conserved for omp20, ompW, and omp27.
[0190] The size of the EcoRI fragments hybridized by the omp20
probe in each serotype was as follows: serotypes-1, 2, 7 and 9 gave
a 5.8 kb fragment; serotype-5 gave a 6.1 kb fragment; serotype-8
gave a 5.0 kb fragment.
[0191] The size of the EcoRI fragments hybridized by the ompW probe
in each serotype was as follows: serotype-1 gave a 1.15 kb
fragment; serotype-2 gave a 1.1 kb fragment; serotype-5 gave a 1.0
kb fragment; serotype-7 gave a 0.9 kb fragment; serotype-8 gave a
1.05 kb fragment; and serotype-9 gave a 1.2 kb fragment.
[0192] The size of the EcoRI fragments hybridized by the omp27
probe in each serotype was as follows: serotypes-1, 2 and 9 gave an
approximately 9.5 kb fragment; serotypes-5, 7 and 8 gave an
approximately 10.5 kb fragment.
[0193] The size of the EcoRI fragments hybridized by the ompA1
probe was 2.3 kb in all serotypes. Weaker hybridizing fragments of
0.55 kb and 0.85 kb were also detected.
[0194] The size of the EcoRI fragments hybridized by the ompA2
probe was 0.85 kb in all serotypes. Weaker hybridizing fragments of
0.55 kb and 2.3 kb were also detected.
8. EXAMPLE
Expression of Recombinant APP Proteins
8.1. Host Strain
[0195] The E. coli host used for recombinant protein expression was
E. coli LW14. The genotype of this strain is .lambda.
IN(rrnD-rrnE)1 gaIE::Tn 10 .lambda.c/857.DELTA.H1 bio. This strain
was provided by SmithKline Beecham Pharmaceuticals, King of
Prussia, Pa., USA, and contains the temperature-sensitive .lambda.
repressor Xc/857 which inhibits expression from .lambda. promoters
at 30.degree. C. At 42.degree. C., the repressor is inactivated and
expression from .lambda. promoters is enabled, yielding high-level
transcription and protein synthesis. E. coli LW14 was propagated at
30.degree. C.
8.2. Plasmid Expression Vectors
[0196] The expression vector used for recombinant protein synthesis
was pEA181, alternatively designated pEA181KanRBS3. This vector is
6.766 kb in size, encodes kanamycin resistance (kan), and contains
the strong .lambda. promoter p.sub.L. The vector contains an NdeI
site just downstream of an optimized ribosome-binding site; the
presence of this NdeI site allows for the precise placement of the
Met start codon of a protein for optimal expression. The vector
also encodes an NS1 leader fusion protein to enable enhanced
expression of poorly expressed proteins. This vector was provided
by SmithKline Beecham Pharmaceuticals (see also U.S. Pat. No.
4,925,799, and Rosenberg et al., 1983, Meth. Enzymol.
101:123-138).
[0197] The coding sequences of each of the five APP proteins were
amplified by PCR using 5' specific primers designed to yield an
NdeI site (CATATG) overlapping the Met start codon (ATG), and 3'
specific primers designed for cloning into the 3' XbaI site in
pEA181. The respective PCR products were initially cloned into the
pGEM.RTM.-T Easy PCR cloning vector (Promega Corp), and transformed
into commercially available competent E. coli DH5.alpha. (MAX
Efficiency DH5.alpha. Competent Cells, GIBCO/BRL). The PCR products
were excised from these plasmid clones as NdeI-XbaI or NdeI-SpeI
fragments and cloned into NdeI/XbaI cut PEA181. Due to the presence
of the strong .lambda. promoter p.sub.L. pEA181 derivatives could
be transformed only into E. coli LW14 which repressed expression
from the vector by the activity of the temperature-sensitive lambda
repressor .lambda.c/857. Transformation and propagation of
transformants bearing pEA181 and its derivatives were done at
30.degree. C. E. coli LW14 was made competent by the method of
Hanahan for the preparation of frozen competent cells (Hanahan,
1985, In: DNA Cloning: A Practical Approach (Glover, D:, ed.) Vol
1, pp. 109-135, IRL Press, Oxford, England).
[0198] Due to difficulties with expression of mature OmpW in E.
coil LW14, a leader peptide allowing enhanced protein synthesis was
employed. This leader peptide, termed a "protective peptide" or
"pp", protects recombinant proteins from proteolytic degradation,
as based upon information from Sung et al., 1986, Proc. NatI. Acad.
Sci. USA 83:561-565; Sung et al., 1987, Meth. Enzymol. 153:385-389;
and U.S. Pat. No. 5,460,954, which references are incorporated
herein by reference. The protective peptide, which consists of the
amino acid sequence Met-Asn-Thr-Thr-Thr-Thr-Thr-Thr-Ser-Arg (SEQ ID
NO:96), was fused to the N-terminus of each of the APP proteins by
designing PCR primers to contain the protective peptide coding
sequence upstream from the APP coding sequence. Amplification of
the separate APP coding sequences with such primers generated
sequences encoding the N-terminal protective peptide fused to the
first amino acid in the mature (i.e., lacking the native signal
sequence) APP protein. An NdeI site was positioned at the Met codon
in the protective peptide so that it could be ligated into the NdeI
site of pEA181. The primer pairs for the amplification of the
protective peptide-APP protein coding sequences were as follows:
for ompW, MW3 (SEQ ID NO:71) and RA52 (SEQ ID NO:80); for ompA1,
RA78 (SEQ ID NO:88) and RA71 (SEQ ID NO:87); for ompA2, RA78 (SEQ
ID NO:88) and RA69 (SEQ ID NO:86); for omp20, ER78 (SEQ ID NO:54)
and ER73 (SEQ ID NO:51); and for omp27, ER92 (SEQ ID NO: 67) and
ER94 (SEQ ID NO: 68).
8.3. Expression of Recombinant Proteins
[0199] The E. coli LW14 transformants bearing pEA181 derivatives
encoding protective peptide fusions with the respective mature APP
proteins OmpA1, OmpA2, Omp20, OmpW and Omp27, were propagated
overnight at 30.degree. C. in LB Km.sup.50 (Luria Broth with 50
.mu.g/ml kanamycin sulfate). The cultures were diluted 1:100 into
2.times.YT Km.sup.50 medium (1.6% tryptone, 1% yeast extract, 0.5%
NaCl, 1.25 mM NaOH, containing 50 pg/ml kanamycin sulfate) and were
grown at 30.degree. C. until A.sub.600 was 0.8 to 1.0. The cultures
were then shifted to 42.degree. C. in a water bath incubator and
incubated for 3 to 4 hr.
[0200] Wet cells of E. coli LW14 transformants from a 5 liter
fermentation grown in 2.times.YT Km.sup.50 medium, and expressing
either pp-OmpA1, pp-OmpA2, or pp-OmpW, were harvested by
centrifugation. The cells were suspended in 0.1M Tris-HCl, pH 8.0,
and lysed in a high pressure homogenizer. Inclusion bodies were
collected by centrifugation (12,000 RCF, 30 min), and washed once
or twice with 2.times.RIPA/TET which was in a 5:4 ratio.
2.times.RIPA is 20 mM Tris (pH 7.4), 0.3 M NaCl, 2.0% sodium
deoxycholate, and 2% (v/v) Igepal CA-630.TM. (Sigma).
[0201] TET is 0.1 M Tris (pH 8.0), 50 mM EDTA, and 2% (v/v) Triton
X-100. The inclusion bodies were dissolved in 5 M
guanidine-hydrochloride, adjusted to >1.4 mg/ml protein in 2.5 M
guanidine-hydrochloride, and filter-sterilized (0.2 .mu.m). This
preparation was used for the vaccination trials as described
below.
[0202] Wet cells of E. coli LW14 transformants from a 5 liter
fermentation grown in 2.times.YT Km.sup.50 medium, and expressing
pp-Omp20, were harvested by centrifugation. The cells were
suspended in 25% sucrose -50 mM Tris-HCl, pH 8.0with lysozyme
(cells dispersed in 0.5 ml sucrose buffer for every 50 ml culture;
for each ml of sucrose buffer, 0.125 ml lysozyme solution at 10
mg/ml was added), and sonicated. Inclusion bodies were collected by
centrifugation (12,000 RCF, 30 min), washed with 2.times.RIPA/TET
as above, collected again by centrifugation, and washed with 0.1M
glycine and Zwittergent 3-14 (Calbiochem) at pH 11. The pH was
adjusted to 7.0, and the inclusion bodies were collected by
centrifugation (12,000 RCF, 30 min), dissolved in 3.5 M guanidine
hydrochloride (final protein concentration, 6.36 mg/ml ), and
filter-sterilized (0.2 .mu.m). This preparation was used for the
vaccination trials as described below.
[0203] Wet cells of E. coli LW14 transformants from a 1600 ml flask
culture grown in 2.times.YT Km.sup.50 medium, as described above,
and expressing pp-Omp27, were harvested by centrifugation. The
cells were suspended in 25% sucrose -50 mM Tris-HCl, pH 8.0, with
lysozyme, as above, sonicated, washed with 2.times.RIPA/TET as
above, and collected by centrifugation. The inclusion bodies were
collected by centrifugation (.12,000 RCF, 30 min), dissolved in 5 M
guanidine hydrochloride (final protein concentration, 2.46 mg/ml),
and filter-sterilized (0.2 .mu.m). This preparation was used for
the vaccination trials as described below.
9. EXAMPLE
Immunological Characterization of Recombinant APP Proteins
9.1. Materials and Methods
9.1.1. Preparation and Quantification of APP Whole Cell Antigens
for Western Blot
[0204] Whole bacterial cell antigens were prepared as described
above in Section 6.1.4, except that HP growth medium was
substituted for the MM3 medium and the cells were suspended in 10
ml of DPBS instead of 5 ml of DPBS. The protein concentration of
each preparation was determined using a BCA Protein Assay kit
(Pierce). In brief, each sample was diluted 1/10, 1/20, 1/40, and
1/80 in sterile deionized, distilled water (ddH.sub.2O). BSA
(protein standard) was diluted to concentrations ranging from 200
to 800 .mu.g/ml. A 20 .mu.l volume of sample or standard was added
to triplicate wells in a 96-well microtitre plate, and 200 .mu.l of
Reagent B diluted 1/50 in Reagent A was added to each well. The
plate was incubated at 37.degree. C. for 30 min. Sample absorbance
was determined at 560 nm. The protein concentration for each sample
was calculated by extrapolation using the BSA standard curve.
9.1.2. Antibodies
[0205] The secondary antibodies used for Western blots included
alkaline phosphatase-conjugated goat anti-porcine IgG (H+L) and
goat anti-mouse IgG (H+L) (Kirkegaard and Perry Laboratories).
These antibodies were used to visualize APP protein-specific
antibody in serum or supernatant samples by Western blot analysis.
Both antibodies were diluted 1/1000 in dilution buffer (PBS, 0.05%
Tween 20, 5% skim milk powder) prior to use.
9.1.3. Vaccination Protocol
[0206] Recombinantly-expressed protein preparations, prepared as
described above in Section 8.3 were diluted to 80 .mu.g/ml in DPBS,
and then combined 1:1 with 2.times. concentrated SEAM-1 adjuvant
(80 .mu.g/ml Quil A, 16 .mu.g/ml cholesterol, 5% squalene, 1% Span
85, 0.1% vitamin A acetate, 0.1% ethanol, and 0.01% thimerosol).
Male CF1 mice were injected s.c. with 0.25 ml of protein/adjuvant
preparation equivalent to 10 .mu.g recombinant protein, 10 .mu.g
Quil A and 2 .mu.g of cholesterol. Negative (adjuvant) control
groups received 0.25 ml of DPBS mixed 1:1 with adjuvant. The mice
were vaccinated a second time with the same protein preparation at
20-22 days post-primary vaccination. Two weeks after the second
vaccination, animals were anesthetized with CO.sub.2 and bled by
either the brachial artery or through cardiac puncture. The serum
was separated from each blood sample, and serum pools from mice
within the same group were stored at -20.degree. C.
9.1.4. Western Blot Analysis
[0207] A volume of APP whole bacterial cell lysate (from APP
serotype 1, 2, 5, 7, or 9; prepared as described above in Section
9.1.1) corresponding to 10 .mu.g of protein was mixed with water to
a final volume of 10 .mu.l, and 2 .mu.l of 5.times. reducing sample
buffer (Pierce) was added. In a similar manner, an aliquot of
recombinant protein (see Section 8.3 above) (protein load was
variable) was also prepared. Samples were heated for 5 min at
100.degree. C., and the entire volume was loaded into separate
wells of a 15-well, 1.5 mm thick, 14% Tris-glycine gel (Novex).
Pre-stained, broad-range molecular weight markers (5 .mu.l/well)
(BioRad) were also included in each gel. Separated proteins in
selected gels were stained with Coomassie Blue.
[0208] Proteins separated by SDS-PAGE were transferred to PVDF
membranes (BioRad) at 200 mA constant current for 1.5 hr. The blots
were either: (i) incubated directly in blocking buffer (5% skim
milk powder and 0.05% Tween 20 in PBS); or (ii) dried at rm temp,
stored frozen at -20.degree. C. until needed, then rehydrated in
methanol, rinsed in water, and subsequently incubated in blocking
buffer. Membranes were incubated in blocking buffer (also used as
dilution buffer) overnight with gentle agitation. The blocking
buffer was removed, and diluted serum or supernatant sample was
added to the membrane, followed by a 1 hr incubation at rm temp.
After removal of the test sample, membranes were washed 3 times for
5-10 min each time with PBST (PBS with 0.05% Tween 20). Alkaline
phosphatase-conjugated anti-murine or anti-porcine IgG (H+L)
antibodies were diluted, added to washed membrane, and incubated
for 1 hr at rm temp. The membranes were washed with PBST, and the
substrate BCIP/NBT (Kirkegaard and Perry Laboratories) was added to
the membranes and incubated with gentle agitation until a suitable
color reaction developed. The membranes were then rinsed with water
to stop the reaction and dried at rm temp.
9.2. Results
[0209] The antigenic characteristics of the novel APP proteins were
determined using the following three methods. The first method used
pig antibody probes, i.e., convalescent pig sera or ASC
supernatants, obtained from animals experimentally infected with
either APP serotype-1 or serotype-5, or serotype-5 followed by
rechallenge with serotype-7, as immunological probes in Western
blots containing the recombinantly-expressed APP proteins (TABLE
3). The second method used sera from mice immunized with the
recombinantly-expressed APP proteins to probe Western blots
containing APP antigens (whole bacterial cell pellets) (TABLE 4).
The third method used sera from mice immunized with the
recombinantly-expressed APP proteins to probe Western blots
containing the recombinantly-expressed APP proteins (TABLE 5). The
results of each method are described below.
9.2.1. Recognition of Recombinant APP Proteins by Pig Antibody
Probes Generated Against APP Serotypes
[0210] Antibody probes (sera or ASC supernatants) were obtained
from pigs following experimental challenge with either APP
serotype-1, or serotype-5, or serotype-5 followed by rechallenge
with serotype-7. The sera were used to originally identify the
novel APP proteins (FIGS. 1-4). TABLE 3 summarizes the reactivity
of the antibody probes with the recombinantly-expressed APP
proteins by Western blotting. ASC probes generated following
challenge with serotype-5 and rechallenge with serotype-7
recognized OmpW, OmpA1, OmpA2, and Omp20 recombinant proteins. The
ASC probes did not react immunologically with recombinant Omp27. In
contrast, sera derived from animals that were challenged with
serotype-1, or serotype-5, or serotype-5 followed by rechallenge
with serotype-7, only recognized OmpA1 and OmpA2 recombinant
proteins. An ASC probe obtained from a non-challenged control pig
(No. 780) did not react with any of the recombinantly-expressed
proteins. However, an additional non-challenged control pig (No.
779) reacted with all recombinantly-expressed proteins. In
addition, serum from a non-challenged control pig (No. 1421)
reacted with OmpA1 and OmpA2. These latter two animals were
suspected of having been subclinically infected with APP.
9.2.2. Recognition of APP Proteins by Mouse Antisera Generated
Against the Recombinant APP Proteins
[0211] Antisera from mice immunized with the
recombinantly-expressed APP proteins were used to probe Western
blots containing APP antigens from bacterial cell pellets. Results
are summarized in TABLE 4. Mice immunized with recombinant pp-OmpW,
or pp-OmpA1, or pp-OmpA2 produced serum antibodies that recognized
specific bands consistent with the predicted molecular weights of
the particular APP protein. However, sera from mice immunized with
either recombinant Omp20 or Omp27 did not react specifically with
the particular native protein in any serotype tested.
9.2.3. Recognition of Recombinant APP Proteins by Mouse Antisera
Generated Against the Recombinant APP Proteins
[0212] Antisera from mice immunized with the
recombinantly-expressed novel proteins were used to probe Western
blots containing the recombinant APP proteins. Results are
summarized in TABLE 5. Sera from mice immunized with recombinant
OmpW, either as a GST or pp fusion protein, recognized recombinant
OmpW, OmpA1, and OmpA2 proteins. Sera from mice immunized with
either recombinant OmpA1 or recombinant OmpA2 reacted strongly with
both OmpA1 and OmpA2 immunogens. Sera from mice immunized with
recombinant Omp20 reacted strongly with recombinant Omp20, and to a
lesser degree with recombinant OmpA1 and OmpA2. In contrast, sera
from mice vaccinated with recombinant Omp27 did not recognize
recombinant Omp27, but did react with recombinant OmpA1 and OmpA2.
Sera from control mice vaccinated with PBS reacted very weakly with
recombinant OmpA1 and OmpA2, and did not recognize recombinant
OmpW, Omp20 or Omp27.
[0213] In summary, pigs that had been experimentally infected with
APP produced local antibodies (ASC probes) that recognized OmpW,
OmpA1, OmpA2, and Omp20 recombinant proteins, whereas serum
antibodies only reacted with recombinant OmpA1 and OmpA2, as
demonstrated by Western blotting (TABLE 3). Serum thus appears to
be much more restricted in terms of immunological reactivity than
the ASC probes. Neither serum nor ASC probes recognized
recombinantly-expressed Omp27. It is possible that Omp27 is not
recognized in a Western blot assay due to the denaturing conditions
of the assay.
[0214] Immunological characterization of recombinant OmpW, OmpA1,
and OmpA2 indicates that these proteins can induce serum antibodies
that recognize the native proteins (based upon the predicted
molecular weight) found in serotypes 1, 2, 5, 7, 8 and 9 (TABLE 4),
as well as recognize the recombinantly-derived forms of these
proteins (TABLE 5), and where recombinant Omp20 was also
recognized.
[0215] Sera from mice immunized with recombinant Omp20 or Omp27.
were used to probe Western blots containing whole bacterial cell
antigens derived from in-vitro grown APP serotypes 1, 2, 5, 7, 8,
and 9 (TABLE 4). These sera did not recognize bands consistent with
their native form in any of the APP serotypes examined. It is
possible that Omp20 and Omp27 represent antigens of APP that are
only expressed in vivo, and thus would not be present in bacterial
cell pellets prepared in the laboratory. Alternatively, these two
proteins may have been denatured by the Western blotting procedure
and rendered unrecognizable to specific antibodies.
3TABLE 3 Reactivity Of Porcine Convalescent Sera And ASC
Supernatants Against Recombinant APP Proteins Recombinant
Proteins.sup.1 Ab source.sup.2 Animal No. APP challenge.sup.3 OmpW
OmpA1 OmpA2 Omp20 Omp27 Serum 1157 Serotype 1 -.sup.4 + + - - Serum
642 Serotype 5 - + + - - Serum 803 Serotype 5 & 7 - + + - -
Serum 1421 None - + + - - ASC probe 803 Serotype 5 & 7 + + + +
- ASC probe 808 Serotype 5 & 7 + + + + - ASC probe 779 None + +
+ + ND.sup.5 ASC probe 780 None - - - - ND .sup.1Each of the
recombinant proteins contained the protective peptide (pp), lacked
the native signal sequence, and was derived from inclusion bodies
(IB). .sup.2Serum, diluted 1/125, or ASC probes, diluted 1/4, were
tested for antibodies specific for each of the recombinant
proteins. .sup.3Animals were challenged with the APP serotype
indicated. Animals No. 803 and 808 were challenged with serotype-5,
allowed to recover from infection, and were then rechallenged with
serotype-7. .sup.4(+) indicates the presence of a band
corresponding to the indicated recombinant protein; (-) indicates
that the serum or ASC probe did not react with the specified
recombinant protein. .sup.5ND = not determined.
[0216]
4TABLE 4 Reactivity Of Serum From Mice Immunized With Recombinant
APP Proteins to APP Whole Cell Preparations APP Antigen.sup.1
Immunogen.sup.2 Serotype-1 Serotype-2 Serotype-5 Seratype-7
Serotype-8 Serotype-9 OmpW-GST.sup.3 +.sup.4 + + + + + pp-OmpW + +
+ + + + pp-OmpA1 + + + + + + pp-OmpA2 + + + + + + pp-Omp20 - - - -
- - pp-Omp27 - - - - - - PBS - - - - - - .sup.1Proteins in whole
bacterial cell preparations (10 .mu.g per lane) were separated by
SDS-PAGE, transferred to PVDF membranes, and probed with mouse
serum (1/50). .sup.2Mice were immunized twice s.c. with either a
recombinant APP: protein preparation or PBS (control). .sup.3All
recombinant APP proteins used for mouse immunizations were
solubilized inclusion body preparations. All APP proteins contained
the protective peptide except for OmpW which was utilized as either
an OmpW-GST fusion protein or a pp-OmpW fusion protein. .sup.4(+)
indicates the presence of a band corresponding to the recombinant
protein used to immunize the animal; (-) indicates the absence of a
specific band.
[0217]
5TABLE 5 Reactivity Of Serum From Mice Immunized With Recombinant
APP Proteins Against The Recombinant APP Proteins Antigen.sup.1
Immunogen.sup.2 OmpW.sup.3 OmpA1 OmpA2 Omp20 Omp27 OmpW-GST.sup.4
+.sup.5 + + - ND.sup.6 pp-OmpW + + + - ND pp-OmpA1 - ++ ++ - ND
pp-OmpA2 - ++ ++ - ND pp-Omp20 - + + ++ ND pp-Omp27 - + + - - PBS
+/- +/- - - .sup.1Recombinant proteins separated by SDS-PAGE were
transferred to PVDF membranes and probed with mouse serum (1/50).
.sup.2Mice were immunized twice s.c. with a recombinant protein
preparation or PBS (control). .sup.3Each of the recombinant APP
proteins in the SDS-PAGE gels contained the protective peptide (pp)
and lacked the native signal sequence. .sup.4All recombinant APP
proteins used for mouse immunizations were from solubilized
inclusion body preparations. All proteins contained the protective
peptide except for OmpW which was utilized as either an OmpW-GST
fusion protein or a pp-OmpW fusion protein, and all proteins lacked
the native signal sequence. .sup.5(+) indicates that the test serum
reacted with the specified recombinant protein; (-) indicates the
absence of a specific band; for the PBS control, (+/-) indicates
that these bands were visible but very faint as compared to serum
from an immunized animal. (++) indicates a very strong
immunoreactivity. .sup.6ND = not determined.
EXAMPLE 10
Animal Study to Test Efficacy of Various Antigen Combinations
10.1 Materials and Methods
[0218] Fifty apparently healthy, crossbred pigs (approximately 6.5
weeks of age) were obtained from a herd with no history of APP
disease or vaccination. Animals were randomly assigned by litter
and by ApxII cytolytic neutralization antibody titer to five groups
of 10 pigs (98% of the animals had serum neutralization titers
<1:200 prior to the initiation of study). Pigs were acclimated
for one week prior to initiation of study.
[0219] Animals were vaccinated with 2 ml of the appropriate vaccine
(APP proteins with pp and without signal sequence) or placebo by
the intramuscular route (IM; left neck muscle) on day 0, when pigs
were approximately 7.5 weeks of age. After 3 weeks, animals were
boosted with a second 2 ml dose (IM; right neck muscle). TABLE 6
identifies the vaccines used for first and second vaccinations of
the 5 groups of pigs.
6TABLE 6 Vaccine Group Vaccine Components A 75 .mu.g pp-OmpW 75
.mu.g pp-OmpA1 75 .mu.g pp-OmpA2 75 .mu.g pp-Omp20 75 .mu.g
pp-Omp27 75 .mu.g rApxI 75 .mu.g rApxII 75 .mu.g rOmIA (5)
Adjuvanted with 500 .mu.g Quil A/200 .mu.g cholesterol B 75 .mu.g
rApxI 75 .mu.g rApxII 75 .mu.g rOmIA (5) Adjuvanted with 500 .mu.g
Quil A/200 .mu.g cholesterol C 75 .mu.g pp-OmpW 75 .mu.g pp-OmpA1
75 .mu.g pp-OmpA2 75 .mu.g pp-Omp20 75 .mu.g pp-Omp27 Adjuvanted
with 500 .mu.g Quil A/200 .mu.g cholesterol D Commercial Vaccine
(whole cell APP bacterin containing serotypes 1, 5, and 7), with
Emulsigen .RTM. adjuvant E Phosphate Buffered Saline adjuvanted
with 500 .mu.g Quil A/200 .mu.g cholesterol
[0220] All pigs were observed for approximately 1 hr following
vaccinations for vomiting, depression, diarrhea,
ataxia-incoordination, increased respiration, and trembling. In
addition, daily observations were made for 3 days following first
and second vaccinations. Rectal temperatures were recorded one day
prior to vaccination, immediately prior to vaccination, 6 hr
following vaccination, and 1 day post-vaccination.
[0221] Two weeks following the second vaccination, pigs were
challenged intranasally with a live virulent culture of APP
serotype-1 (ATCC strain 27088) which causes approximately 50%
mortality in non-immune pigs. A dose of 1.0 ml (0.5 ml per nostril)
of culture containing 1.5.times.10.sup.7 cfu/ml was used. All
animals were anesthetized prior to challenge with an i.m. injection
consisting of a combination of 50 mg telazol, 50 mg xylazine, and
50 mg ketamine per ml at the rate of 1.0 ml/50 pounds of body
weight.
10.2. Results
[0222] No significant elevations in temperature were seen following
first or second vaccinations with the recombinant proteins of the
present invention. Significant post-vaccinal site reactions were
observed in animals that received the commercial vaccine as
compared to all other groups (TABLE 7). None of the animals that
received the novel APP proteins alone (Group C), and only 1 animal
that received a second vaccination of the novel APP proteins and
ApxI/ApxII/OmIA(5) combination (Group A), exhibited post-vaccinal
site reactions.
7TABLE 7 Second Vaccination First Vaccination Site Site Reactions
(cm.sup.3)* Reactions (cm.sup.3)* Vaccine Group (% Affected
Animals) (% Affected Animals) A 0 (0) 2 (10) B 2 (10) 17 (30) C 0
(0) 0 (0) D 16 (30) 68 (70) E 0 (0) 0 (0) *Numbers represent group
average
[0223] Group A, which was vaccinated with all of the novel APP
proteins plus ApxI/ApxII/OmIA(5), had lower mortality than any
other group, including the commercial vaccine (30% vs. 60%) (TABLE
8). The amount of lung damage (% lesions) was also less in Group A
as compared to Groups B, C, and.controls, but similar to the lung
damage seen in animals that received the commercial vaccine (Group
D).
8TABLE 8 Vaccine Group Average Lesion (%) Mortality (%) A 58 30 B
73 70 C 67 70 D 55 60 E 73 60
[0224] These results indicate that a vaccine comprised of the novel
APP proteins of the present invention in combination with toxin
antigens provides protection against a heterologous APP challenge,
which protection is equivalent or superior to that of a commercial
vaccine.
11. EXAMPLE
Preparation of Plasmids and Deposit Materials
[0225] Separate plasmid constructs were prepared encoding each of
the APP proteins for deposit with the American Type Culture
Collection (ATCC). Each construct contains the total ORF encoding
the particular APP protein with its native signal sequence. The
ORFs were inserted in the TA cloning site of pCR2.1Topo in the
opposite orientation relative to the lactose promoter. The ORFs
were obtained by PCR from APP serotype-7 genomic DNA using the
primers listed below in TABLE 9. The host cells were E. coli Top1
0. Both host cells and vector are available from Invitrogen
(Carlsbad, Calif.). The 5' primers all begin at the ATG start codon
of the respective ORF.
[0226] The strains prepared as above, and listed in TABLE 9 below,
were deposited with the ATCC at 10801 University Blvd, Manassas,
Va., 20110, USA, on Oct. 15, 1998, and were assigned the listed
accession numbers.
9TABLE 9 5' Primer ATCC (SEQ 3' Primer Construct Strain Accession
Protein ID NO) (SEQ ID NO) Name Name No. Omp20 ER218 (89) ER73 (51)
pER416 Pz416 98926 Omp27 ER219 (90) ER94 (68) pER417 Pz417 98927
OmpW ER220 (91) RA52 (80) pER418 Pz418 98928 OmpA1 ER221 (92) RA71
(87) pER419 Pz419 98929 OmpA2 ER222 (93) RA69 (86) pER420 Pz420
98930
[0227] All patents, patent applications, and publications cited
above are incorporated herein by reference in their entirety.
[0228] The present invention is not to be limited in scope by the
specific embodiments described, which are intended as single
illustrations of individual aspects of the invention. Functionally
equivalent compositions and methods are within the scope of the
invention.
Sequence CWU 1
1
96 1 1018 DNA Actinobacillus pleuropneumoniae CDS (272)..(787) 1
ggttggaaaa caccttatga agtttacttc aaaaaatcgt tgcacttggt ttgacaattc
60 aagactaaaa atgaccggtc gtgagctaaa accgcatgac cgtactgtgg
atgtgacgat 120 tcgtcgtatt cgtaaacact ttgaagatca ccctaataca
ccggaaatca ttgtaaccat 180 tcatggtgaa ggttaccgtt tttgcggcga
gttagagtag taattaaacg cctataagcg 240 tttagcatct tctttctaaa
aaggacattt t atg aaa aat tta aca gtt tta 292 Met Lys Asn Leu Thr
Val Leu 1 5 gca tta gca ggt tta ttc tct gcg tcg gca ttt gcc gca ccg
gtc gga 340 Ala Leu Ala Gly Leu Phe Ser Ala Ser Ala Phe Ala Ala Pro
Val Gly 10 15 20 aat acc ttt acc ggc gta ggc gta ggc gtt gat ctc
acc acg gta aaa 388 Asn Thr Phe Thr Gly Val Gly Val Gly Val Asp Leu
Thr Thr Val Lys 25 30 35 tat aaa gtg gac ggt gtg aaa ggt aaa caa
tca acc ggt cct gcg tta 436 Tyr Lys Val Asp Gly Val Lys Gly Lys Gln
Ser Thr Gly Pro Ala Leu 40 45 50 55 gtc gta gat tac ggt atg gat tac
ggt gac aat ttt gtc ggt gtt gta 484 Val Val Asp Tyr Gly Met Asp Tyr
Gly Asp Asn Phe Val Gly Val Val 60 65 70 caa ggt aaa gta aaa gta
ggc agt aca aaa gta ttt agc gat gta aaa 532 Gln Gly Lys Val Lys Val
Gly Ser Thr Lys Val Phe Ser Asp Val Lys 75 80 85 caa aaa act aaa
tat act gtc gct tat caa caa ggt tat cgt gta gct 580 Gln Lys Thr Lys
Tyr Thr Val Ala Tyr Gln Gln Gly Tyr Arg Val Ala 90 95 100 tct gat
tta ctt ccg tat gtc aaa gtc gat gtg gcg caa agt aaa gtc 628 Ser Asp
Leu Leu Pro Tyr Val Lys Val Asp Val Ala Gln Ser Lys Val 105 110 115
ggc gat acc aat ttc cgt ggt tac ggt tac ggt gcc ggt gct aaa tat 676
Gly Asp Thr Asn Phe Arg Gly Tyr Gly Tyr Gly Ala Gly Ala Lys Tyr 120
125 130 135 gcc gta tca agt aat gta gaa gtg ggt gcg gaa tat acg cgc
agc aat 724 Ala Val Ser Ser Asn Val Glu Val Gly Ala Glu Tyr Thr Arg
Ser Asn 140 145 150 tta aga aaa agc ggt gct aaa tta aaa ggt aat gaa
ttt act gcg aac 772 Leu Arg Lys Ser Gly Ala Lys Leu Lys Gly Asn Glu
Phe Thr Ala Asn 155 160 165 cta ggt tac cgt ttc taattatttt
tcccttatga caagcggtcg tttcttgcaa 827 Leu Gly Tyr Arg Phe 170
aaaatttgcg aaaaacgacc gcttattttt ttattaatac tttatttact gagccatttt
887 ttcagctacg gttagaaaac cgtctgcagt cgcatagatt tcttcaaagc
cttgcgcttg 947 tagaatacgg tcggacactt cacgaaatgc gccctctcca
cctgcctttt ctaatatcca 1007 atccgctttt g 1018 2 172 PRT
Actinobacillus pleuropneumoniae 2 Met Lys Asn Leu Thr Val Leu Ala
Leu Ala Gly Leu Phe Ser Ala Ser 1 5 10 15 Ala Phe Ala Ala Pro Val
Gly Asn Thr Phe Thr Gly Val Gly Val Gly 20 25 30 Val Asp Leu Thr
Thr Val Lys Tyr Lys Val Asp Gly Val Lys Gly Lys 35 40 45 Gln Ser
Thr Gly Pro Ala Leu Val Val Asp Tyr Gly Met Asp Tyr Gly 50 55 60
Asp Asn Phe Val Gly Val Val Gln Gly Lys Val Lys Val Gly Ser Thr 65
70 75 80 Lys Val Phe Ser Asp Val Lys Gln Lys Thr Lys Tyr Thr Val
Ala Tyr 85 90 95 Gln Gln Gly Tyr Arg Val Ala Ser Asp Leu Leu Pro
Tyr Val Lys Val 100 105 110 Asp Val Ala Gln Ser Lys Val Gly Asp Thr
Asn Phe Arg Gly Tyr Gly 115 120 125 Tyr Gly Ala Gly Ala Lys Tyr Ala
Val Ser Ser Asn Val Glu Val Gly 130 135 140 Ala Glu Tyr Thr Arg Ser
Asn Leu Arg Lys Ser Gly Ala Lys Leu Lys 145 150 155 160 Gly Asn Glu
Phe Thr Ala Asn Leu Gly Tyr Arg Phe 165 170 3 1188 DNA
Actinobacillus pleuropneumoniae CDS (376)..(1020) 3 ctaacgcata
aagtaaatgt gccggttcaa tgtagttatt atcttttccg atagctaacg 60
attgggcttc tgcaagggct tcttgcaatt tggtagtaaa tttttcgaaa ttcatatttt
120 tactcctaaa tttcattaat ctgtatcgag cagaatttat accgcttcaa
cgttttaata 180 aatggagcta gtcgaactta tttcaagtga aaatgtgaaa
aagatcgcaa aaaataaatt 240 agtacctcgt tgtaggtact aaaatggcgt
atatttgatt cttgtcaata aaagttagcc 300 gaattgttct tagaatgtta
ttaacgtaac gaattggtta cttttttatt tttaagaaaa 360 tattaagagg tcaaa
atg aaa aaa gca gta tta gcg gca gta tta ggc ggt 411 Met Lys Lys Ala
Val Leu Ala Ala Val Leu Gly Gly 1 5 10 gcg tta tta gcg ggt tcg gca
atg gca cat caa gcg ggc gat gtg att 459 Ala Leu Leu Ala Gly Ser Ala
Met Ala His Gln Ala Gly Asp Val Ile 15 20 25 ttc cgt gcc ggt gcg
atc ggt gtg att gca aat tca agt tcg gat tat 507 Phe Arg Ala Gly Ala
Ile Gly Val Ile Ala Asn Ser Ser Ser Asp Tyr 30 35 40 caa acc ggg
gcg gac gta aac tta gat gta aat aat aat att cag ctt 555 Gln Thr Gly
Ala Asp Val Asn Leu Asp Val Asn Asn Asn Ile Gln Leu 45 50 55 60 ggt
tta acc ggt acc tat atg tta agt gat aat tta ggt ctt gaa tta 603 Gly
Leu Thr Gly Thr Tyr Met Leu Ser Asp Asn Leu Gly Leu Glu Leu 65 70
75 tta gcg gca aca ccg ttt tct cac aaa atc acc ggt aag tta ggt gca
651 Leu Ala Ala Thr Pro Phe Ser His Lys Ile Thr Gly Lys Leu Gly Ala
80 85 90 aca gat tta ggc gaa gtg gca aaa gta aaa cat ctt ccg ccg
agc ctt 699 Thr Asp Leu Gly Glu Val Ala Lys Val Lys His Leu Pro Pro
Ser Leu 95 100 105 tac tta caa tat tat ttc ttt gat tct aat gcg aca
gtt cgt cca tac 747 Tyr Leu Gln Tyr Tyr Phe Phe Asp Ser Asn Ala Thr
Val Arg Pro Tyr 110 115 120 gtt ggt gcc ggt tta aac tat act cgc ttt
ttc agt gct gaa agt tta 795 Val Gly Ala Gly Leu Asn Tyr Thr Arg Phe
Phe Ser Ala Glu Ser Leu 125 130 135 140 aaa ccg caa tta gta caa aac
tta cgt gtt aaa aaa cat tcc gtc gca 843 Lys Pro Gln Leu Val Gln Asn
Leu Arg Val Lys Lys His Ser Val Ala 145 150 155 ccg att gcg aat tta
ggt gtt gat gtg aaa tta acg gat aat cta tca 891 Pro Ile Ala Asn Leu
Gly Val Asp Val Lys Leu Thr Asp Asn Leu Ser 160 165 170 ttc aat gcg
gca gct tgg tac aca cgt att aaa act act gcc gat tat 939 Phe Asn Ala
Ala Ala Trp Tyr Thr Arg Ile Lys Thr Thr Ala Asp Tyr 175 180 185 gat
gtt ccg gga tta ggt cat gta agt aca ccg att act tta gat cct 987 Asp
Val Pro Gly Leu Gly His Val Ser Thr Pro Ile Thr Leu Asp Pro 190 195
200 gtt gta tta ttc tca ggt att agc tac aaa ttc taagtatttt
gaaactgtta 1040 Val Val Leu Phe Ser Gly Ile Ser Tyr Lys Phe 205 210
215 tgagaaaggg agcgttaatc gctccctttt tgttgtaaaa aatccttgaa
aaacgaccgc 1100 ttgttaagca caaaaatgta ggatcatttt agtgagcaat
tcacgagtcg gctcaataaa 1160 ttttgtttct aaaaattcat ccggctgg 1188 4
215 PRT Actinobacillus pleuropneumoniae 4 Met Lys Lys Ala Val Leu
Ala Ala Val Leu Gly Gly Ala Leu Leu Ala 1 5 10 15 Gly Ser Ala Met
Ala His Gln Ala Gly Asp Val Ile Phe Arg Ala Gly 20 25 30 Ala Ile
Gly Val Ile Ala Asn Ser Ser Ser Asp Tyr Gln Thr Gly Ala 35 40 45
Asp Val Asn Leu Asp Val Asn Asn Asn Ile Gln Leu Gly Leu Thr Gly 50
55 60 Thr Tyr Met Leu Ser Asp Asn Leu Gly Leu Glu Leu Leu Ala Ala
Thr 65 70 75 80 Pro Phe Ser His Lys Ile Thr Gly Lys Leu Gly Ala Thr
Asp Leu Gly 85 90 95 Glu Val Ala Lys Val Lys His Leu Pro Pro Ser
Leu Tyr Leu Gln Tyr 100 105 110 Tyr Phe Phe Asp Ser Asn Ala Thr Val
Arg Pro Tyr Val Gly Ala Gly 115 120 125 Leu Asn Tyr Thr Arg Phe Phe
Ser Ala Glu Ser Leu Lys Pro Gln Leu 130 135 140 Val Gln Asn Leu Arg
Val Lys Lys His Ser Val Ala Pro Ile Ala Asn 145 150 155 160 Leu Gly
Val Asp Val Lys Leu Thr Asp Asn Leu Ser Phe Asn Ala Ala 165 170 175
Ala Trp Tyr Thr Arg Ile Lys Thr Thr Ala Asp Tyr Asp Val Pro Gly 180
185 190 Leu Gly His Val Ser Thr Pro Ile Thr Leu Asp Pro Val Val Leu
Phe 195 200 205 Ser Gly Ile Ser Tyr Lys Phe 210 215 5 1171 DNA
Actinobacillus pleuropneumoniae CDS (157)..(930) 5 tatttgagct
taggctttaa taaagctcga atcctaagcc aggaaatata gaaagtacat 60
taaatataat ttagtattgt attaatagag gataaagcca caaactggca agcaagaatt
120 ggttttactt tttaacctca ctaaaaggag acaact atg aaa cat agc aaa ttc
174 Met Lys His Ser Lys Phe 1 5 aaa tta ttt aaa tat tat tta att agc
ttt cct ttt att act ttt gca 222 Lys Leu Phe Lys Tyr Tyr Leu Ile Ser
Phe Pro Phe Ile Thr Phe Ala 10 15 20 agt aat gtt aat gga gcc gaa
att gga ttg gga gga gcc cgt gag agt 270 Ser Asn Val Asn Gly Ala Glu
Ile Gly Leu Gly Gly Ala Arg Glu Ser 25 30 35 agt att tac tat tct
aaa cat aaa gta gca aca aat ccc ttt tta gca 318 Ser Ile Tyr Tyr Ser
Lys His Lys Val Ala Thr Asn Pro Phe Leu Ala 40 45 50 ctt gat ctt
tct tta ggt aat ttt tat atg aga ggg act gca gga att 366 Leu Asp Leu
Ser Leu Gly Asn Phe Tyr Met Arg Gly Thr Ala Gly Ile 55 60 65 70 agc
gaa ata gga tat gaa caa tct ttc act gac aat ttc agc gta tca 414 Ser
Glu Ile Gly Tyr Glu Gln Ser Phe Thr Asp Asn Phe Ser Val Ser 75 80
85 ctg ttt gtt aac cca ttt gat ggt ttt tca att aaa gga aaa gac ttg
462 Leu Phe Val Asn Pro Phe Asp Gly Phe Ser Ile Lys Gly Lys Asp Leu
90 95 100 tta cct gga tat caa agt att caa act cgc aaa act caa ttt
gcc ttt 510 Leu Pro Gly Tyr Gln Ser Ile Gln Thr Arg Lys Thr Gln Phe
Ala Phe 105 110 115 ggt tgg gga tta aat tat aat ttg gga ggt tta ttc
ggc tta aat gat 558 Gly Trp Gly Leu Asn Tyr Asn Leu Gly Gly Leu Phe
Gly Leu Asn Asp 120 125 130 act ttt ata tcc ttg gaa gga aaa agc gga
aaa cgt ggt gcg agt agt 606 Thr Phe Ile Ser Leu Glu Gly Lys Ser Gly
Lys Arg Gly Ala Ser Ser 135 140 145 150 aat gtc agc tta ctt aaa tcg
ttt aat atg acg aaa aat tgg aaa gtt 654 Asn Val Ser Leu Leu Lys Ser
Phe Asn Met Thr Lys Asn Trp Lys Val 155 160 165 tca cca tat att ggc
tca agt tat tat tca tct aaa tat aca gat tat 702 Ser Pro Tyr Ile Gly
Ser Ser Tyr Tyr Ser Ser Lys Tyr Thr Asp Tyr 170 175 180 tac ttt ggt
att aaa caa tcc gaa tta ggt aat aaa att aca tcc gta 750 Tyr Phe Gly
Ile Lys Gln Ser Glu Leu Gly Asn Lys Ile Thr Ser Val 185 190 195 tat
aaa cct aaa gca gct tat gca aca cac ata ggt att aat act gat 798 Tyr
Lys Pro Lys Ala Ala Tyr Ala Thr His Ile Gly Ile Asn Thr Asp 200 205
210 tat gct ttc acg aac aat ctt ggc atg ggt tta tct gtc ggt tgg aat
846 Tyr Ala Phe Thr Asn Asn Leu Gly Met Gly Leu Ser Val Gly Trp Asn
215 220 225 230 aaa tat tct aaa gaa att aag caa tct cct atc ata aaa
cga gac tct 894 Lys Tyr Ser Lys Glu Ile Lys Gln Ser Pro Ile Ile Lys
Arg Asp Ser 235 240 245 caa ttt act tca tct ctt agc ctt tat tat aag
ttc taaaatagaa 940 Gln Phe Thr Ser Ser Leu Ser Leu Tyr Tyr Lys Phe
250 255 tattctaggg agaatactca ttctttatct ttataaagtt aattgtttct
ccctgtttct 1000 atattattta gttacttgtt caaaagctac attggttatt
ttgtcatttt ataaaagata 1060 ataaggtggt tattttgaaa attaagaaat
atattaaata taccctattt actttccttt 1120 taggcatatc atatttatat
tttgggggcg aaaacgaaaa ttatcaagag a 1171 6 258 PRT Actinobacillus
pleuropneumoniae 6 Met Lys His Ser Lys Phe Lys Leu Phe Lys Tyr Tyr
Leu Ile Ser Phe 1 5 10 15 Pro Phe Ile Thr Phe Ala Ser Asn Val Asn
Gly Ala Glu Ile Gly Leu 20 25 30 Gly Gly Ala Arg Glu Ser Ser Ile
Tyr Tyr Ser Lys His Lys Val Ala 35 40 45 Thr Asn Pro Phe Leu Ala
Leu Asp Leu Ser Leu Gly Asn Phe Tyr Met 50 55 60 Arg Gly Thr Ala
Gly Ile Ser Glu Ile Gly Tyr Glu Gln Ser Phe Thr 65 70 75 80 Asp Asn
Phe Ser Val Ser Leu Phe Val Asn Pro Phe Asp Gly Phe Ser 85 90 95
Ile Lys Gly Lys Asp Leu Leu Pro Gly Tyr Gln Ser Ile Gln Thr Arg 100
105 110 Lys Thr Gln Phe Ala Phe Gly Trp Gly Leu Asn Tyr Asn Leu Gly
Gly 115 120 125 Leu Phe Gly Leu Asn Asp Thr Phe Ile Ser Leu Glu Gly
Lys Ser Gly 130 135 140 Lys Arg Gly Ala Ser Ser Asn Val Ser Leu Leu
Lys Ser Phe Asn Met 145 150 155 160 Thr Lys Asn Trp Lys Val Ser Pro
Tyr Ile Gly Ser Ser Tyr Tyr Ser 165 170 175 Ser Lys Tyr Thr Asp Tyr
Tyr Phe Gly Ile Lys Gln Ser Glu Leu Gly 180 185 190 Asn Lys Ile Thr
Ser Val Tyr Lys Pro Lys Ala Ala Tyr Ala Thr His 195 200 205 Ile Gly
Ile Asn Thr Asp Tyr Ala Phe Thr Asn Asn Leu Gly Met Gly 210 215 220
Leu Ser Val Gly Trp Asn Lys Tyr Ser Lys Glu Ile Lys Gln Ser Pro 225
230 235 240 Ile Ile Lys Arg Asp Ser Gln Phe Thr Ser Ser Leu Ser Leu
Tyr Tyr 245 250 255 Lys Phe 7 1922 DNA Actinobacillus
pleuropneumoniae CDS (614)..(1705) 7 acggtaacta cttattcttc
tcatgttgca acgccaattc ttgcagagaa attaatcccg 60 atgttacaaa
aaggcgactt aggggagccg acacctgctg ctgaaatcga caacgtttac 120
ttacgtgata tcaacgatgc aatccgtaac catccggttg aattaatcgg tcaagagtta
180 cgtggttata tgacggatat gaaacgtatt tcatcgcaag gttaattaaa
aattaatcaa 240 aagcctactt cgcaagaagt gggctttttg ttattcaagc
cgcttaccgc tatcaatggt 300 aagtgatatg cataatagct tataaattat
aagttgtttt aagcaaatat atctctatcg 360 gtaagtaaaa aattaattgt
agatcattaa aaaacggata aaaaaatcta ttttttgagt 420 gaattacgca
taaaaaatga tctaaattat aggtttagtt gtatttttca atttttattt 480
ggtagaatac aactgtaata aaagcttaat tattttgaga tacacataaa ataatttacg
540 gctttattca ttatcccttt taggttaggg atttgtcttt aatagatgac
gataaattta 600 gaggatcatc aaa atg aaa aaa tca tta gtt gct tta aca
gta tta tcg 649 Met Lys Lys Ser Leu Val Ala Leu Thr Val Leu Ser 1 5
10 gct gca gcg gta gct caa gca gcg cca caa caa aat act ttc tac gca
697 Ala Ala Ala Val Ala Gln Ala Ala Pro Gln Gln Asn Thr Phe Tyr Ala
15 20 25 ggt gcg aaa gca ggt tgg gcg tca ttc cat gat ggt atc gaa
caa tta 745 Gly Ala Lys Ala Gly Trp Ala Ser Phe His Asp Gly Ile Glu
Gln Leu 30 35 40 gat tca gct aaa aac aca gat cgc ggt aca aaa tac
ggt atc aac cgt 793 Asp Ser Ala Lys Asn Thr Asp Arg Gly Thr Lys Tyr
Gly Ile Asn Arg 45 50 55 60 aat tca gta act tac ggc gta ttc ggc ggt
tac caa att tta aac caa 841 Asn Ser Val Thr Tyr Gly Val Phe Gly Gly
Tyr Gln Ile Leu Asn Gln 65 70 75 gac aaa tta ggt tta gcg gct gaa
tta ggt tat gac tat ttc ggt cgt 889 Asp Lys Leu Gly Leu Ala Ala Glu
Leu Gly Tyr Asp Tyr Phe Gly Arg 80 85 90 gtg cgc ggt tct gaa aaa
cca aac ggt aaa gcg gac aag aaa act ttc 937 Val Arg Gly Ser Glu Lys
Pro Asn Gly Lys Ala Asp Lys Lys Thr Phe 95 100 105 cgt cac gct gca
cac ggt gcg aca atc gca tta aaa cct agc tac gaa 985 Arg His Ala Ala
His Gly Ala Thr Ile Ala Leu Lys Pro Ser Tyr Glu 110 115 120 gta tta
cct gac tta gac gtt tac ggt aaa gta ggt atc gca tta gta 1033 Val
Leu Pro Asp Leu Asp Val Tyr Gly Lys Val Gly Ile Ala Leu Val 125 130
135 140 aac aat aca tat aaa aca ttc aat gca gca caa gag aaa gtg aaa
act 1081 Asn Asn Thr Tyr Lys Thr Phe Asn Ala Ala Gln Glu Lys Val
Lys Thr 145 150 155 cgt cgt ttc caa agt tct tta att tta ggt gcg ggt
gtt gag tac gca 1129 Arg Arg Phe Gln Ser Ser Leu Ile Leu Gly Ala
Gly Val Glu Tyr Ala 160 165 170 att ctt cct gaa tta gcg gca cgt gtt
gaa tac caa tgg tta aac aac 1177 Ile Leu Pro Glu Leu Ala Ala Arg
Val Glu Tyr Gln Trp Leu Asn Asn 175 180 185 gca ggt aaa gca agc tac
tct act tta aat cgt atg ggt gca act gac 1225 Ala Gly Lys Ala Ser
Tyr Ser Thr Leu Asn Arg Met Gly Ala Thr Asp 190 195 200 tac cgt tcg
gat atc agt tcc gta tct gca ggt tta agc tac cgt ttc
1273 Tyr Arg Ser Asp Ile Ser Ser Val Ser Ala Gly Leu Ser Tyr Arg
Phe 205 210 215 220 ggt caa ggt gcg gta ccg gtt gca gct ccg gca gtt
gaa act aaa aac 1321 Gly Gln Gly Ala Val Pro Val Ala Ala Pro Ala
Val Glu Thr Lys Asn 225 230 235 ttc gca ttc agc tct gac gta tta ttc
gca ttc ggt aaa tca aac tta 1369 Phe Ala Phe Ser Ser Asp Val Leu
Phe Ala Phe Gly Lys Ser Asn Leu 240 245 250 aaa ccg gct gcg gca aca
gca tta gat gca atg caa acc gaa atc aat 1417 Lys Pro Ala Ala Ala
Thr Ala Leu Asp Ala Met Gln Thr Glu Ile Asn 255 260 265 aac gca ggt
tta tca aat gct gcg atc caa gta aac ggt tac acg gac 1465 Asn Ala
Gly Leu Ser Asn Ala Ala Ile Gln Val Asn Gly Tyr Thr Asp 270 275 280
cgt atc ggt aaa gaa gct tca aac tta aaa ctt tca caa cgt cgt gcg
1513 Arg Ile Gly Lys Glu Ala Ser Asn Leu Lys Leu Ser Gln Arg Arg
Ala 285 290 295 300 gaa aca gta gct aac tac atc gtt tct aaa ggt gct
ccg gca gct aac 1561 Glu Thr Val Ala Asn Tyr Ile Val Ser Lys Gly
Ala Pro Ala Ala Asn 305 310 315 gta act gca gta ggt tac ggt gaa gca
aac cct gta acc ggc gca aca 1609 Val Thr Ala Val Gly Tyr Gly Glu
Ala Asn Pro Val Thr Gly Ala Thr 320 325 330 tgt gac aaa gtt aaa ggt
cgt aaa gca tta atc gct tgc tta gca ccg 1657 Cys Asp Lys Val Lys
Gly Arg Lys Ala Leu Ile Ala Cys Leu Ala Pro 335 340 345 gat cgt cgt
gtt gaa gtt caa gtt caa ggt act aaa gaa gta act atg 1705 Asp Arg
Arg Val Glu Val Gln Val Gln Gly Thr Lys Glu Val Thr Met 350 355 360
taatttagtt aattttctaa agttaaatta gtaaccctct tgcttattta agcaagaggg
1765 ttattttttt gttccatttt aattagtgct actcttcctg tgtttatatt
tgtgtttatg 1825 ataaactctt cataactttt attcacttat agatgaaaat
gaaatacagc ttaacccctt 1885 tccatacctt tcatttagcg gcaaatgcaa caaaatc
1922 8 364 PRT Actinobacillus pleuropneumoniae 8 Met Lys Lys Ser
Leu Val Ala Leu Thr Val Leu Ser Ala Ala Ala Val 1 5 10 15 Ala Gln
Ala Ala Pro Gln Gln Asn Thr Phe Tyr Ala Gly Ala Lys Ala 20 25 30
Gly Trp Ala Ser Phe His Asp Gly Ile Glu Gln Leu Asp Ser Ala Lys 35
40 45 Asn Thr Asp Arg Gly Thr Lys Tyr Gly Ile Asn Arg Asn Ser Val
Thr 50 55 60 Tyr Gly Val Phe Gly Gly Tyr Gln Ile Leu Asn Gln Asp
Lys Leu Gly 65 70 75 80 Leu Ala Ala Glu Leu Gly Tyr Asp Tyr Phe Gly
Arg Val Arg Gly Ser 85 90 95 Glu Lys Pro Asn Gly Lys Ala Asp Lys
Lys Thr Phe Arg His Ala Ala 100 105 110 His Gly Ala Thr Ile Ala Leu
Lys Pro Ser Tyr Glu Val Leu Pro Asp 115 120 125 Leu Asp Val Tyr Gly
Lys Val Gly Ile Ala Leu Val Asn Asn Thr Tyr 130 135 140 Lys Thr Phe
Asn Ala Ala Gln Glu Lys Val Lys Thr Arg Arg Phe Gln 145 150 155 160
Ser Ser Leu Ile Leu Gly Ala Gly Val Glu Tyr Ala Ile Leu Pro Glu 165
170 175 Leu Ala Ala Arg Val Glu Tyr Gln Trp Leu Asn Asn Ala Gly Lys
Ala 180 185 190 Ser Tyr Ser Thr Leu Asn Arg Met Gly Ala Thr Asp Tyr
Arg Ser Asp 195 200 205 Ile Ser Ser Val Ser Ala Gly Leu Ser Tyr Arg
Phe Gly Gln Gly Ala 210 215 220 Val Pro Val Ala Ala Pro Ala Val Glu
Thr Lys Asn Phe Ala Phe Ser 225 230 235 240 Ser Asp Val Leu Phe Ala
Phe Gly Lys Ser Asn Leu Lys Pro Ala Ala 245 250 255 Ala Thr Ala Leu
Asp Ala Met Gln Thr Glu Ile Asn Asn Ala Gly Leu 260 265 270 Ser Asn
Ala Ala Ile Gln Val Asn Gly Tyr Thr Asp Arg Ile Gly Lys 275 280 285
Glu Ala Ser Asn Leu Lys Leu Ser Gln Arg Arg Ala Glu Thr Val Ala 290
295 300 Asn Tyr Ile Val Ser Lys Gly Ala Pro Ala Ala Asn Val Thr Ala
Val 305 310 315 320 Gly Tyr Gly Glu Ala Asn Pro Val Thr Gly Ala Thr
Cys Asp Lys Val 325 330 335 Lys Gly Arg Lys Ala Leu Ile Ala Cys Leu
Ala Pro Asp Arg Arg Val 340 345 350 Glu Val Gln Val Gln Gly Thr Lys
Glu Val Thr Met 355 360 9 1319 DNA Actinobacillus pleuropneumoniae
CDS (197)..(1303) 9 atgtaatatt aggactggaa agttcgaaat tacaaattga
tattacaaat tgattgtagt 60 tttgcttttt atccttgata attaactctc
ttttttctct agtgagacga gagcattaaa 120 tcaaaacttt ggtgccataa
gcggtgctga agtgattttg ttttattaat cgatgacaat 180 ttagaggatc atcaaa
atg aaa aaa tca tta gtt gct tta gca gta tta tca 232 Met Lys Lys Ser
Leu Val Ala Leu Ala Val Leu Ser 1 5 10 gct gca gca gta gct caa gca
gct cca caa caa aat act ttc tac gca 280 Ala Ala Ala Val Ala Gln Ala
Ala Pro Gln Gln Asn Thr Phe Tyr Ala 15 20 25 ggt gcg aaa gtt ggt
caa tca tca ttt cac cac ggt gtt aac caa tta 328 Gly Ala Lys Val Gly
Gln Ser Ser Phe His His Gly Val Asn Gln Leu 30 35 40 aaa tct ggt
cac gat gat cgt tat aat gat aaa aca cgt aag tat ggt 376 Lys Ser Gly
His Asp Asp Arg Tyr Asn Asp Lys Thr Arg Lys Tyr Gly 45 50 55 60 atc
aac cgt aac tct gta act tac ggt gta ttc ggc ggt tac caa atc 424 Ile
Asn Arg Asn Ser Val Thr Tyr Gly Val Phe Gly Gly Tyr Gln Ile 65 70
75 tta aac caa aac aat ttc ggt tta gcg act gaa tta ggt tat gat tac
472 Leu Asn Gln Asn Asn Phe Gly Leu Ala Thr Glu Leu Gly Tyr Asp Tyr
80 85 90 tac ggt cgt gta cgt ggt aac gat ggt gaa ttc cgt gca atg
aaa cac 520 Tyr Gly Arg Val Arg Gly Asn Asp Gly Glu Phe Arg Ala Met
Lys His 95 100 105 tct gct cac ggt tta aac ttt gcg tta aaa cca agc
tac gaa gta tta 568 Ser Ala His Gly Leu Asn Phe Ala Leu Lys Pro Ser
Tyr Glu Val Leu 110 115 120 cct gac tta gac gtt tac ggt aaa gta ggt
gtt gcg gtt gtt cgt aac 616 Pro Asp Leu Asp Val Tyr Gly Lys Val Gly
Val Ala Val Val Arg Asn 125 130 135 140 gac tat aaa tcc tat ggt gca
gaa aac act aac gaa cca aca gaa aaa 664 Asp Tyr Lys Ser Tyr Gly Ala
Glu Asn Thr Asn Glu Pro Thr Glu Lys 145 150 155 ttc cat aaa tta aaa
gca tca act att tta ggt gca ggt gtt gag tac 712 Phe His Lys Leu Lys
Ala Ser Thr Ile Leu Gly Ala Gly Val Glu Tyr 160 165 170 gca att ctt
cct gaa tta gcg gca cgt gtt gaa tac caa tac tta aac 760 Ala Ile Leu
Pro Glu Leu Ala Ala Arg Val Glu Tyr Gln Tyr Leu Asn 175 180 185 aaa
gcg ggt aac tta aat aaa gca tta gtt cgt tca ggc aca caa gat 808 Lys
Ala Gly Asn Leu Asn Lys Ala Leu Val Arg Ser Gly Thr Gln Asp 190 195
200 gtg gac ttc caa tat gct cct gat atc cac tct gta aca gca ggt tta
856 Val Asp Phe Gln Tyr Ala Pro Asp Ile His Ser Val Thr Ala Gly Leu
205 210 215 220 tca tac cgt ttc ggt caa ggc gct gta gca cca gtt gtt
gag cca gaa 904 Ser Tyr Arg Phe Gly Gln Gly Ala Val Ala Pro Val Val
Glu Pro Glu 225 230 235 gtt gta act aaa aac ttc gca ttc agc tca gac
gtt tta ttt gat ttc 952 Val Val Thr Lys Asn Phe Ala Phe Ser Ser Asp
Val Leu Phe Asp Phe 240 245 250 ggt aaa tca agc tta aaa cca gca gca
gca aca gct tta gac gca gct 1000 Gly Lys Ser Ser Leu Lys Pro Ala
Ala Ala Thr Ala Leu Asp Ala Ala 255 260 265 aac act gaa atc gct aac
tta ggt tta gca act cca gct atc caa gtt 1048 Asn Thr Glu Ile Ala
Asn Leu Gly Leu Ala Thr Pro Ala Ile Gln Val 270 275 280 aac ggt tat
aca gac cgt atc ggt aaa gaa gct tca aac tta aaa ctt 1096 Asn Gly
Tyr Thr Asp Arg Ile Gly Lys Glu Ala Ser Asn Leu Lys Leu 285 290 295
300 tca caa cgc cgt gca gaa act gta gct aac tac tta gtt tct aaa ggt
1144 Ser Gln Arg Arg Ala Glu Thr Val Ala Asn Tyr Leu Val Ser Lys
Gly 305 310 315 caa aac cct gca aac gta act gca gta ggt tac ggt gaa
gca aac cca 1192 Gln Asn Pro Ala Asn Val Thr Ala Val Gly Tyr Gly
Glu Ala Asn Pro 320 325 330 gta acc ggc gca aca tgt gac aaa gtt aaa
ggt cgt aaa gca tta atc 1240 Val Thr Gly Ala Thr Cys Asp Lys Val
Lys Gly Arg Lys Ala Leu Ile 335 340 345 gct tgc tta gca ccg gat cgt
cgt gtt gaa gtt caa gta caa ggt gct 1288 Ala Cys Leu Ala Pro Asp
Arg Arg Val Glu Val Gln Val Gln Gly Ala 350 355 360 aaa aac gta gct
atg taatatagtg ggtttt 1319 Lys Asn Val Ala Met 365 10 369 PRT
Actinobacillus pleuropneumoniae 10 Met Lys Lys Ser Leu Val Ala Leu
Ala Val Leu Ser Ala Ala Ala Val 1 5 10 15 Ala Gln Ala Ala Pro Gln
Gln Asn Thr Phe Tyr Ala Gly Ala Lys Val 20 25 30 Gly Gln Ser Ser
Phe His His Gly Val Asn Gln Leu Lys Ser Gly His 35 40 45 Asp Asp
Arg Tyr Asn Asp Lys Thr Arg Lys Tyr Gly Ile Asn Arg Asn 50 55 60
Ser Val Thr Tyr Gly Val Phe Gly Gly Tyr Gln Ile Leu Asn Gln Asn 65
70 75 80 Asn Phe Gly Leu Ala Thr Glu Leu Gly Tyr Asp Tyr Tyr Gly
Arg Val 85 90 95 Arg Gly Asn Asp Gly Glu Phe Arg Ala Met Lys His
Ser Ala His Gly 100 105 110 Leu Asn Phe Ala Leu Lys Pro Ser Tyr Glu
Val Leu Pro Asp Leu Asp 115 120 125 Val Tyr Gly Lys Val Gly Val Ala
Val Val Arg Asn Asp Tyr Lys Ser 130 135 140 Tyr Gly Ala Glu Asn Thr
Asn Glu Pro Thr Glu Lys Phe His Lys Leu 145 150 155 160 Lys Ala Ser
Thr Ile Leu Gly Ala Gly Val Glu Tyr Ala Ile Leu Pro 165 170 175 Glu
Leu Ala Ala Arg Val Glu Tyr Gln Tyr Leu Asn Lys Ala Gly Asn 180 185
190 Leu Asn Lys Ala Leu Val Arg Ser Gly Thr Gln Asp Val Asp Phe Gln
195 200 205 Tyr Ala Pro Asp Ile His Ser Val Thr Ala Gly Leu Ser Tyr
Arg Phe 210 215 220 Gly Gln Gly Ala Val Ala Pro Val Val Glu Pro Glu
Val Val Thr Lys 225 230 235 240 Asn Phe Ala Phe Ser Ser Asp Val Leu
Phe Asp Phe Gly Lys Ser Ser 245 250 255 Leu Lys Pro Ala Ala Ala Thr
Ala Leu Asp Ala Ala Asn Thr Glu Ile 260 265 270 Ala Asn Leu Gly Leu
Ala Thr Pro Ala Ile Gln Val Asn Gly Tyr Thr 275 280 285 Asp Arg Ile
Gly Lys Glu Ala Ser Asn Leu Lys Leu Ser Gln Arg Arg 290 295 300 Ala
Glu Thr Val Ala Asn Tyr Leu Val Ser Lys Gly Gln Asn Pro Ala 305 310
315 320 Asn Val Thr Ala Val Gly Tyr Gly Glu Ala Asn Pro Val Thr Gly
Ala 325 330 335 Thr Cys Asp Lys Val Lys Gly Arg Lys Ala Leu Ile Ala
Cys Leu Ala 340 345 350 Pro Asp Arg Arg Val Glu Val Gln Val Gln Gly
Ala Lys Asn Val Ala 355 360 365 Met 11 20 PRT Actinobacillus
pleuropneumoniae SITE (18) Xaa=unknown amino acid 11 Ala Pro Val
Gly Asn Thr Phe Thr Gly Val Lys Val Tyr Val Asp Leu 1 5 10 15 Thr
Xaa Val Ala 20 12 35 PRT Actinobacillus pleuropneumoniae SITE (30)
Xaa=Asn or Val 12 His Gln Ala Gly Asp Val Ile Phe Arg Ala Gly Ala
Ile Gly Val Ile 1 5 10 15 Ala Asn Ser Ser Ser Asp Tyr Gln Thr Gln
Ala Asp Val Xaa Leu Asp 20 25 30 Val Asn Asn 35 13 30 PRT
Actinobacillus pleuropneumoniae 13 Ala Glu Ile Gly Leu Gly Gly Ala
Arg Glu Ser Ser Ile Tyr Tyr Ser 1 5 10 15 Lys His Lys Val Ala Thr
Asn Pro Phe Leu Ala Leu Asp Leu 20 25 30 14 19 PRT Actinobacillus
pleuropneumoniae SITE (2) Xaa=Asp or Glu 14 Ala Xaa Pro Glu Asn Thr
Phe Tyr Pro Gly Ala Lys Val Xaa Xaa Ser 1 5 10 15 Xaa Phe His 15 20
DNA Artificial Sequence Description of Artificial Sequence Primer
15 aaaaatttgc gaaaaacgac 20 16 20 DNA Artificial Sequence
Description of Artificial Sequence Primer 16 acttctacat tacttgatac
20 17 20 DNA Artificial Sequence Description of Artificial Sequence
Primer 17 tccgtatgtc aaagtcgatg 20 18 20 DNA Artificial Sequence
Description of Artificial Sequence Primer 18 taaacaatca accggtcctg
20 19 20 DNA Artificial Sequence Description of Artificial Sequence
Primer 19 ttaccttgta caacaccgac 20 20 20 DNA Artificial Sequence
Description of Artificial Sequence Primer 20 aaaagcagta ttagcggcag
20 21 20 DNA Artificial Sequence Description of Artificial Sequence
Primer 21 ttgatgtgcc attgccgaac 20 22 20 DNA Artificial Sequence
Description of Artificial Sequence Primer 22 gttttaaact ttcagcactg
20 23 20 DNA Artificial Sequence Description of Artificial Sequence
Primer 23 agttcgtcca tacgttggtg 20 24 20 DNA Artificial Sequence
Description of Artificial Sequence Primer 24 aatatatctc tatcggtaag
20 25 20 DNA Artificial Sequence Description of Artificial Sequence
Primer 25 ctaaacctat aatttagatc 20 26 20 DNA Artificial Sequence
Description of Artificial Sequence Primer 26 tgtttccgca cgacgttgtg
20 27 20 DNA Artificial Sequence Description of Artificial Sequence
Primer 27 aagtaaacgg ttacacggac 20 28 20 DNA Artificial Sequence
Description of Artificial Sequence Primer 28 atcaaccgta attcagtaac
20 29 20 DNA Artificial Sequence Description of Artificial Sequence
Primer 29 atagtcataa cctaattcag 20 30 20 DNA Artificial Sequence
Description of Artificial Sequence Primer 30 gtatgggtgc aactgactac
20 31 20 DNA Artificial Sequence Description of Artificial Sequence
Primer 31 aacacgtgcc gctaattcag 20 32 20 DNA Artificial Sequence
Description of Artificial Sequence Primer 32 tgcttgagct accgctgcag
20 33 20 DNA Artificial Sequence Description of Artificial Sequence
Primer 33 gcgaaagttg gtcaatcatc 20 34 20 DNA Artificial Sequence
Description of Artificial Sequence Primer 34 ataacgatca tcgtgaccag
20 35 20 DNA Artificial Sequence Description of Artificial Sequence
Primer 35 tgcgtctaaa gctgttgctg 20 36 20 DNA Artificial Sequence
Description of Artificial Sequence Primer 36 atcaagctta aaaccagcag
20 37 20 DNA Artificial Sequence Description of Artificial Sequence
Primer 37 ctataaatcc tatggtgcag 20 38 20 DNA Artificial Sequence
Description of Artificial Sequence Primer 38 tgcacctaaa atagttgatg
20 39 34 DNA Artificial Sequence Description of Artificial Sequence
Primer 39 ggggatccgc dccdgtdggh aatacnttta cngg 34 40 43 DNA
Artificial Sequence Description of Artificial Sequence Primer 40
ggggatccat ytattatwsw aaacataaag tdgcdacnaa tcc 43 41 30 DNA
Artificial Sequence Description of Artificial Sequence Primer 41
ggggatccgt accgcgatct gtgtttttag 30 42 27 DNA Artificial Sequence
Description of Artificial Sequence Primer 42 ggggatccgg tgctccggca
gctaacg 27 43 35 DNA Artificial Sequence Description of Artificial
Sequence Primer 43 ggggatccat acttacgtgt tttatcatta taacg 35 44 27
DNA Artificial Sequence Description of Artificial Sequence Primer
44 ggggatccgg tcaaaaccct gcaaacg 27 45 33 DNA Artificial Sequence
Description
of Artificial Sequence Primer 45 tttgcccggg ctcttttatt gatttaagtt
act 33 46 24 DNA Artificial Sequence Description of Artificial
Sequence Primer 46 gactaacgca ggaccggttg attg 24 47 31 DNA
Artificial Sequence Description of Artificial Sequence Primer 47
ggggatccgt gggtgcggaa tatacgcgca g 31 48 25 DNA Artificial Sequence
Description of Artificial Sequence Primer 48 caacgtggat ccgaattcaa
gcttc 25 49 24 DNA Artificial Sequence Description of Artificial
Sequence Primer 49 gtcaccgtaa tccataccgt aatg 24 50 26 DNA
Artificial Sequence Description of Artificial Sequence Primer 50
gattgtttac ctttcacacc gtccac 26 51 27 DNA Artificial Sequence
Description of Artificial Sequence Primer 51 tcacccggga aaaatatcta
gaaacgg 27 52 28 DNA Artificial Sequence Description of Artificial
Sequence Primer 52 ccgtggtgag atcaacgcct acgcctac 28 53 38 DNA
Artificial Sequence Description of Artificial Sequence Primer 53
cctttcacac cgtccacttt atattttacc gtggtgag 38 54 68 DNA Artificial
Sequence Description of Artificial Sequence Primer 54 gcgcatatga
acaccaccac caccaccacc tctcgtgcac cggtcggaaa tacctttacc 60 ggcgtagg
68 55 24 DNA Artificial Sequence Description of Artificial Sequence
Primer 55 caagactaaa aatgaccggt cgtg 24 56 25 DNA Artificial
Sequence Description of Artificial Sequence Primer 56 tgaatttacg
accacgtaaa tgttt 25 57 25 DNA Artificial Sequence Description of
Artificial Sequence Primer 57 ctatgtgaaa gcaaaagcgg attgg 25 58 23
DNA Artificial Sequence Description of Artificial Sequence Primer
58 ccgtccggtt gtttgactaa cgc 23 59 24 DNA Artificial Sequence
Description of Artificial Sequence Primer 59 tcgaacaagc acaccagccg
gatg 24 60 25 DNA Artificial Sequence Description of Artificial
Sequence Primer 60 ggcggaatac ggtaactact tattc 25 61 27 DNA
Artificial Sequence Description of Artificial Sequence Primer 61
cgcataaaaa atgatctaaa ttatagg 27 62 24 DNA Artificial Sequence
Description of Artificial Sequence Primer 62 ggggaattca acgattttgc
ttgc 24 63 26 DNA Artificial Sequence Description of Artificial
Sequence Primer 63 gaattcttgc tcgtttgaat tagaag 26 64 25 DNA
Artificial Sequence Description of Artificial Sequence Primer 64
ggtaattttt atatgagagg gactg 25 65 25 DNA Artificial Sequence
Description of Artificial Sequence Primer 65 caaacagtga tacgctgaaa
ttgtc 25 66 25 DNA Artificial Sequence Description of Artificial
Sequence Primer 66 gcaacacaca taggtattaa tactg 25 67 66 DNA
Artificial Sequence Description of Artificial Sequence Primer 67
gcgcatatga acaccaccac caccaccacc tctcgtgccg aaattggatt gggaggagcc
60 cgtgag 66 68 28 DNA Artificial Sequence Description of
Artificial Sequence Primer 68 gtattctctc tagaatattc tattttag 28 69
27 DNA Artificial Sequence Description of Artificial Sequence
Primer 69 gccacatgaa gaattattat ttgagct 27 70 25 DNA Artificial
Sequence Description of Artificial Sequence Primer 70 acgtgaaaaa
taatctcttg ataat 25 71 60 DNA Artificial Sequence Description of
Artificial Sequence Primer 71 cgccatatga acaccaccac caccaccacc
tctcgtcatc aggcgggaga tgtgattttc 60 72 23 DNA Artificial Sequence
Description of Artificial Sequence Primer 72 caycargcdg ghgatgtdat
ytt 23 73 23 DNA Artificial Sequence Description of Artificial
Sequence Primer 73 gcdgadccdg araayacdtt yta 23 74 22 DNA
Artificial Sequence Description of Artificial Sequence Primer 74
accggtacct atatgttaag tg 22 75 22 DNA Artificial Sequence
Description of Artificial Sequence Primer 75 ataggtaccg gttaaaccaa
gc 22 76 23 DNA Artificial Sequence Description of Artificial
Sequence Primer 76 gttgccgcta ataattcaag acc 23 77 23 DNA
Artificial Sequence Description of Artificial Sequence Primer 77
aayacnttyt ayccdggngc naa 23 78 24 DNA Artificial Sequence
Description of Artificial Sequence Primer 78 nacdckdckr tcnggngcna
rrca 24 79 21 DNA Artificial Sequence Description of Artificial
Sequence Primer 79 datytcnacd ckdckrtcng g 21 80 30 DNA Artificial
Sequence Description of Artificial Sequence Primer 80 cgctctagag
attttttaca acaaaaaggg 30 81 23 DNA Artificial Sequence Description
of Artificial Sequence Primer 81 gmnaayacnt tytaygyngg ngc 23 82 24
DNA Artificial Sequence Description of Artificial Sequence Primer
82 aayacnttyt aygynggngc naar 24 83 23 DNA Artificial Sequence
Description of Artificial Sequence Primer 83 cargmnaaya cnttytaygy
ngg 23 84 23 DNA Artificial Sequence Description of Artificial
Sequence Primer 84 gcnccncarg mnaayacntt yta 23 85 23 DNA
Artificial Sequence Description of Artificial Sequence Primer 85
cgcgcnccnc argmnaayac ntt 23 86 29 DNA Artificial Sequence
Description of Artificial Sequence Primer 86 cggtcgactg atttaagtta
ctaaaaccc 29 87 29 DNA Artificial Sequence Description of
Artificial Sequence Primer 87 cggtcgacgg gttactaatt taactttag 29 88
67 DNA Artificial Sequence Description of Artificial Sequence
Primer 88 gcgtcgacca tatgaacacc accaccacca ccacctctcg tgcgccacaa
caaaatactt 60 tytacgc 67 89 32 DNA Artificial Sequence Description
of Artificial Sequence Primer 89 atgaaaaatt taacagtttt agcattagca
gg 32 90 38 DNA Artificial Sequence Description of Artificial
Sequence Primer 90 atgaaacata gcaaattcaa attatttaaa tattattt 38 91
32 DNA Artificial Sequence Description of Artificial Sequence
Primer 91 atgaaaaaag cagtattagc ggcagtatta gg 32 92 36 DNA
Artificial Sequence Description of Artificial Sequence Primer 92
atgaaaaaat cattagttgc tttaacagta ttatcg 36 93 36 DNA Artificial
Sequence Description of Artificial Sequence Primer 93 atgaaaaaat
cattagttgc tttagcagta ttatca 36 94 13 PRT Artificial Sequence
Description of Artificial SequenceN-terminal consensus sequence of
OmpA-related proteins from Pasteurellaceae 94 Ala Pro Gln Xaa Asn
Thr Phe Tyr Xaa Gly Ala Lys Ala 1 5 10 95 10 PRT Artificial
Sequence Description of Artificial SequenceC-terminal consensus
sequence of OmpA-related proteins from Pasteurellaceae 95 Cys Leu
Ala Pro Asp Arg Arg Val Glu Ile 1 5 10 96 10 PRT Artificial
Sequence Description of Artificial Sequenceprotective peptide 96
Met Asn Thr Thr Thr Thr Thr Thr Ser Arg 1 5 10
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