U.S. patent application number 11/021823 was filed with the patent office on 2005-10-20 for novel therapeutic compositions for treating infection by lawsonia spp..
Invention is credited to Good, Robert Trygve, King, Kendall Wayne, Rosey, Everett Lee, Strugnell, Richard Anthony.
Application Number | 20050232939 11/021823 |
Document ID | / |
Family ID | 3825422 |
Filed Date | 2005-10-20 |
United States Patent
Application |
20050232939 |
Kind Code |
A1 |
Rosey, Everett Lee ; et
al. |
October 20, 2005 |
Novel therapeutic compositions for treating infection by Lawsonia
spp.
Abstract
The present invention relates generally to therapeutic
compositions for the treatment and/or prophylaxis of intestinal
disease conditions in animals and birds caused or exacerbated by
Lawsonia intracellularis or similar or otherwise related
microorganism. In particular, the present invention provides a
novel gene derived from Lawsonia intracellularis, which encodes an
immunogenic polypeptide that is particularly useful as an antigen
in a vaccine preparation for conferring humoral immunity against
Lawsonia intracellularis and related pathogens in animal hosts,
wherein said polypeptide is selected from the group consisting of
flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM, and ytfN
polypeptides, or a homologue, analogue or derivative of any one or
more of said polypeptides. The present invention is also directed
to methods for the treatment and/or prophylaxis of such intestinal
disease conditions and to diagnostic agents and procedures for
detecting Lawsonia intracellularis or similar or otherwise related
microorganisms.
Inventors: |
Rosey, Everett Lee;
(Preston, CT) ; King, Kendall Wayne; (Waterford,
CT) ; Good, Robert Trygve; (Romsey, AU) ;
Strugnell, Richard Anthony; (Hawthorn, AU) |
Correspondence
Address: |
KOHN & ASSOCIATES PLLC
30500 NORTHWESTERN HWY
STE 410
FARMINGTON HILLS
MI
48334
US
|
Family ID: |
3825422 |
Appl. No.: |
11/021823 |
Filed: |
December 22, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11021823 |
Dec 22, 2004 |
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10010160 |
Nov 9, 2001 |
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6846487 |
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60249596 |
Nov 17, 2000 |
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Current U.S.
Class: |
424/190.1 ;
435/252.3; 435/320.1; 435/69.3; 530/350; 536/23.7 |
Current CPC
Class: |
C07K 14/195 20130101;
A61K 39/00 20130101; C07K 14/205 20130101; A61K 2039/53
20130101 |
Class at
Publication: |
424/190.1 ;
435/069.3; 435/252.3; 435/320.1; 530/350; 536/023.7 |
International
Class: |
A61K 039/02; C07H
021/04; C12N 015/74; C07K 014/195; C12N 001/21 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 10, 2000 |
AU |
PR1381/00 |
Claims
1. An isolated or recombinant immunogenic polypeptide which
comprises, mimics or cross-reacts with a B-cell or T-cell epitope
of a Lawsonia spp. polypeptide selected from the group consisting
of flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM, and ytfN
polypeptides.
2. The isolated or recombinant immunogenic polypeptide of claim 1
capable of eliciting the production of antibodies against Lawsonia
spp. when administered to an avian or porcine animal.
3. The isolated or recombinant immunogenic polypeptide of claim 1
capable of conferring a protective immune response against Lawsonia
spp. when administered to an avian or porcine animal.
4. The isolated or recombinant immunogenic polypeptide of claim 2
wherein the Lawsonia spp. is L. intracellularis.
5. The isolated or recombinant immunogenic polypeptide of claim 3
wherein the Lawsonia spp. is L. intracellularis.
6. An isolated or recombinant polypeptide selected from the group
consisting of: (i) a polypeptide of Lawsonia spp. which comprises
an amino acid sequence which has at least about 60% sequence
identity overall to an amino acid sequence selected from the group
consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, and 18; (ii)
a polypeptide of Lawsonia spp. which comprises an amino acid
sequence which has at least about 60% sequence identity overall to
an amino acid sequence encoded by Lawsonia intracellularis DNA
contained within a plasmid selected from the group consisting of
AGAL Accession Nos: NM00/16476 (plasmid pGTE#1 glnH); NM00/16477
(plasmid pGTE#2 flhB); NM00/16478 (plasmid pGTE#3 fliR); NM00/16479
(plasmid pGTE#4 motA/B); NM00/16480 (plasmid pGTE#5 tlyC);
NM00/16481 (plasmid pGTE#6 ntrC); NM00/16482 (plasmid pGTE#7 ytfM);
and NM01/23286 (plasmid pGTE#8 ytfN); (iii) a polypeptide which
comprises at least about 5 contiguous amino acids of an amino acid
sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6,
8, 10, 12, 14, 16, and 18; (iv) a polypeptide which comprises at
least about 5 contiguous amino acids of an amino acid sequence
encoded by Lawsonia intracellularis DNA contained within a plasmid
selected from the group consisting of AGAL Accession Nos:
NM00/16476 (plasmid pGTE#1 glnH); NM00/16477 (plasmid pGTE#2 flhB);
NM00/16478 (plasmid pGTE#3 fliR); NM00/16479 (plasmid pGTE#4
motA/B); NM00/16480 (plasmid pGTE#5 tlyC); NM00/16481 (plasmid
pGTE#6 ntrC); NM00/16482 (plasmid pGTE#7 ytfM); and NM01/23286
(plasmid pGTE#8 ytfN); and (v) a polypeptide which comprises an
amino acid sequence encoded by a nucleotide sequence of Lawsonia
spp. having at least about 60% sequence identity overall to a
nucleotide sequence selected from the group consisting of SEQ ID
NOs: 1, 3, 5, 7, 9, 11, 13, 15, and 17; (vi) a polypeptide which
comprises an amino acid sequence encoded by a nucleotide sequence
of Lawsonia spp. having at least about 60% sequence identity
overall to the nucleotide sequence of Lawsonia intracellularis DNA
contained within a plasmid selected from the group consisting of
AGAL Accession Nos: NM00/16476 (plasmid pGTE#1 glnH); NM00/16477
(plasmid pGTE#2 flhB); NM00/16478 (plasmid pGTE#3 fliR); NM00/16479
(plasmid pGTE#4 motA/B); NM00/16480 (plasmid pGTE#5 tlyC);
NM00/16481 (plasmid pGTE#6 ntrC); NM00/16482 (plasmid pGTE#7 ytfM);
and NM01/23286 (plasmid pGTE#8 ytfN); (vii) a polypeptide encoded
by at least about 15 contiguous nucleotides of a nucleotide
sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5,
7, 9, 11, 13, 15, and 17; (viii) a polypeptide encoded by at least
about 15 contiguous nucleotides of a nucleotide sequence of
Lawsonia intracellularis DNA contained within a plasmid selected
from the group consisting of AGAL Accession Nos: NM00/16476
(plasmid pGTE#1 glnH); NM00/16477 (plasmid pGTE#2 flhB); NM00/16478
(plasmid pGTE#3 fliR); NM00/16479 (plasmid pGTE#4 motB); NM00/16480
(plasmid pGTE#5 tlyC); NM00/16481 (plasmid pGTE#6 ntrC); NM00/16482
(plasmid pGTE#7 ytfM); and NM01/23286 (plasmid pGTE#8 ytfN); and
(ix) a homologue, analogue or derivative of any one of (i) to (vii)
which mimics a B-cell or T-cell epitope of Lawsonia spp.
7. The isolated or recombinant polypeptide of claim 6 capable of
eliciting the production of antibodies against Lawsonia spp. in a
porcine or avian animal.
8. The isolated or recombinant polypeptide of claim 6 capable of
conferring a protective immune response against Lawsonia spp. in a
porcine or avian animal.
9. The isolated or recombinant polypeptide of claim 8, capable of
inducing humoral immunity against Lawsonia spp. in a porcine or
avian animal.
10. The isolated or recombinant polypeptide of claim 9, capable of
inducing humoral immunity against Lawsonia spp. in a porcine
animal.
11. The isolated or recombinant polypeptide of claim 7 wherein the
Lawsonia spp. is Lawsonia intracellularis.
12. The isolated or recombinant polypeptide of claim 8 wherein the
Lawsonia spp. is Lawsonia intracellularis.
13. The isolated or recombinant polypeptide of claim 6 comprising
an amino acid sequence selected from the group consisting of: (i)
an amino acid sequence selected from the group consisting of SEQ ID
NOs: 2, 4, 6, 8, 10, 12, 14, 16, and 18; and (ii) an amino acid
sequence encoded by Lawsonia intracellularis DNA contained within a
deposited plasmid selected from the group consisting of AGAL
Accession Nos: NM00/16476 (plasmid pGTE#1 glnH); NM00/16477
(plasmid pGTE#2 flhB); NM00/16478 (plasmid pGTE#3 fliR); NM00/16479
(plasmid pGTE#4 motA/B); NM00/16480 (plasmid pGTE#5 tlyC);
NM00/16481 (plasmid pGTE#6 ntrC); NM00/16482 (plasmid pGTE#7 ytfM);
and NM01/23286 (plasmid pGTE#8 ytfN).
14. The isolated or recombinant polypeptide of claim 13 capable of
eliciting the production of antibodies against Lawsonia
intracellularis when administered to an avian or porcine
animal.
15. The isolated or recombinant polypeptide of claim 13 capable of
inducing a protective immune response against Lawsonia
intracellularis in a porcine or avian animal.
16. The isolated or recombinant polypeptide of claim 15 capable of
inducing a protective immune response against Lawsonia
intracellularis in a porcine animal.
17. A vaccine composition for the prophylaxis or treatment of
infection of an animal by Lawsonia spp., said vaccine composition
comprising an immunogenic component which comprises the isolated or
recombinant immunogenic polypeptide according to claim 1 in
combination with one or more carriers, diluents or adjuvants
suitable for veterinary or pharmaceutical use.
18. The vaccine composition according to claim 17 wherein the
Lawsonia spp. is Lawsonia intracellularis.
19. The vaccine composition according to claim 17 wherein the
immunogenic component comprises an isolated or recombinant
polypeptide having an amino acid sequence selected from the group
consisting of: (i) an amino acid sequence selected from the group
consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, and 18; and
(ii) an amino acid sequence encoded by Lawsonia intracellularis DNA
contained within a deposited plasmid selected from the group
consisting of AGAL Accession Nos: NM00/16476 (plasmid pGTE#1 glnH);
NM00/16477 (plasmid pGTE#2 flhB); NM00/16478 (plasmid pGTE#3 fliR);
NM00/16479 (plasmid pGTE#4 motA/B); NM00/16480 (plasmid pGTE#5
tlyC); NM00/16481 (plasmid pGTE#6 ntrC); NM00/16482 (plasmid pGTE#7
ytfM); and NM01/23286 (plasmid pGTE#8 ytfN);
20. The vaccine composition of claim 17, wherein the immunogenic
component is a recombinant polypeptide expressed in a cell that has
been transfected with a vector comprising a nucleotide sequence
selected from the group consisting of: (i) a protein-encoding
nucleotide sequence having at least about 60% sequence identity
overall to a nucleotide sequence selected from the group consisting
of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, and 17; (ii) a
protein-encoding nucleotide sequence having at least about 60%
sequence identity overall to the protein-encoding sequence of
Lawsonia intracellularis DNA contained within a plasmid selected
from the group consisting of AGAL Accession Nos: NM00/16476
(plasmid pGTE#1 glnH); NM00/16477 (plasmid pGTE#2 flhB); NM00/16478
(plasmid pGTE#3 fliR); NM00/16479 (plasmid pGTE#4 motA/B);
NM00/16480 (plasmid pGTE#5 tlyC); NM00/16481 (plasmid pGTE#6 ntrC);
NM00/16482 (plasmid pGTE#7 ytfM); and NM01/23286 (plasmid pGTE#8
ytfN); (iii) a protein-encoding nucleotide sequence which comprises
at least about 15 contiguous nucleotides of a sequence selected
from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15,
and 17; (iv) a protein-encoding nucleotide sequence which comprises
at least about 15 contiguous nucleotides of the protein-encoding
sequence of Lawsonia intracellularis DNA contained within a plasmid
selected from the group consisting of AGAL Accession Nos:
NM00/16476 (plasmid pGTE#1 glnH); NM00/16477 (plasmid pGTE#2 flhB);
NM00/16478 (plasmid pGTE#3 fliR); NM00/16479 (plasmid pGTE#4
motA/B); NM00/16480 (plasmid pGTE#5 tlyC); NM00/16481 (plasmid
pGTE#6 ntrC); NM00/16482 (plasmid pGTE#7 ytfM); and NM01/23286
(plasmid pGTE#8 ytfN); (v) a protein-encoding nucleotide sequence
which hybridizes under at least low stringency conditions to the
complement of a nucleotide sequence selected from the group
consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, and 17; (vi) a
protein-encoding nucleotide sequence which hybridizes under at
least low stringency conditions to the non-coding strand of
Lawsonia intracellularis DNA contained within a p plasmid selected
from the group consisting of AGAL Accession Nos: NM00/16476
(plasmid pGTE#1 glnH); NM00/16477 (plasmid pGTE#2 flhB); NM00/16478
(plasmid pGTE#3 fliR); NM00/16479 (plasmid pGTE#4 motA/B);
NM00/16480 (plasmid pGTE#5 tlyC); NM00/16481 (plasmid pGTE#6 ntrC);
NM00/16482 (plasmid pGTE#7 ytfM); and NM01/23286 (plasmid pGTE#8
ytfN); and (vii) a homologue, analogue or derivative of any one of
(i) to (vi) which encodes a polypeptide which mimics a B-cell or
T-cell epitope of Lawsonia spp.
21. A combination vaccine composition for the prophylaxis or
treatment of infection of an animal by Lawsonia spp., said vaccine
composition comprising: (i) a first immunogenic component which
comprises the isolated or recombinant polypeptide having according
to claim 1; (ii) a second immunogenic component different from said
first immunogenic component and comprising a polypeptide selected
from the group consisting of the Lawsonia intracellularis FIgE,
hemolysin, OmpH, SodC, flhB, fliR, ntrC, glnH, motA, motB, tlyC,
ytfM, and ytfN polypeptides; and (iii) one or more carriers,
diluents or adjuvants suitable for veterinary or pharmaceutical
use.
22-50. (canceled)
Description
RELATED APPLICATION DATA
[0001] This application claims benefit of priority from Australian
Patent Application No. PR1381 filed on Nov. 10, 2000, and from U.S.
Patent Application Ser. No. 60/249,596 filed on Nov. 17, 2000.
FIELD OF THE INVENTION
[0002] The present invention relates generally to therapeutic
compositions for the treatment and/or prophylaxis of intestinal
disease conditions in animals and birds caused or exacerbated by
Lawsonia intracellularis or similar or otherwise related
microorganism. In particular, the present invention provides a
novel gene derived from L. intracellularis which encodes an
immunogenic polypeptide. The polypeptide described herein, selected
from the group consisting of flhB, fliR, ntrC, glnH, motA, motB,
tlyC, ytfM, and ytfN polypeptides, or a homologue, analogue or
derivative of any one or more of said polypeptides, is particularly
useful as an antigen in vaccine preparation for conferring humoral
immunity against L. intracellularis and related pathogens in animal
hosts. The present invention is also directed to methods for the
treatment and/or prophylaxis of such intestinal disease conditions
and to diagnostic agents and procedures for detecting L.
intracellularis or similar or otherwise related microorganisms.
[0003] General
[0004] Bibliographic details of the publications numerically
referred to in this specification are collected at the end of the
description. All patents, patent applications, and publications
cited herein are incorporated by reference in their entirety.
[0005] Reference hereinafter to "Lawsonia intracellularis" or its
abbreviation "L. intracellularis includes all microorganisms
similar to or otherwise related to this microorganism, as described
by Stills (1991) or Jones et al. (1997) or Lawson et al. (1993) or
McOrist et al. (1995).
[0006] References herein to "AGAL" shall be taken to mean a
reference to the Australian Government Analytical Laboratories
located at 1 Suakin Street, Pymble, New South Wales 2073,
Australia. All biological deposits referred to herein in respect of
the plasmids assigned AGAL Accession Nos: NM00/16476 (plasmid
pGTE#1 glnH); NM00/16477 (plasmid pGTE#2 flhB); NM00/16478 (plasmid
pGTE#3 fliR); NM00/16479 (plasmid pGTE#4 motA/B); NM00/16480
(plasmid pGTE#5 tlyC); NM00/16481 (plasmid pGTE#6 ntrC); and
NM00/16482 (plasmid pGTE#7 ytfM); and NM01/23286 (plasmid pGTE#8
ytfN) have been made under the provisions of the Budapest Treaty on
the International Recognition of the Deposit of Microorganisms for
the Purposes of Patent Procedure.
[0007] As used herein, the word "flhB", or the term "flhB gene",
shall be taken to refer to a gene encoding the antigenic flhB
polypeptide of the present invention, which gene comprises the
nucleotide sequence set forth in SEQ ID NO: 1 or the nucleotide
sequence of the L. intracellularis gene contained in the plasmid
pGTE#2 which has been deposited under AGAL Accession No.
NM00/16477. The word "flhB" or the term "flhB gene" shall further
be taken to include a degenerate or complementary nucleotide
sequence to SEQ ID NO: 1 or the nucleotide sequence of the L.
intracellularis gene contained in the plasmid pGTE#2 which has been
deposited under AGAL Accession No. NM00/16477. It shall also be
understood that the term "flhB polypeptide" refers to a polypeptide
of the invention which comprises the amino acid sequence set forth
in SEQ ID NO: 2 or a polypeptide encoded by the L. intracellularis
gene contained in the plasmid pGTE#2 which has been deposited under
AGAL Accession No. NM00/16477. The term "flhB" polypeptide" shall
further be taken to include a polypeptide which is
functionally-related to or immunologically cross-reactive with the
polypeptide of SEQ ID NO: 2 or a polypeptide encoded by the L.
intracellularis gene contained in the plasmid pGTE#2 which has been
deposited under AGAL Accession No. NM00/16477.
[0008] As used herein, the word "fliR", or the term "fliR gene",
shall be taken to refer to a gene encoding the antigenic fliR
polypeptide of the present invention, which gene comprises the
nucleotide sequence set forth in SEQ ID NO: 3 or the nucleotide
sequence of the L. intracellularis gene contained in the plasmid
pGTE#3 which has been deposited under AGAL Accession No.
NM00/16478. The word "fliR" or the term "fliR gene" shall further
be taken to include a degenerate or complementary nucleotide
sequence to SEQ ID NO: 3, or the nucleotide sequence of the L.
intracellularis gene contained in the plasmid pGTE#3 which has been
deposited under AGAL Accession No. NM00/16478. It shall also be
understood that the term "fliR polypeptide" refers to a polypeptide
of the invention which comprises the amino acid sequence set forth
in SEQ ID NO: 4 or a polypeptide encoded by the L. intracellularis
gene contained in the plasmid pGTE#3 which has been deposited under
AGAL Accession No. NM00/16478. The term "fliR polypeptide" shall
further be taken to include a polypeptide which is
functionally-related to or immunologically cross-reactive with the
polypeptide of SEQ ID NO: 4 or a polypeptide encoded by the L.
intracellularis gene contained in the plasmid pGTE#3 which has been
deposited under AGAL Accession No. NM00/16478.
[0009] As used herein, the word "ntrC", or the term "ntrC gene",
shall be taken to refer to a gene encoding the antigenic ntrC
polypeptide of the present invention, which gene comprises the
nucleotide sequence set forth in SEQ ID NO: 5 or the nucleotide
sequence of the L. intracellularis gene contained in the plasmid
pGTE#6 which has been deposited under AGAL Accession No.
NM00/16481. The word "ntrC" or the term "ntrC gene" shall further
be taken to include a degenerate or complementary nucleotide
sequence to SEQ ID NO: 5, or the nucleotide sequence of the L.
intracellularis gene contained in the plasmid pGTE#6 which has been
deposited under AGAL Accession No. NM00/16481. It shall also be
understood that the term "ntrC polypeptide" refers to a polypeptide
of the invention which comprises the amino acid sequence set forth
in SEQ ID NO: 6 or a polypeptide encoded by the L. intracellularis
gene contained in the plasmid pGTE#6 which has been deposited under
AGAL Accession No. NM00/16481. The term "ntrC polypeptide" shall
further be taken to include a polypeptide which is
functionally-related to or immunologically cross-reactive with the
polypeptide of SEQ ID NO: 6 or a polypeptide encoded by the L.
intracellularis gene contained in the plasmid pGTE#6 which has been
deposited under AGAL Accession No. NM00/16481.
[0010] As used herein, the word "glnH", or the term "glnH gene",
shall be taken to refer to a gene encoding the antigenic glnH
polypeptide of the present invention, which gene comprises the
nucleotide sequence set forth in SEQ ID NO: 7 or the nucleotide
sequence of the L. intracellularis gene contained in the plasmid
pGTE#1 which has been deposited under AGAL Accession No.
NM00/16476. The word "glnH" or the term "glnH gene" shall further
be taken to include a degenerate or complementary nucleotide
sequence to SEQ ID NO: 7, or the nucleotide sequence of the L.
intracellularis gene contained in the plasmid pGTE#1 which has been
deposited under AGAL Accession No. NM00/16476. It shall also be
understood that the term "glnH polypeptide" refers to a polypeptide
of the invention which comprises the amino acid sequence set forth
in SEQ ID NO: 8 or a polypeptide encoded by the L. intracellularis
gene contained in the plasmid pGTE#1 which has been deposited under
AGAL Accession No. NM00/16476. The term "glnH polypeptide" shall
further be taken to include a polypeptide which is
functionally-related to or immunologically cross-reactive with the
polypeptide of SEQ ID NO: 8 or a polypeptide encoded by the L.
intracellularis gene contained in the plasmid pGTE#1 which has been
deposited under AGAL Accession No. NM00/16476.
[0011] As used herein, the word "motA", or the term "motA gene",
shall be taken to refer to a gene encoding the antigenic motA
polypeptide of the present invention, which gene comprises the
nucleotide sequence set forth in SEQ ID NO: 9, or to the nucleotide
sequence of the L. intracellularis gene contained in the plasmid
pGTE#4 which has been deposited under AGAL Accession No. NM00/16479
and which has homology to SEQ ID NO: 9. The word "motA" or the term
"motA gene" shall further be taken to include a degenerate or
complementary nucleotide sequence to SEQ ID NO: 9, or the
nucleotide sequence of the L. intracellularis gene contained in the
plasmid pGTE#4 which has been deposited under AGAL Accession No.
NM00/16479 and which has homology to SEQ ID NO: 9. It shall also be
understood that the term "motA polypeptide" refers to a polypeptide
of the invention which comprises the amino acid sequence set forth
in SEQ ID NO: 10 or a polypeptide encoded by the L. intracellularis
gene contained in the plasmid pGTE#4 which has been deposited
underAGAL Accession No. NM00/16479 and which has homology to SEQ ID
NO: 9. The term "motA polypeptide" shall further be taken to
include a polypeptide which is functionally-related to or
immunologically cross-reactive with the polypeptide of SEQ ID NO:
10 or a polypeptide encoded by the L. intracellularis gene
contained in the plasmid pGTE#4 which has been deposited under AGAL
Accession No. NM00/16479 and having homology to SEQ ID NO: 9.
[0012] As used herein, the word "motB", or the term "motB gene",
shall be taken to refer to a gene encoding the antigenic motB
polypeptide of the present invention, which gene comprises the
nucleotide sequence set forth in SEQ ID NO: 11 or the nucleotide
sequence of the L. intracellularis gene contained in the plasmid
pGTE#4 which has been deposited under AGAL Accession No. NM00/16479
and having homology to SEQ ID NO: 11. The word "motB" or the term
"motB gene" shall further be taken to include a degenerate or
complementary nucleotide sequence to SEQ ID NO: 11, or the
nucleotide sequence of the L. intracellularis gene contained in the
plasmid pGTE#4 which has been deposited under AGAL Accession No.
NM00/16479 and having homology to SEQ ID NO: 11. It shall also be
understood that the term "motB polypeptide" refers to a polypeptide
of the invention which comprises the amino acid sequence set forth
in SEQ ID NO: 12 or a polypeptide encoded by the L. intracellularis
gene contained in the plasmid pGTE#4 which has been deposited under
AGAL Accession No. NM00/16479 and having homology to SEQ ID NO: 11.
The term "motB polypeptide" shall further be taken to include a
polypeptide which is functionally-related to or immunologically
cross-reactive with the polypeptide of SEQ ID NO: 12 or a
polypeptide encoded by the L. intracellularis gene contained in the
plasmid pGTE#4 which has been deposited under AGAL Accession No.
NM00/16479 and having homology to SEQ ID NO: 11.
[0013] As used herein, the word "tlyC", or the term "tlyC gene",
shall be taken to refer to a gene encoding the antigenic tlyC
polypeptide of the present invention, which gene comprises the
nucleotide sequence set forth in SEQ ID NO: 13 or the nucleotide
sequence of the L. intracellularis gene contained in the plasmid
pGTE#5 which has been deposited under AGAL Accession No.
NM00/16480. The word "tlyC" or the term "tlyC gene" shall further
be taken to include a degenerate or complementary nucleotide
sequence to SEQ ID NO: 13, or the nucleotide sequence of the L.
intracellularis gene contained in the plasmid pGTE#5 which has been
deposited under AGAL Accession No. NM00/16480. It shall also be
understood that the term "tlyC polypeptide" refers to a polypeptide
of the invention which comprises the amino acid sequence set forth
in SEQ ID NO: 14 or a polypeptide encoded by the L. intracellularis
gene contained in the plasmid pGTE#5 which has been deposited under
AGAL Accession No. NM00/16480. The term "tlyC polypeptide" shall
further be taken to include a polypeptide which is
functionally-related to or immunologically cross-reactive with the
polypeptide of SEQ ID NO: 14 or a polypeptide encoded by the L.
intracellularis gene contained in the plasmid pGTE#5 which has been
deposited under AGAL Accession No. NM00/16480.
[0014] As used herein, the word "ytfM", or the term "ytfM gene",
shall be taken to refer to a gene encoding the antigenic ytfM
polypeptide of the present invention, which gene comprises the
nucleotide sequence set forth in SEQ ID NO: 15 or the nucleotide
sequence of the L. intracellularis gene contained in the plasmid
pGTE#7 which has been deposited under AGAL Accession No.
NM00/16482. The word "ytfM" or the term "ytfM gene" shall further
be taken to include a degenerate or complementary nucleotide
sequence to SEQ ID NO: 15, or the nucleotide sequence of the L.
intracellularis gene contained in the plasmid pGTE#7 which has been
deposited under AGAL Accession No. NM00/16482. It shall also be
understood that the term "ytfM polypeptide" refers to a polypeptide
of the invention which comprises the amino acid sequence set forth
in SEQ ID NO: 16 or a polypeptide encoded by the L. intracellularis
gene contained in the plasmid pGTE#7 which has been deposited under
AGAL Accession No. NM00/16482. The term "ytfM polypeptide" shall
further be taken to include a polypeptide which is
functionally-related to or immunologically cross-reactive with the
polypeptide of SEQ ID NO: 16 or a polypeptide encoded by the L.
intracellularis gene contained in the plasmid pGTE#7 which has been
deposited under AGAL Accession No. NM00/16482.
[0015] As used herein, the word "ytfN", or the term "ytfN gene",
shall be taken to refer to a gene encoding the antigenic ytfn
polypeptide of the present invention, which gene comprises the
nucleotide sequence set forth in SEQ ID NO: 17 or the nucleotide
sequence of the L. intracellularis gene contained in the plasmid
pGTE#8 which has been deposited under AGAL Accession No.
NM01/23286. The word "ytfN" or the term "ytfN gene" shall further
be taken to include a degenerate or complementary nucleotide
sequence to SEQ ID NO: 17 or the nucleotide sequence of the L.
intracellularis gene contained in the plasmid pGTE#8 which has been
deposited under AGAL Accession No. NM01/23286. It shall also be
understood that the term "ytfN polypeptide" refers to a polypeptide
of the invention which comprises the amino acid sequence set forth
in SEQ ID NO: 18 or a polypeptide encoded by the L. intracellularis
gene contained in the plasmid pGTE#8 which has been deposited under
AGAL Accession No. NM01/23286. The term "ytfN polypeptide" shall
further be taken to include a polypeptide which is
functionally-related to or immunologically cross-reactive with the
polypeptide of SEQ ID NO: 18 or a polypeptide encoded by the L.
intracellularis gene contained in the plasmid pGTE#8 which has been
deposited under AGAL Accession No. NM01/23286.
[0016] As used herein the words "from" or "of", and the term
"derived from" shall be taken to indicate that a specified product,
in particular a macromolecule such as a polypeptide, protein, gene
or nucleic acid molecule, antibody molecule, 1 g fraction, or other
macromolecule, or a biological sample comprising said
macromolecule, may be obtained from a particular source, organism,
tissue, organ or cell, albeit not necessarily directly from that
source, organism, tissue, organ or cell.
[0017] Throughout this specification, unless the context requires
otherwise, the word "comprise", or variations such as "comprises"
or "comprising", will be understood to imply the inclusion of a
stated step or element or integer or group of steps or elements or
integers but not the exclusion of any other step or element or
integer or group of elements or integers.
[0018] Those skilled in the art will appreciate that the invention
described herein is susceptible to variations and modifications
other than those specifically described. It is to be understood
that the invention includes all such variations and modifications.
The invention also includes all of the steps, features,
compositions and compounds referred to or indicated in this
specification, individually or collectively, and any and all
combinations or any two or more of said steps, features,
compositions and compounds.
[0019] The present invention is not to be limited in scope by the
specific embodiments described herein, which are intended for the
purposes of exemplification only. Functionally equivalent products,
compositions and methods are clearly within the scope of the
invention, as described herein.
BACKGROUND OF THE INVENTION
[0020] The meat-producing sector of the agricultural industry is
dependent upon the health of its livestock and there is a need to
maintain disease-free livestock for human consumption. The industry
is subject to rapid economic downturn in response to disease
conditions adversely affecting livestock and the quality of meat
products derived therefrom, including those diseases which may
potentially be transmitted to humans. It is important, therefore,
to have well defined treatments and prophylactic and diagnostic
procedures available to deal with infections or potential
infections in livestock animals and humans.
[0021] Meat products derived from porcine and avian species are
significant commercial products in the agriculture industry. In
particular, pigs form a major component of the meat industry.
However, pigs are sensitive to a wide spectrum of intestinal
diseases collectively referred to as porcine proliferative
enteropathy (PPE). These diseases have previously been known as
intestinal adenomatosis complex (Barker and van Drumel, 1985),
porcine intestinal adenomatosis (PIA), necrotic enteritis (Rowland
and Lawson, 1976), proliferative haemorrhagic enteropathy (Love and
Love, 1977), regional Ileitis (Jonsson and Martinsson, 1976),
haemorrhagic bowel syndrome (O'Neil, 1970), porcine proliferative
enteritis and Campylobacter spp--induced enteritis (Straw,
1990).
[0022] There are two main forms of PPE: a non-haemorrhagic form
represented by intestinal adenomatosis which frequently causes
growth retardation and mild diarrhoea; and a haemorrhagic form,
which is often fatal, represented by proliferative haemorrhagic
enteropathy (PHE), where the distal small intestine lumen becomes
engorged with blood. PPE has been reported in a number of animal
species including pigs (McOrist et al, 1993), hamsters (Stills,
1991), ferrets (Fox et al, 1989), guinea pigs (Elwell et al, 1981),
rabbits (Schodeb and Fox, 1990) as well as avian species (Mason et
al, 1998).
[0023] PPE is a significant cost component associated with the pig
industry, especially in terms of stock losses, medication costs,
reduced growth rates of pigs and increased feed costs. PPE also
contributes to downstream indirect costs in, for example,
additional labour costs and environmental costs in dealing with
antibiotic residue contamination, and in control measures to
prevent the organism from being passed on or carried to other
animals or humans.
[0024] L. intracellularis is a causative agent of PPE (McOrist et
al, 1995). L. intracellularis is an intracellular, possibly
obligate intracellular, bacterium. It can only be cultured in vitro
with tissue culture cells (Jones et al., 1997; Lawson et al., 1993;
McOrist et al, 1995; International Patent Application No.
PCT/US96/09576). L. intracellularis is located in the cytoplasm of
the villus cells and intestinal crypt cells of infected animals.
Pigs suffering from PPE are characterised by irregularities in the
villus cells and intestinal crypt structure with epithelial cell
dysplasia, wherein crypt abscesses form as the villi and intestinal
crypts become branched and fill with inflammatory cells.
[0025] Current control strategies for PPE rely on the use of
antibacterials. However, such a strategy is considered to only be
short to medium term, especially since governmental regulatory
pressures tend to discourage animal husbandry practices which
involve the use of prophylactic antibiotics. There is a need,
therefore, to develop effective, safe and low cost alternatives to
the use of antibiotics and, in particular, to develop vaccine
preparations capable of conferring protective immunity against L.
intracellularis infection in livestock animals.
[0026] The most effective vaccine preparations are generally
comprised of a highly antigenic component, such as a polypeptide or
other macromolecule which is derived from the pathogenic organism
against which the vaccine is directed, wherein said antigenic
component produces little or no contraindications when administered
to a susceptible host animal, and produces little or no antigenic
cross-reactivity with desirable organisms, such as non-pathogenic
organisms that are a part of the normal flora of the intestinal
tract or other tissues of said host animal. In summary, an
effective vaccine preparation must be immunogenic, specific and
safe.
[0027] Accordingly, there is a need to identify highly immunogenic
antigens produced by the bacterium L. intracellularis.
[0028] International Patent Application No. PCT/AU96/00767
describes several L. intracellularis partial genetic sequences, and
partial polypeptides encoded thereby. However, there is a need to
further identify polypeptide immunogens produced by the bacterium
L. intracellularis and immunogenic peptides derived therefrom,
including those immunogens which are genus- or species-specific,
for use in improved vaccine compositions. The presently-described
invention provides such immunogens.
SUMMARY OF THE INVENTION
[0029] One aspect of the present invention is directed to an
isolated or recombinant immunogenic polypeptide which comprises,
mimics or cross-reacts with a B-cell or T-cell epitope of a
polypeptide derived from Lawsonia spp, in particular a polypeptide
selected from the group consisting of flhB, fliR, ntrC, glnH, motA,
motB, tlyC, ytfM, and ytfN polypeptides, or a homologue, analogue
or derivative of any one or more of said polypeptides.
[0030] Preferably, the isolated or recombinant immunogenic
polypeptide is selected from the group consisting of the
following:
[0031] (i) a polypeptide which comprises an amino acid sequence
which has at least about 60% sequence identity overall to an amino
acid sequence selected from the group consisting of SEQ ID NOs: 2,
4, 6, 8, 10, 12, 14, 16, and 18;
[0032] (ii) a polypeptide which comprises an amino acid sequence
which has at least about 60% sequence identity overall to an amino
acid sequence encoded by L. intracellularis DNA contained within a
plasmid selected from the group consisting of AGAL Accession Nos:
NM00/16476 (plasmid pGTE#1 glnH); NM00/16477 (plasmid pGTE#2 flhB);
NM00/16478 (plasmid pGTE#3 fliR); NM00/16479 (plasmid pGTE#4
motA/B); NM00/16480 (plasmid pGTE#5 tlyC); NM00/16481 (plasmid
pGTE#6 ntrC); NM00/16482 (plasmid pGTE#7 ytfM); and NM01/23286
(plasmid pGTE#8 ytfN);
[0033] (iii) a polypeptide which comprises at least about 5
contiguous amino acids of an amino acid sequence selected from the
group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, and
18;
[0034] (iv) a polypeptide which comprises at least about 5
contiguous amino acids of an amino acid sequence encoded by L.
intracellularis DNA contained within a plasmid selected from the
group consisting of AGAL Accession Nos: NM00/16476 (plasmid pGTE#1
glnH); NM00/16477 (plasmid pGTE#2 flhB); NM00/16478 (plasmid pGTE#3
fliR); NM00/16479 (plasmid pGTE#4 motA/B); NM00/16480 (plasmid
pGTE#5 tlyC); NM00/16481 (plasmid pGTE#6 ntrC); NM00/16482 (plasmid
pGTE#7 ytfM); and NM01/23286 (plasmid pGTE#8 ytfN); and
[0035] (v) a homologue, analogue or derivative of any one of (I) to
(iv) which mimics a B-cell or T-cell epitope of Lawsonia spp.
[0036] In an alternative preferred embodiment, the isolated or
recombinant immunogenic polypeptide is selected from the group
consisting of the following:
[0037] (i) a polypeptide which comprises an amino acid sequence
encoded by a nucleotide sequence having at least about 60% sequence
identity overall to a nucleotide sequence selected from the group
consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, and 17;
[0038] (ii) a polypeptide which comprises an amino acid sequence
encoded by a nucleotide sequence having at least about 60% sequence
identity overall to the nucleotide sequence of L. intracellularis
DNA contained within a plasmid selected from the group consisting
of AGAL Accession Nos: NM00/16476 (plasmid pGTE#1 glnH); NM00/16477
(plasmid pGTE#2 flhB); NM00/16478 (plasmid pGTE#3 fliR); NM00/16479
(plasmid pGTE#4 motA/B); NM00/16480 (plasmid pGTE#5 tlyC);
NM00/16481 (plasmid pGTE#6 ntrC); NM00/16482 (plasmid pGTE#7 ytfM);
and NM01/23286 (plasmid pGTE#8 ytfN);
[0039] (iii) a polypeptide encoded by at least about 15 contiguous
nucleotides of a nucleotide sequence selected from the group
consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, and 17;
[0040] (iv) a polypeptide encoded by at least about 15 contiguous
nucleotides of a nucleotide sequence of L. intracellularis DNA
contained within a plasmid selected from the group consisting of
AGAL Accession Nos: NM00/16476 (plasmid pGTE#1 glnH); NM00/16477
(plasmid pGTE#2 flhB); NM00/16478 (plasmid pGTE#3 fliR); NM00/16479
(plasmid pGTE#4 motA/B); NM00/16480 (plasmid pGTE#5 tlyC);
NM00/16481 (plasmid pGTE#6 ntrC); NM00/16482 (plasmid pGTE#7 ytfM);
and NM01/23286 (plasmid pGTE#8 ytfN); and
[0041] (v) a homologue, analogue or derivative of any one of (i) to
(iv) which mimics a B-cell or T-cell epitope of Lawsonia spp.
[0042] In a particularly preferred embodiment, the polypeptide of
the present invention comprises or consists of an amino acid
sequence selected from the group consisting of:
[0043] (i) an amino acid sequence selected from the group
consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, and 18;
and
[0044] (ii) an amino acid sequence encoded by L. intracellularis
DNA contained within a deposited plasmid selected from the group
consisting of AGAL Accession Nos: NM00/16476 (plasmid pGTE#1 glnH);
NM00/16477 (plasmid pGTE#2 flhB); NM00/16478 (plasmid pGTE#3 fliR);
NM00/16479 (plasmid pGTE#4 motA/B); NM00/16480 (plasmid pGTE#5
tlyC); NM00/16481 (plasmid pGTE#6 ntrC); NM00/16482 (plasmid pGTE#7
ytfM); and NM01/23286 (plasmid pGTE#8 ytfN).
[0045] A further aspect of the present invention provides a vaccine
composition for the prophylaxis or treatment of infection in an
animal, such as a pig or bird, by L. intracellularis or a similar
or otherwise related microorganism, said vaccine composition
comprising an immunologically effective amount of an immunogenic
component which comprises an isolated or recombinant polypeptide
selected from the group consisting of flhB, fliR, ntrC, glnH, motA,
motB, tlyC, ytfM, and ytfN polypeptides as described herein and one
or more carriers, diluents and/or adjuvants suitable for veterinary
or pharmaceutical use.
[0046] A further aspect of the invention extends to an
immunologically interactive molecule, such as an antibody or
antibody fragment, which is capable of binding to an immunogenic
polypeptide of the invention selected from the group consisting of
flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM, and ytfN
polypeptides.
[0047] A further aspect of the invention provides a method of
diagnosing infection of an animal by L. intracellularis or a
related microorganism, said method comprising the steps of
contacting a biological sample derived from said animal with an
immunologically interactive molecule of the present invention for a
time and under conditions sufficient for a complex, such as an
antigen:antibody complex, to form, and then detecting said complex
formation.
[0048] A further aspect of the present invention contemplates a
method of determining whether or not an animal has suffered from a
past infection, or is currently infected, by L. intracellularis or
a related microorganism, said method comprising contacting a tissue
or fluid sample, such as blood or serum derived from said animal,
with an immunogenic polypeptide selected from the group consisting
of flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM, and ytfN
polypeptides, or a peptide derived therefrom, for a time and under
conditions sufficient for a complex, such as an antigen:antibody
complex, to form, and then detecting said complex formation.
[0049] A further aspect of the present invention provides an
isolated nucleic acid molecule which comprises a sequence of
nucleotides that encodes, or is complementary to a nucleic acid
molecule that encodes, a polypeptide selected from the group
consisting of flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM, and
ytfN polypeptides, including any and all genes selected from the
group consisting of flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM,
and ytfN genes as defined hereinabove.
[0050] In a preferred embodiment, the isolated nucleic acid
molecule comprises a nucleotide sequence encoding a polypeptide
that is immunologically cross-reactive with L. intracellularis or
other causative agent of PPE, wherein said nucleotide sequence is
selected from the group consisting of:
[0051] (i) a nucleotide sequence having at least about 60% sequence
identity overall to a nucleotide sequence selected from the group
consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, and 17;
[0052] (ii) a nucleotide sequence having at least about 60%
sequence identity overall to L. intracellularis DNA contained
within a plasmid selected from the group consisting of AGAL
Accession Nos: NM00/16476 (plasmid pGTE#1 glnH); NM00/16477
(plasmid pGTE#2 flhB); NM00/16478 (plasmid pGTE#3 fliR); NM00/16479
(plasmid pGTE#4 motA/B); NM00/16480 (plasmid pGTE#5 tlyC);
NM00/16481 (plasmid pGTE#6 ntrC); NM00/16482 (plasmid pGTE#7 ytfM);
and NM01/23286 (plasmid pGTE#8 ytfN);
[0053] (iii) a nucleotide sequence which comprises at least about
15 contiguous nucleotides of a sequence selected from the group
consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, and 17;
[0054] (iv) a nucleotide sequence which comprises at least about 15
contiguous nucleotides of L. intracellularis DNA contained within a
plasmid selected from the group consisting of AGAL Accession Nos:
NM00/16476 (plasmid pGTE#1 glnH); NM00/16477 (plasmid pGTE#2 flhB);
NM00/16478 (plasmid pGTE#3 fliR); NM00/16479 (plasmid pGTE#4
motA/B); NM00/16480 (plasmid pGTE#5 tlyC); NM00/16481 (plasmid
pGTE#6 ntrC); NM00/16482 (plasmid pGTE#7 ytfM); and NM01/23286
(plasmid pGTE#8 ytfN);
[0055] (v) a nucleotide sequence which hybridizes under at least
low stringency, more preferably moderate stringency, and most
preferably high stringency conditions to a nucleotide sequence
selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9,
11, 13, 15, and 17 or a complementary nucleotide sequence
thereto;
[0056] (vi) a nucleotide sequence which hybridizes under at least
low stringency, more preferably moderate stringency, and most
preferably high stringency conditions to L. intracellularis DNA
contained within a plasmid selected from the group consisting of
AGAL Accession Nos: NM00/16476 (plasmid pGTE#1 glnH); NM00/16477
(plasmid pGTE#2 flhB); NM00/16478 (plasmid pGTE#3 fliR); NM00/16479
(plasmid pGTE#4 motA/B); NM00/16480 (plasmid pGTE#5 tlyC);
NM00/16481 (plasmid pGTE#6 ntrC); NM00/16482 (plasmid pGTE#7 ytfM);
and NM01/23286 (plasmid pGTE#8 ytfN); and
[0057] (vii) a homologue, analogue or derivative of any one of (i)
to (vi) which encodes a polypeptide which mimics a B-cell or T-cell
epitope of Lawsonia spp.
[0058] In a particularly preferred embodiment, the isolated nucleic
acid molecule of the present invention comprises or consists of a
nucleotide sequence selected from the group consisting of:
[0059] (i) a nucleotide sequence selected from the group consisting
of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, and 17; or a degenerate
variant thereof;
[0060] (ii) a nucleotide sequence of the L. intracellularis DNA
contained within a deposited plasmid selected from the group
consisting of AGAL Accession Nos: NM00/16476 (plasmid pGTE#1 glnH);
NM00/16477 (plasmid pGTE#2 flhB); NM00/16478 (plasmid pGTE#3 fliR);
NM00/16479 (plasmid pGTE#4 motA/B); NM00/16480 (plasmid pGTE#5
tlyC); NM00/16481 (plasmid pGTE#6 ntrC); NM00/16482 (plasmid pGTE#7
ytfM); and NM01/23286 (plasmid pGTE#8 ytfN); and
[0061] (iii) a nucleotide sequence that encodes the same
polypeptide as (i) or (ii), wherein said polypeptide is selected
from the group consisting of flhB, fliR, ntrC, glnH, motA, motB,
tlyC, ytfvl, and ytfN;
[0062] (iv) a nucleotide sequence that is complementary to (i) or
(ii) or (iii); and
[0063] (v) a nucleotide sequence that hybridises under high
stringency conditions to the complement of a sequence selected from
the group consisting of: SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15 and
17, wherein said nucleotide sequence is the complement of a
sequence that encodes a polypeptide that is immunologically
cross-reactive to a polypeptide selected from the group consisting
of flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM, and ytfN.
[0064] A still further aspect of the invention provides a
diagnostic method of detecting L. intracellularis or related
microorganism in a biological sample derived from an animal
subject, said method comprising the steps of hybridising one or
more polynucleotide or oligonucleotide probes or primers derived
from a gene selected from the group consisting of flhB, fliR, ntrC,
glnH, motA, motB, tlyC, ytfM, and ytfN genes, or a homologue,
analogue or derivative thereof, to said sample, and then detecting
said hybridisation using a detection means. The detection means
according to this aspect of the invention is any nucleic acid-based
hybridisation or amplification reaction.
[0065] A further aspect of the invention provides an isolated probe
or primer derived from a gene selected from the group consisting of
flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM, and ytfN genes. In
a particularly preferred embodiment, the probe or primer of the
invention is useful for isolating the ytfM and/or ytfN genes
described herein. More preferably, the probe or primer of the
invention comprises a nucleotide sequence selected from the group
consisting of SEQ ID NO: 19 to SEQ ID NO: 68 or a complementary
nucleotide sequence thereto.
BRIEF DESCRIPTION OF THE DRAWINGS
[0066] FIG. 1 is a copy of a photographic representation showing
expression of recombinant YtfN protein. The 5' portion of the gene
up to the BgIII site was cloned into pET-30a. A plasmid with the
fragment inserted in the proper orientation was transformed into E.
coli BL21 (DE3) cells, and a single clone was propagated. Induction
was at OD.sub.625=2.9 with 0.1 mM IPTG. Lane 1, whole cell lysate
(WCL) from uninduced cells; lanes 2 and 3, WCL at 2.25 and 3 hrs
post-induction, respectively. Arrow indicates the position of
recombinant YtfN protein.
DETAILED DESCRIPTION OF THE INVENTION
[0067] In work leading up to the present invention, the inventors
sought to identify immunogenic proteins of L. intracellularis for
use in vaccines for the prophylaxis and treatment of PPE in
animals, including pigs and birds.
[0068] Accordingly, one aspect of the present invention is directed
to an isolated or recombinant immunogenic polypeptide which
comprises, mimics or cross-reacts with a B-cell or T-cell epitope
of a polypeptide derived from Lawsonia spp, selected from the group
consisting of flhB, fliR, ntrC, glnH, motA, motB, tlyc, ytfM, and
ytfN, or a homologue, analogue or derivative of any one or more of
said polypeptides.
[0069] Epitopes of Lawsonia spp. may be B cell epitopes or T-cell
epitopes. It is well-known that antibody-binding sites (B-cell
epitopes) involve linear as well as conformational epitopes (van
Regenmortel, 1992). B-cell epitopes are predominantly
conformational. In contrast, T-cells recognize predominantly linear
epitope sequences in combination with MHC class II molecules.
[0070] A precise identification and careful selection of epitopes
of Lawsonia spp. facilitates the development of diagnostic reagents
and vaccine compositions for the effective treatment or prophylaxis
of Lawsonia infections. Epitope identification and characterization
(i.e., determination of the molecular weight, amino acid sequence,
and structure of epitopes of Lawsonia spp.) may be performed using
art-recognised techniques. For the detection of conformational
epitopes, degrading and denaturing of the epitope molecule must be
avoided in order to conserve the three-dimensional structure,
because the antigen-antibody reaction will be diminished if the
secondary structure of the epitope is altered significantly. In
practice, the characterisation and isolation of linear
non-conformational epitopes is easier, because any immunoreactive
regions are contained within a single polypeptide or peptide
fragment which is capable of being purified under a range of
conditions.
[0071] Both non-conformational and conformational epitopes may be
identified by virtue of their ability to bind detectable amounts of
antibodies (such as IgM or IgG) from sera of animals immunised
against or infected with Lawsonia spp. and, in particular L.
intracellularis, or an isolated polypeptide derived therefrom or,
alternatively, by virtue of their ability to bind detectable
amounts of antibodies in a purified Ig fraction derived from such
sera. The antibodies may be derived from or contained within pools
of polyclonal sera, or may be monoclonal antibodies. Antibody
fragments or recombinant antibodies, such as those expressed on the
surface of a bacteriophage or virus particle, such as in a phage
display library, may also be employed.
[0072] The determination of T-cell epitopes is performed by
analysing the ability of the epitope peptides to induce the
proliferation of peripheral blood lymphocytes or T-cell clones. The
identification of T-cell epitopes is accomplished using a variety
of methods as known in the art, including the use of whole and
fragmented native or recombinant antigenic protein, as well as the
more commonly employed "overlapping peptide" method. In the latter
method, overlapping peptides which span the entire sequence of a
polypeptide derived from Lawsonia spp. are synthesized and tested
for their capacity to stimulate T-cell cytotoxic or proliferative
responses in vitro.
[0073] Structure determination of both conformational non-linear
and non-conformational linear epitopes may be performed by nuclear
magnetic resonance spectroscopy (NMR) and X-ray crystallographic
analysis. The determination of epitopes using X-ray techniques
requires the protein-antibody complex to be crystallized, whereas
NMR allows analysis of the complex in a liquid state. NMR measures
the amount of amino acids as well as the neighbourhood of protons
of different amino acid residues, wherein the alternating effect of
two protons along the carbon backbone is characteristic of a
particular epitope.
[0074] A successful method to recognize non-conformational linear
epitopes is the immunoblot and in particular, the Western blot.
Peptides may be generated from a complete Lawsonia spp. polypeptide
by digestion with site-specific proteases, such as trypsin or
chymotrypsin, and the peptides generated thereby can be separated
using standard electrophoretic or chromatographic procedures. For
example, after electrophoresis according to molecular weight using
SDS/PAGE (SDS/polyacrylamide gel electrophoresis) and/or according
to isoelectric point using IEF (isoelectric focussing) or
alternatively, by two-dimensional electrophoresis, the peptides can
be transferred to immobilizing nylon or nitrocellulose membranes
and incubated with sera raised against the intact polypeptides.
Peptides that comprise immunogenic regions (i.e., B-cell or T-cell
epitopes) are bound by the antibodies in the sera and the bound
antibodies may be detected using secondary antibodies, such as
anti-IgG antibodies, that have been labelled radioactively or
enzymatically. The epitopes may then be characterised by
purification based upon their size, charge or ability to bind
specifically to antibodies against the intact polypeptide, using
one or more techniques, such as size-exclusion chromatography,
ion-exchange chromatography, affinity chromatography or ELISA among
others. After purification of the epitope, only one band or spot
should be detectable with gel electrophoresis. The N-terminal or
total sequencing of the polypeptide or peptide fragment offers the
possibility to compare the amino acid sequence with known proteins
in databases.
[0075] Several computer-driven algorithms have now been devised to
search for T-cell epitopes in proteins (Margalit et al., 1987;
Vajda and C. DeLisi, 1990; Altuvia et al., 1994; Parker et
a/0.1994; DeGroot et al., 1995; Gabriel et al., 1995; Meister et
al., 1995). These algorithms search the amino acid sequence of a
given protein for characteristics believed to be common to
immunogenic peptides, locating regions that are likely to induce a
cellular immune response in vitro. Computer-driven algorithms can
identify regions of a Lawsonia spp. polypeptide that contain
epitopes and are less variable among different isolates.
Alternatively, computer-driven algorithms can rapidly identify
regions of each isolate's more variable proteins that should be
included in a multivalent vaccine.
[0076] The AMPHI algorithm (Margalit et al., 1987), which is based
on the periodicity of T cell epitopes, has been widely used for the
prediction of T-cell antigenic sites from sequence information
alone. Essentially, AMPHI describes a common structural pattern of
MHC binding motifs, since MHC binding motifs (i.e., patterns of
amino acids that appear to be common to most of the peptides that
bind to a specific MHC molecule) appear to exhibit the same
periodicity as an alpha helix. Identification of T-cell epitopes by
locating MHC binding motifs in an amino acid sequence provides an
effective means of identifying immunogenic epitopes in diagnostic
assays.
[0077] The EpiMer algorithm (Meister et al., 1995; Gabriel et al.,
1995; DeGroot et al., 1995) locates clustered MHC binding motifs in
amino acid sequences of proteins, based upon the correlation
between MHC binding motif-dense regions and peptides that may have
the capacity to bind to a variety of MHC molecules (promiscuous or
multi-determinant binders) and to stimulate an immune response in
these various MHC contexts as well (promiscuous or
multi-determinant epitopes). The EpiMer algorithm uses a library of
MHC binding motifs for multiple class I and class II HLA alleles to
predict antigenic sites within a protein that have the potential to
induce an immune response in subjects with a variety of genetic
backgrounds. EpiMer locates matches to each MHC-binding motif
within the primary sequence of a given protein antigen. The
relative density of these motif matches is determined along the
length of the antigen, resulting in the generation of a
motif-density histogram. Finally, the algorithm identifies protein
regions in this histogram with a motif match density above an
algorithm-defined cutoff density value, and produces a list of
subsequences representing these clustered, or motif-rich regions.
The regions selected by EpiMer may be more likely to act as
multi-determinant binding peptides than randomly chosen peptides
from the same antigen, due to their concentration of MHC-binding
motif matches. The selection of regions that are MHC binding
motif-dense increases the likelihood that the predicted polypeptide
or peptide fragment contains a "valid" motif, and furthermore, that
the reiteration of identical motifs may contribute to binding.
[0078] Additional MHC binding motif-based algorithms have been
described by Parker et al. (1994) and Altuvia et al. (1994). In
these algorithms, binding to a given MHC molecule is predicted by a
linear function of the residues at each position, based on
empirically defined parameters, and in the case of the Altuvia et
al. (1994) algorithm, known crystallographic structures may also be
taken into consideration.
[0079] Recombinant methods offer the opportunity to obtain well
characterized epitopes of high purity for the production of
diagnostic reagents and epitope-specific vaccine formulations
(Mohapatra et al., 1995). Based upon the amino acid sequence of a
linear epitope and identification of the corresponding nucleotide
sequence encoding same, polymerase chain reaction (PCR) may be
performed to amplify the epitope-encoding region from cDNA. After
cloning and expression in a suitable vector/host system, a large
amount of epitopes of high purity can be extracted. Accordingly,
the present invention clearly extends to both isolated
non-recombinant polypeptides and recombinant polypeptides in an
impure or isolated form.
[0080] The term "polypeptide" as used herein shall be taken to
refer to any polymer consisting of amino acids linked by covalent
bonds and includes within its scope the full-length amino acids
disclosed herein, and any parts or fragments thereof such as, for
example, peptides consisting of about 5-50 amino acid residues in
length, preferably about 5-30 amino acid residues in length, more
preferably about 5-20 amino acid residues in length, and even more
preferably about 5-10 amino acid residues in length. Also included
within the scope of the definition of a "polypeptide" are amino
acid sequence variants, containing one or more preferably
conservative amino acid substitutions, deletions, or insertions,
which do not alter at least one essential property of said
polypeptide such as, for example, its immunogenicity, use as a
diagnostic reagent, or effectiveness as a vaccine against Lawsonia
spp, amongst others. Accordingly, a polypeptide may be isolated
from a source in nature, or chemically synthesized. Furthermore, a
polypeptide may be derived from a full-length protein by chemical
or enzymatic cleavage, using reagents such as CNBr, trypsin, or
chymotrypsin, amongst others.
[0081] Conservative amino acid substitutions are well-known in the
art. For example, one or more amino acid residues of a native
flagellar hook protein of the present invention can be substituted
conservatively with an amino acid residue of similar charge, size
or polarity, with the resulting polypeptide retaining an ability to
function in a vaccine or as a diagnostic reagent as described
herein. Rules for making such substitutions include those described
by Dayhof (1978). More specifically, conservative amino acid
substitutions are those that generally take place within a family
of amino acids that are related in their side chains.
Genetically-encoded amino acids are generally divided into four
groups: (1) acidic=aspartate, glutamate; (2) basic=lysine,
arginine, and histidine; (3) non-polar=alanine, valine, leucine,
isoleucine, proline, phenylalanine, methionine, and tryptophan; and
(4) uncharged polar=glycine, asparagine, glutamine, cysteine,
serine, threonine, and tyrosine. Phenylalanine, tyrosine and
tryptophan are also jointly classified as aromatic amino acids. One
or more replacements within any particular group such as, for
example, the substitution of leucine for isoleucine or valine or
alternatively, the substitution of aspartate for glutamate or
threonine for serine, or of any other amino acid residue with a
structurally-related amino acid residue, will generally have an
insignificant effect on the function of the resulting
polypeptide.
[0082] The present Invention is not limited by the source of the
subject immunogen and clearly extends to isolated and recombinant
polypeptides which are derived from a natural or a non-natural
occurring source.
[0083] The term "recombinant polypeptide" as used herein shall be
taken to refer to a polypeptide which is produced in vitro or in a
host cell by the expression of a genetic sequence encoding said
polypeptide, which genetic sequence is under the control of a
suitable promoter, wherein a genetic manipulation has been
performed in order to achieve said expression. Accordingly, the
term "recombinant polypeptide" clearly encompasses polypeptides
produced by the expression of genetic sequences contained in viral
vectors, cosmids or plasmids that have been introduced into
prokaryotic or eukaryotic cells, tissues or organs. Genetic
manipulations which may be used in this context will be known to
those skilled in the art and include, but are not limited to,
nucleic acid isolation, restriction endonuclease digestion,
exonuclease digestion, end-filling using the Klenow fragment of E.
coli DNA polymerase I or T4 DNA polymerase enzymes, blunt-ending of
DNA molecules using T4 DNA polymerase or ExoIII enzymes,
site-directed mutagenesis, ligation, and amplification reactions.
As will be known to those skilled in the art, additional techniques
such as nucleic acid hybridisations and nucleotide sequence
analysis may also be utilised in the preparation of recombinant
polypeptides, in confirming the identity of a nucleic acid molecule
encoding a desired recombinant polypeptide and a genetic construct
comprising the nucleic acid molecule.
[0084] Wherein the polypeptide of the present invention is a
recombinant polypeptide, it may be produced in and, if desirable,
isolated from a recombinant viral vector expression system or host
cell. As will be known to those skilled in the relevant art, a cell
for production of a recombinant polypeptide is selected on the
basis of several parameters including the genetic constructs used
to express the polypeptide under consideration, as well as the
stability and activity of said polypeptide. It will also be known
to those skilled in the art that the stability or activity of a
recombinant polypeptide may be determined, at least in part, by
post-translational modifications to the polypeptide such as, for
example, glycosylatlon, acylation or alkylation reactions, amongst
others, which may vary between cell lines used to produce the
recombinant polypeptide.
[0085] Accordingly, in a more particularly preferred embodiment,
the present invention extends to a recombinant polypeptide or a
derivative, homologue or analogue thereof as present in a virus
particle, or as produced in prokaryotic or eukaryotic host cell, or
in a virus or cell culture thereof.
[0086] The present invention also extends to a recombinant
polypeptide according to any of the foregoing embodiments which is
produced in a bacterial cell belonging to the genus Lawsonia, in
particular a cell of L. intracellularis, or a culture thereof.
[0087] The term "isolated polypeptide" refers to a polypeptide of
the present invention which has been purified to some extent,
preferably to at least about 20% by weight of protein, preferably
to at least about 50% by weight of protein, more preferably to at
least about 60% by weight of protein, still more preferably to at
least about 70% by weight of protein and even more preferably to at
least about 80% by weight of protein or greater, from its natural
source or, in the case of non-naturally-occurring polypeptides,
from the culture medium or cellular environment in which it was
produced. Such isolation may be performed to improve the
immunogenicity of the polypeptide of the present invention, or to
improve the specificity of the immune response against that
polypeptide, or to remove toxic or undesirable contaminants
therefrom. The necessary or required degree of purity of an
isolated polypeptide will vary depending upon the purpose for which
the polypeptide is intended, and for many applications it will be
sufficient for the polypeptide preparation to contain no
contaminants which would reduce the immunogenicity of the
polypeptide when administered to a host animal, in particular a
porcine or avian animal being immunized against PPE or,
alternatively, which would inhibit immuno-specific binding in an
immunoassay for the diagnosis of PPE or a causative agent
thereof.
[0088] The purity of an isolated polypeptide of the present
invention may be determined by any means known to those skilled in
the art, including the degree of homogeneity of a protein
preparation as assessed by SDS/polyacrylamide gel electrophoresis,
2-dimensional electrophoresis, or amino acid composition analysis
or sequence analysis.
[0089] Preferably, the polypeptide of the present invention will be
substantially homogeneous or substantially free of nonspecific
proteins, as assessed by SDS/polyacrylamide gel electrophoresis,
2-dimensional electrophoresis, or amino acid composition analysis
or sequence analysis.
[0090] The polypeptide of the present invention can be purified for
use as a component of a vaccine composition by any one or a
combination of methods known to those of ordinary skill in the art,
including, for example, reverse phase chromatography, HPLC,
ion-exchange chromatography, and affinity chromatography, among
others.
[0091] In a preferred embodiment, the isolated or recombinant
polypeptide of the invention functions is secretable into the
periplasmic space of a cell, preferably into the periplasm of a
prokaryotic cell, such as, for example, Escherichia coli. or L.
intracellularis, or, alternatively, is immunologically
cross-reactive with a L. intracellularis polypeptide selected from
the group consisting of flhB, fliR, ntrC, glnH, motA, motB, tlyC,
ytfM, and ytfN.
[0092] In a particularly preferred embodiment, the isolated or
recombinant polypeptide of the invention is derived from Lawsonia
spp. or other pathogenic agent associated with the onset and/or
development of PPE and more preferably, the subject polypeptide is
derived from L. intracellularis.
[0093] A B-cell or T-cell epitope of a polypeptide selected from
the group consisting of flhB, fliR, ntrC, glnH, motA, motB, tlyC,
ytfM, and ytfN polypeptides, or a homologue, analogue or derivative
of any one or more of said polypeptides, may comprise one or more
of the following:
[0094] (i) the primary amino acid sequence of any one of said
polypeptides, as determined by an art-accepted methodology to
comprise a continuous non-conformational epitope;
[0095] (ii) the secondary structure which any one of said
polypeptides adopt, as determined by an art-accepted methodology to
comprise a continuous conformational epitope;
[0096] (iii) the tertiary structure which any one of said
polypeptides adopt in contact with another region of the same
polypeptide molecule, as determined by an art-accepted methodology
to comprise a discontinuous conformational epitope; or
[0097] (iv) the quaternary structure which any one of said
polypeptides adopt in contact with a region of another polypeptide
molecule, as determined by an art-accepted methodology to comprise
a discontinuous conformational epitope.
[0098] Accordingly, immunogenic polypeptides or derivatives,
homologues or analogues thereof comprising the same, or
substantially the same primary amino acid sequence are hereinafter
defined as "immunogens which comprise a B-cell or T-cell epitope"
or similar term.
[0099] Immunogenic polypeptides or derivatives, homologues, or
analogues thereof comprising different primary amino acid sequences
may comprise immunologically identical immunogens, because they
possess conformational B-cell or T-cell epitopes that are
recognised by the immune system of a host species to be identical.
Such immunogenic polypeptides or derivatives, homologues or
analogues thereof are hereinafter defined as "immunogens which
mimic or cross-react with a B-cell or T-cell epitope", or similar
term.
[0100] Accordingly, the present invention extends to an immunogen
which comprises, mimics, or cross-reacts with a B-cell or T-cell
epitope of an isolated or recombinant polypeptide according to any
one of the foregoing embodiments or a derivative, homologue or
analogue thereof. In a particularly preferred embodiment, the
present invention provides an immunogen which comprises, mimics, or
cross-reacts with a B-cell or T-cell epitope of an isolated or
recombinant polypeptide which in its native form is obtainable from
a species of Lawsonia such as, but not limited to L.
intracellularis and which polypeptide preferably has the same
biological function as a polypeptide selected from the group
consisting of flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM, and
ytfN, as hereinbefore defined.
[0101] Preferably, such immunogenic polypeptides will not comprise
a primary amino acid sequence which is highly-conserved between L.
intracellularis and another non-pathogenic microorganism which is
normally resident in the gut or other organ of an animal, in
particular a porcine or avian animal. The significance of this
exclusion to those embodiments of the invention wherein specificity
is essential to performance (eg vaccine and diagnostic
applications) will be apparent to those skilled in the art.
[0102] To improve the immunogenicity of a subject polypeptide of
the present invention one or more amino acids not corresponding to
the original protein sequence can be added to the amino or carboxyl
terminus of the polypeptide. Such extra amino acids are useful for
coupling the polypeptide to another peptide or polypeptide, to a
large carrier protein or to a solid support. Amino acids that are
useful for these purposes include but are not limited to tyrosine,
lysine, glutamic acid, aspartic acid, cysteine and derivatives
thereof. Additional protein modification techniques can be used
such as, e.g., NH.sub.2-acetylation or COOH-terminal amidation, to
provide additional means for coupling the polypeptide to another
polypeptide or peptide molecule, or to a solid support. Procedures
for coupling polypeptides to each other, or to carrier proteins or
solid supports, are well known in the art. Polypeptides containing
the above-mentioned extra amino acid residues at either the
carboxyl- or amino-termini and either uncoupled or coupled to a
carrier or solid support, are consequently within the scope of the
present invention.
[0103] Furthermore, the polypeptide can be immobilised to a
polymeric carrier or support material.
[0104] In an alternative embodiment, the immunogenicity of a
polypeptide of the present invention may be improved using
molecular biology techniques to produce a fusion protein containing
one or more polypeptides of the present invention fused to a
carrier molecules such as a highly immunogenic protein. For
example, a fusion protein containing a polypeptide of the present
invention fused to the highly immunogenic B subunit of cholera
toxin can be used to increase the immune response to the
polypeptide. The present invention also contemplates fusion
proteins comprising a cytokine, such as an interleukin, fused to
the subject polypeptide of the present invention, and genes
encoding same.
[0105] Preferably, the polypeptide of the present invention, or a
derivative, homologue or analogue thereof, when administered to a
mammal, induces an immune response in said mammal. More preferably,
the polypeptide of the present invention, when administered to a
mammal, in particular a porcine animal (e.g., a pig) induces a
protective immune response against Lawsonia spp., and preferably
against L. intracellularis, therein. As used herein, the phrase
"induction of a protective immune response", and the like, refers
to the ability of the administered polypeptide of the present
invention to prevent or detectably slow the onset, development, or
progression of symptoms associated with Lawsonia infection, and
preferably, to prevent or detectably slow the onset, development,
or progression of symptoms associated with PPE in pigs.
[0106] Preferably, the isolated or recombinant immunogenic
polypeptide is selected from the group consisting of the
following:
[0107] (i) a polypeptide which comprises an amino acid sequence
which has at least about 60% sequence identity overall to an amino
acid sequence selected from the group consisting of SEQ ID NOs: 2,
4, 6, 8, 10, 12, 14, 16, and 18;
[0108] (ii) a polypeptide which comprises an amino acid sequence
which has at least about 60% sequence identity overall to an amino
acid sequence encoded by L. intracellularis DNA contained within a
plasmid selected from the group consisting of AGAL Accession Nos:
NM00/16476 (plasmid pGTE#1 glnH); NM00/16477 (plasmid pGTE#2 flhB);
NM00/16478 (plasmid pGTE#3 fliR); NM00/16479 (plasmid pGTE#4
motA/B); NM00/16480 (plasmid pGTE#5 tlyC); NM00/16481 (plasmid
pGTE#6 ntrC); NM00/16482 (plasmid pGTE#7 ytfM); and NM01/23286
(plasmid pGTE#8 ytfN);
[0109] (iii) a polypeptide which comprises at least about 5
contiguous amino acids, of an amino acid sequence selected from the
group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, and
18;
[0110] (iv) a polypeptide which comprises at least about 5
contiguous amino acids of an amino acid sequence encoded by L.
intracellularis DNA contained within a plasmid selected from the
group consisting of AGAL Accession Nos: NM00/16476 (plasmid pGTE#1
glnH); NM00/16477 (plasmid pGTE#2 flhB); NM00/16478 (plasmid pGTE#3
fliR); NM00/16479 (plasmid pGTE#4 mot/AB); NM00/16480 (plasmid
pGTE#5 tlyC); NM00/16481 (plasmid pGTE#6 ntrC); NM00/16482 (plasmid
pGTE#7 ytfM); and NM01/23286 (plasmid pGTE#8 ytfN);
[0111] (v) a homologue, analogue or derivative of any one of (i) to
(iv) which mimics a B-cell or T-cell epitope of Lawsonia spp.
[0112] In an alternative preferred embodiment, the isolated or
recombinant immunogenic polypeptide is selected from the group
consisting of the following:
[0113] (i) a polypeptide which comprises an amino acid sequence
encoded by a nucleotide sequence having at least about 60% sequence
identity overall to a nucleotide sequence selected from the group
consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, and 17;
[0114] (ii) a polypeptide which comprises an amino acid sequence
encoded by a nucleotide sequence having at least about 60% sequence
identity overall to the nucleotide sequence of L. intracellularis
DNA contained within a plasmid selected from the group consisting
of AGAL Accession Nos: NM00/16476 (plasmid pGTE#1 glnH); NM00/16477
(plasmid pGTE#2 flhB); NM00/16478 (plasmid pGTE#3 fliR); NM00/16479
(plasmid pGTE#4 motA/B); NM00/16480 (plasmid pGTE#5 tlyC);
NM00/16481 (plasmid pGTE#6 ntrC); NM00/16482 (plasmid pGTE#7 ytfM);
and NM01/23286 (plasmid pGTE#8 ytfN);
[0115] (iii) a polypeptide encoded by at least about 15 contiguous
nucleotides of a nucleotide sequence selected from the group
consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, and 17;
[0116] (iv) a polypeptide encoded by at least about 15 contiguous
nucleotides of a nucleotide sequence of L. intracellularis DNA
contained within a plasmid selected from the group consisting of
AGAL Accession Nos: NM00/16476 (plasmid pGTE#1 glnH); NM00/16477
(plasmid pGTE#2 flhB); NM00/16478 (plasmid pGTE#3 fliR); NM00/16479
(plasmid pGTE#4 motA/B); NM00/16480 (plasmid pGTE#5 tlyC);
NM00/16481 (plasmid pGTE#6 ntrC); NM00/16482 (plasmid pGTE#7 ytfM);
and NM01/23286 (plasmid pGTE#8 ytfN);
[0117] (v) a homologue, analogue or derivative of any one of (i) to
(iv) which mimics a B-cell or T-cell epitope of Lawsonia spp.
[0118] Preferably, the immunogenic polypeptide encompassed by the
present invention has at least about 70% identity, more preferably
at least about 80% identity, even more preferably at least about
90% identity, and still even more preferably at least about 95%
identity to the amino acid sequence of a polypeptide selected from
the group consisting of flhB, fliR, ntrC, glnH, motA, motB, tlyC,
ytfM, and ytfN polypeptides, as hereinbefore defined.
[0119] In determining whether or not two amino acid sequences fall
within these percentage limits, those skilled in the art will be
aware that it is necessary to conduct a side-by-side comparison or
multiple alignment of sequences. In such comparisons or alignments,
differences will arise in the positioning of non-identical
residues, depending upon the algorithm used to perform the
alignment. In the present context, reference to a percentage
sequence identity or similarity between two or more amino acid
sequences shall be taken to refer to the number of identical and
similar residues respectively, between said sequences as determined
using any standard algorithm known to those skilled in the art. For
example, amino acid sequence identities or similarities may be
calculated using the GAP programme of the Computer Genetics Group,
Inc., University Research Park, Madison, Wis., United States of
America (Devereaux et al., 1984). The GAP programme utilizes the
algorithm of Needleman and Wunsch (1970) to maximise the number of
identical/similar residues and to minimise the number and/or length
of sequence gaps in the alignment. Alternatively or in addition,
where more than two amino acid sequences are being compared, the
ClustalW programme of Thompson et al (1994) can be used.
[0120] Preferably, the isolated or recombinant immunogenic
polypeptide of the invention comprises at least about 10 contiguous
amino acids of a polypeptide selected from the group consisting of
flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM, and ytfN
polypeptides, as hereinbefore defined. More preferably, the
isolated or recombinant immunogenic polypeptide of the invention
comprises at least about 20 contiguous amino acid residues of a
polypeptide selected from the group consisting of flhB, fliR, ntrC,
glnH, motA, motB, tlyC, ytfM, and ytfN polypeptides, as
hereinbefore defined. Even more preferably, the isolated or
recombinant immunogenic polypeptide of the invention comprises at
least about 30 contiguous amino acid residues of a polypeptide
selected from the group consisting of flhB, fliR, ntrC, glnH, motA,
motB, tlyC, ytfM, and ytfN polypeptides, as hereinbefore defined,
and still even more preferably, at least about 40 contiguous amino
acid residues of said flhB, fliR, ntrC, glnH, motA, motB, tlyC,
ytfM, or ytfN polypeptides. The present invention further
encompasses homologues, analogues and derivatives of a polypeptide
selected from the group consisting of flhB, fliR, ntrC, glnH, motA,
motB, tlyC, ytfM, and ytfN polypeptides, as hereinbefore
defined.
[0121] "Homologues" of a polypeptide are those immunogenic
polypeptides that are derived from a full-length L. intracellularis
polypeptides described herein, or have sequence similarity to a
full-length L. intracellularis polypeptide, notwithstanding one or
more amino acid substitutions, deletions and/or additions relative
to the full-length L. intracellularis polypeptide. A homologue may
also retain the biological activity or catalytic activity of the
full-length polypeptide. In such homologues, one or more amino
acids can be replaced by other amino acids having similar
properties such as, for example, hydrophobicity, hydrophilicity,
hydrophobic moment, antigenicity, propensity to form or break
.alpha.-helical structures of .beta.-sheet structures, and so
on.
[0122] Substitutional variants are those in which at least one
residue in the sequence has been removed and a different residue
inserted in its place. Amino acid substitutions are typically of
single residues, but may be clustered depending upon functional
constraints placed upon the polypeptide; insertions will usually be
of the order of about 1-10 amino acid residues. and deletions will
range from about 1-20 residues. Preferably, amino acid
substitutions will comprise conservative amino acid substitutions,
such as those described supra.
[0123] Insertional amino acid sequence variants are those in which
one or more amino acid residues are introduced into a predetermined
site in the protein. Insertions can comprise amino-terminal and/or
carboxyl terminal fusions as well as intra-sequence insertions of
single or multiple amino acids. Generally, insertions within the
amino acid sequence will be smaller than amino or carboxyl terminal
fusions, of the order of about 1 to 4 residues.
[0124] Deletional variants are characterised by the removal of one
or more amino acids from the sequence.
[0125] Amino acid variants of the polypeptide of the present
invention may readily be made using polypeptide synthetic
techniques well known in the art, such as solid phase synthesis and
the like, or by recombinant DNA manipulations. The manipulation of
DNA sequences to produce variant proteins which manifest as
substitutional, insertional or deletional variants are well known
in the art. For example, techniques for making substitution
mutations at predetermined sites in DNA having known sequence are
well known to those skilled in the art, such as by M13 mutagenesis
or other site-directed mutagenesis protocol.
[0126] "Analogues" are defined as those immunogenic polypeptides
that are derived from a full-length L. intracellularis polypeptides
described herein, or have sequence similarity to a full-length L.
intracellularis polypeptide, notwithstanding one or more
non-naturally occurring or modified amino acid residues relative to
the naturally-occurring full-length L. intracellularis polypeptide.
The term "analogue" shall also be taken to include an amino acid
sequence which is not similar to an amino acid sequence of a
full-length L. intracellularis polypeptide set forth herein,
however mimics or cross-reacts with a B-cell or T-cell epitope of
Lawsonia spp. and preferably, mimics or cross-reacts with a B-cell
or T-cell epitope of L. intracellularis, such as, for example, a
polypeptide which is derived from a computational prediction or
empirical data revealing the secondary, tertiary or quaternary
structure of the full-length polypeptide or an epitope thereof.
[0127] For example, mimotopes (polypeptide analogues that
cross-react with a B-cell or T-cell epitope of the Lawsonia
polypeptide of the invention but, however, comprise a different
amino acid sequence to said epitope) may be identified by screening
random amino acid sequences in polypeptide libraries with
antibodies that bind to a desired T-cell or B-cell epitope. As with
techniques for the identification of B-cell or T-cell epitopes as
described supra, the antibodies used to identify such mimotopes may
be polyclonal or monoclonal or recombinant antibodies, in crude or
purified form. Mimotopes of a T-cell epitope may then be assayed
further for their ability to stimulate T-cell cytotoxic or
proliferative responses in vitro. Mimotopes are particularly useful
as analogues of non-linear (i.e., conformational) epitopes of the
polypeptide of the present invention, because conformational
epitopes are generally formed from non-contiguous regions in a
polypeptide, and the mimotopes provide immunogenic equivalents
thereof in the form of a single polypeptide molecule.
[0128] Additionally, the use of polypeptide analogues can result in
polypeptides with increased immunogenic and/or antigenic activity,
that are less sensitive to enzymatic degradation, and which are
more selective. A suitable proline analogue is 2-aminocyclopentane
carboxylic acid (.beta.AC.sup.5c) which has been shown to increase
the immunogenic activity of a native polypeptide more than 20 times
(Mierke et al, 1990; Portoghese et al., 1990; Goodman et al,
1987).
[0129] "Derivatives" of a polypeptide selected from the group
consisting of flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM, and
ytfN polypeptides, as hereinbefore defined, are those peptides or
polypeptides which comprise at least about five contiguous amino
acid residues of any one or more of said flhB, fliR, ntrC, glnH,
motA, motB, tlyC, ytfM, or ytfN polypeptides.
[0130] A "derivative" may further comprise additional
naturally-occurring, altered glycosylated, acylated or
non-naturally occurring amino acid residues compared to the amino
acid sequence of a flhB, or fliR, or ntrC, or glnH, or motA, or
motB, or tlyc, or ytfM, or ytfN polypeptide, as hereinbefore
defined. Alternatively or in addition, a derivative may comprise
one or more non-amino acid substituents such as, for example, a
reporter molecule or other ligand, covalently or non-covalently
bound to the amino acid sequence of a flhB, or fliR, or ntrC, or
glnH, or motA, or motB, or tlyC, or ytfM, or ytfN polypeptide, such
as, for example, a reporter molecule which is bound thereto to
facilitate its detection.
[0131] Other examples of recombinant or synthetic mutants and
derivatives of a polypeptide immunogen of the present invention
include those incorporating single or multiple substitutions in the
amino acid sequence of a polypeptide selected from the group
consisting of flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM, and
ytfN polypeptides. Recombinant or synthetic mutants and derivatives
produced by making deletions from the amino acid sequence of a
polypeptide selected from the group consisting of flhB, fliR, ntrC,
glnH, motA, motB, tlyC, ytfM, and ytfN polypeptides, are also
included within the scope of preferred derivatives. Additionally,
recombinant or synthetic mutants and derivatives produced by making
additions to a polypeptide selected from the group consisting of
flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM, and ytfN
polypeptides, such as, for example, using carbohydrates, lipids
and/or proteins or polypeptides, are also encompassed by the
present invention.
[0132] Naturally-occurring or altered glycosylated or acylated
forms of the flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM, or
ytfN polypeptides are particularly contemplated by the present
invention.
[0133] Additionally, homopolymers or heteropolymers comprising one
or more copies of the reference polypeptides, or one or more
derivatives, homologues or analogues thereof, are clearly within
the scope of the present invention.
[0134] Preferably, homologues, analogues and derivatives of the
flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM, or ytfN
polypeptides of the invention are "immunogenic", defined
hereinafter as the ability of said polypeptide, or a derivative,
homologue or analogue thereof, to elicit B cell and/or T cell
responses in the host, in response to immunization.
[0135] Preferred homologues, analogues and derivatives of the flhB,
fliR, ntrC, glnH, motA, motB, tlyC, ytfM, or ytfN polypeptides of
the invention include any amino acid variant that functions as B
cell or T cell epitope of any one of said polypeptides, wherein
said variant is capable of mediating an immune response, such as,
for example, a mimotope of the immunogenic polypeptide which has
been produced by synthetic means, such as by Fmoc chemistry. The
only requirement of such variant molecules is that they cross-react
immunologically with a polypeptide selected from the group
consisting of flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM, and
ytfN, as hereinbefore defined, or an epitope of said
polypeptide.
[0136] As will be apparent to those skilled in the art, such
homologues, analogues and derivatives of the polypeptides of the
invention molecules will be useful to prepare antibodies that
cross-react with antibodies against said polypeptide and/or to
elicit a protective immune response of similar specificity to that
elicited by said polypeptide. Such molecules will also be useful in
diagnostic and other applications that are immunological in nature
such as, for example, diagnostics which utilise one or more
immunoassay formats (eg. ELISA, RIA and the like).
[0137] Accordingly, the immunogen of the present invention or a
derivative, homologue or analogue thereof is useful in vaccine
compositions that protect an individual against infection by L.
intracellularis and/or as an antigen to elicit polyclonal or
monoclonal antibody production and/or in the detection of
antibodies against L. intracellularis in infected animals,
particularly in porcine and avian animals.
[0138] The polypeptides of the present invention may comprise
leader sequences to facilitate their secretion into the periplasmic
space, either as part of the native protein, or alternatively,
added by recombinant engineering means. Such may have improved
immunogenicity compared to non-secreted or non-secretable
polypeptides of L. intracellularis, or non-secreted or
non-secretable polypeptides of other causative agents of PPE. The
particular advantages of such peptides will be immediately apparent
to those skilled in the production of vaccine compositions, where
the inherent immunogenicity of the immunogen is an important
consideration for a protective immune response to be conferred.
[0139] Moreover, unique regions of the L. intracellularis
polypeptides exemplified herein are promising antigenic peptides
for the formulation of Lawsonia-specific vaccines and diagnostics
for the specific detection of Lawsonia spp. in biological
samples.
[0140] A second aspect of the present invention provides a vaccine
composition for the prophylaxis or treatment of infection in a
mammal or bird by L. intracellularis or similar or otherwise
related microorganism, said vaccine composition comprising:
[0141] (i) an immunogenic component which comprises an isolated or
recombinant polypeptide selected from the group consisting of flhB,
fliR, ntrC, glnH, motA, motB, tlyC, ytfM, and ytfN polypeptides or
an immunogenic homologue, analogue or derivative of any one of said
polypeptides which is immunologically cross-reactive with L.
Intracellularis; and
[0142] (ii) one or more carriers, diluents and/or adjuvants
suitable for veterinary or pharmaceutical use.
[0143] As used herein, the term "immunogenic component" refers to a
polypeptide encoded by DNA from, or derived from, L.
intracellularis or a related microorganism thereto which is capable
of inducing a protective immune response in an animal, in
particular a porcine or avian animal, whether or not said
polypeptide is in an isolated or recombinant form. Accordingly, the
vaccine composition clearly encompasses those vaccine compositions
which comprise attenuated, killed or non-pathogenic isolates or
forms of L. intracellularis or related microorganisms thereto which
comprise or express said polypeptide.
[0144] By "protective immune response" is meant that the
immunogenic component elicits an immune response in the animal to
which the vaccine composition is administered at the humoral and/or
cellular level which is sufficient to prevent infection by L.
intracellularis or a related microorganism thereto and/or which is
sufficient to detectably reduce one or more symptoms or conditions,
or to detectably slow the onset of one or more symptoms or
conditions, associated with infection by L. intracellularis or a
related microorganism thereto in an animal host, as compared to a
control infected animal. The term "effective amount" of an
immunogenic component present in the vaccine composition refers to
that amount of said immunogenic component that is capable of
inducing a protective immune response after a single complete dose
has been administered, or after several divided doses have been
administered.
[0145] Preferably, the polypeptide component of the subject vaccine
composition comprises an amino acid sequence which is both
immunogenic and specific, by virtue of its immunological
cross-reactivity with the causative agent of PPE, L.
intracellularis. In this regard, It will be apparent from the
preceding description that such polypeptide components may comprise
the amino acid sequence of a polypeptide of L. intracellularis as
exemplified herein, or alternatively, an immunologically
cross-reactive homologue, analogue or derivative of said amino acid
sequence, such as, for example, a mimotope of said sequence.
[0146] The immunogenic polypeptide or immunogenic homologue,
analogue or derivative may be a naturally-occurring polypeptide in
isolated or recombinant form according to any of the embodiments
described supra or exemplified herein. Preferably, the immunogenic
polypeptide or immunogenic homologue, analogue or derivative is
derived from Lawsonia spp., in particular L. intracellularis or a
microorganism that is related thereto.
[0147] Preferably, the immunogenic component has undergone at least
one purification step or at least partial concentration from a cell
culture comprising L. intracellularis or a related microorganism
thereto, or from a lysed preparation of L. intracellularis cells or
related microorganism, or from another culture in which the
immunogenic component is recombinantly expressed. The purity of
such a component which has the requisite immunogenic properties is
preferably at least about 20% by weight of protein in a particular
preparation, more preferably at least about 50%, even more
preferably at least about 60%, still more preferably at least about
70% and even more preferably at least about 80% or greater.
[0148] The immunogenic component of the vaccine of the present
invention can comprise a single polypeptide, or a range or
combination of different polypeptides covering different or similar
epitopes. In addition or, alternatively, a single polypeptide can
be provided with multiple epitopes. The latter type of vaccine is
referred to as a polyvalent vaccine. A multiple epitope includes
two or more epitopes located within a polypeptide molecule.
[0149] The formulation of vaccines is generally known in the art
and reference can conveniently be made to Remington's
Pharmaceutical Sciences, 17th ed., Mack Publishing Co., Easton,
Pa., USA.
[0150] A particularly useful form of the vaccine is a recombinant
vaccine produced, for example, in a vaccine vector, such as but not
limited to a mammalian cell transfected with a vaccinia virus
vector, an insect cell transfected with a baculovirus vector, or a
bacterial cell transfected with a plasmid or cosmid, the only
requirement being that the vector expresses the immunogenic
component.
[0151] The present invention clearly extends to recombinant vaccine
compositions in which the immunogenic component at least is
contained within killed vaccine vectors prepared, for example, by
heat, formalin or other chemical treatment, electric shock or high
or low pressure forces. According to this embodiment, the
immunogenic component of the vaccine is generally synthesized in a
live vaccine vector which is killed prior to administration to an
animal.
[0152] Furthermore, the vaccine vector expressing the immunogenic
component may be non-pathogenic or attenuated. Within the scope of
this embodiment are cells that have been transfected with
non-pathogenic or attenuated viruses encoding the immunogenic
component of the vaccine and non-pathogenic or attenuated cells
that directly express the immunogenic component.
[0153] Attenuated or non-pathogenic host cells include those cells
which are not harmful to an animal to which the subject vaccine is
administered. As will be known to those skilled in the art, "live
vaccines" can comprise an attenuated virus vector encoding the
immunogenic component or a host cell comprising same, which is
capable of replicating in an animal to which it is administered,
and using host cell machinery to express the immunogenic component
albeit producing no adverse side-effects therein. Such vaccine
vectors may colonise the gut or other organ of the vaccinated
animal. Such live vaccine vectors are efficacious by virtue of
their ability to continually express the immunogenic component in
the host animal for a time and at a level sufficient to confer
protective immunity against a pathogen which expresses an
immunogenic equivalent of said immunogenic component. The present
invention clearly encompasses the use of such attenuated or
non-pathogenic vectors and live vaccine preparations.
[0154] The vaccine vector may be a virus, bacterial cell or a
eukaryotic cell such as an insect, avian, porcine or other
mammalian cell or a yeast cell or a cell line such as COS, VERO,
HeLa, mouse C127, Chinese hamster ovary (CHO), WI-38, baby hamster
kidney (BHK) or MDCK cell lines. Suitable prokaryotic cells include
Mycobacterium spp., Corynebacterium spp., Salmonella spp.,
Escherichia coli, Bacillus spp. and Pseudomonas spp, amongst
others. Bacterial strains which are suitable for the present
purpose are well-known in the relevant art (Ausubel et al, 1987;
Sambrook et al, 1989).
[0155] Such cells and cell lines are capable of expression of a
genetic sequence encoding a polypeptide of the present invention
from L. intracellularis, or a homologue, analogue or derivative
thereof, in a manner effective to induce a protective immune
response in the animal. For example, a non-pathogenic bacterium can
be prepared containing an expression vector which comprises a
nucleotide sequence encoding a polypeptide selected from the group
consisting of flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM, and
ytfN polypeptides, or a homologue, analogue, or derivative thereof,
wherein said nucleotide sequence is placed operably under the
control of a constitutive or inducible promoter sequence. The
bacterium is then permitted to colonise suitable locations in a
pig's gut, where it replicates and expresses the said polypeptide
in amount sufficient to induce a protective immune response against
L. intracellularis.
[0156] In a further alternative embodiment, the vaccine can be a
DNA or RNA vaccine comprising a DNA or RNA molecule encoding a
polypeptide selected from the group consisting of flhB, fliR, ntrC,
glnH, motA, motB, tlyC, ytfM, and ytfN polypeptides or homologues,
analogues or derivatives thereof, wherein said vaccine is injected
into muscular tissue or other suitable tissue in a pig under
conditions sufficient to permit transient expression of said DNA or
RNA to produce an effective amount of said polypeptide to induce a
protective immune response. In a preferred embodiment, the DNA
vaccine is in the form of a plasmid, in which the DNA is operably
connected with a promoter region capable of expressing the
nucleotide sequence encoding the immunogen in cells of the
immunized animal.
[0157] In the production of a recombinant vaccine, except for a DNA
vaccine described herein, it is therefore necessary to express the
immunogenic component in a suitable vector system. For the present
purpose, the immunogenic component can be expressed by:
[0158] (i) placing an isolated nucleic acid molecule in an
expressible format, said nucleic acid molecule comprising the
coding region of a gene selected from the group consisting of flhB,
fliR, ntrC, glnH, motA, motB, tlyC, ytfM, and ytfN genes, or a
protein-encoding homologue, analogue or derivative thereof;
[0159] (ii) introducing the isolated nucleic acid molecule of (i)
in an expressible format into a suitable vaccine vector; and
[0160] (iii) incubating or growing the vaccine vector for a time
and under conditions sufficient for expression of the immunogenic
component encoded by said nucleic acid molecule to occur.
[0161] It will be apparent from the preceding discussion that the
protein-encoding region of a flhB, fliR, ntrC, glnH, motA, motB,
tlyC, ytfM, or ytfN gene comprises a nucleotide sequence selected
from the group consisting of SEQ ID NOs:1, 3, 5, 79, 11, 13, 15,
and 17, or alternatively or in addition, a protein-encoding
nucleotide sequence of L. intracellularis DNA contained within a
deposited plasmid selected from the group consisting of AGAL
Accession Nos: NM00/16476 (plasmid pGTE#1 glnH); NM00/16477
(plasmid pGTE#2 flhB); NM00/16478 (plasmid pGTE#3 fliR); NM00/16479
(plasmid pGTE#4 motA/B); NM00/16480 (plasmid pGTE#5 tlyC);
NM00/16481 (plasmid pGTE#6 ntrC); NM00/16482 (plasmid pGTE#7 ytfM);
and NM01/23286 (plasmid pGTE#8 ytfN).
[0162] Preferred homologues of the protein-encoding region of a
flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM, or ytfN gene
include those nucleotide sequences selected from the group
consisting of:
[0163] (i) a protein-encoding nucleotide sequence having at least
about 60% sequence identity overall to a nucleotide sequence
selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9,
11, 13, 15, and 17 or a degenerate variant thereof;
[0164] (ii) a protein-encoding nucleotide sequence having at least
about 60% sequence identity overall to the protein-encoding
sequence of L. intracellularis DNA contained within a plasmid
selected from the group consisting of AGAL Accession Nos:
NM00/16476 (plasmid pGTE#1 glnH); NM00/16477 (plasmid pGTE#2 flhB);
NM00/16478 (plasmid pGTE#3 fliR); NM00/16479 (plasmid pGTE#4
motA/B); NM00/16480 (plasmid pGTE#5 tlyC); NM00/16481 (plasmid
pGTE#6 ntrC); NM00/16482 (plasmid pGTE#7 ytfM); and NM01/23286
(plasmid pGTE#8 ytfN);
[0165] (iii) a protein-encoding nucleotide sequence which comprises
at least about 15 contiguous nucleotides of a sequence selected
from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15,
and 17;
[0166] (iv) a protein-encoding nucleotide sequence which comprises
at least about contiguous nucleotides of the protein-encoding
sequence of L. intracellularis DNA contained within a plasmid
selected from the group consisting of AGAL Accession Nos:
NM00/16476 (plasmid pGTE#1 glnH); NM00/16477 (plasmid pGTE#2 flhB);
NM00/16478 (plasmid pGTE#3 fliR); NM00/16479 (plasmid pGTE#4
motA/B); NM00/16480 (plasmid pGTE#5 tlyC); NM00/16481 (plasmid
pGTE#6 ntrC); NM00/16482 (plasmid pGTE#7 ytfM); and NM01/23286
(plasmid pGTE#8 ytfN);
[0167] (v) a protein-encoding nucleotide sequence which hybridizes
under at least low stringency conditions to the complement of a
nucleotide sequence selected from the group consisting of SEQ ID
NOs: 1, 3, 5, 7, 9, 11, 13, 15, and 17; and
[0168] (vi) a protein-encoding nucleotide sequence which hybridizes
under at least low stringency conditions to the non-coding strand
of L. intracellularis DNA contained within a plasmid selected from
the group consisting of AGAL Accession Nos: NM00/16476 (plasmid
pGTE#1 glnH); NM00/16477 (plasmid pGTE#2 flhB); NM00/16478 (plasmid
pGTE#3 fliR); NM00/16479 (plasmid pGTE#4 motB); NM00/16480 (plasmid
pGTE#5 tlyC); NM00/16481 (plasmid pGTE#6 ntrC); NM00/16482 (plasmid
pGTE#7 ytfM); and NM01/23286 (plasmid pGTE#8 ytfN).
[0169] The present invention clearly extends to analogues or
derivatives of any one of (i) to (vi) which encode a polypeptide
which mimics a B-cell or T-cell epitope of Lawsonia spp.
[0170] For the present purpose, a preferred homologue of the
protein-encoding region of a flhB, fliR, ntrC, glnH, motA, motB,
tlyC, ytfM, or ytfN gene will have at least about 80% nucleotide
sequence identity to the coding region of said gene, still more
preferably at least about 90% identity, and yet still more
preferably at least about 95% identity.
[0171] In determining whether or not two nucleotide sequences fall
within these percentage limits, those skilled in the art will be
aware that it is necessary to conduct a side-by-side comparison or
multiple alignment of sequences. In such comparisons or alignments,
differences may arise in the positioning of non-identical residues,
depending upon the algorithm used to perform the alignment. In the
present context, reference to a percentage identity between two or
more nucleotide sequences shall be taken to refer to the number of
identical residues between said sequences as determined using any
standard algorithm known to those skilled in the art. For example,
nucleotide sequences may be aligned and their identity calculated
using the BESTFIT programme or other appropriate programme of the
Computer Genetics Group, Inc., University Research Park, Madison,
Wis., United States of America (Devereaux et al, 1984).
[0172] Preferably, a homologue of the protein-encoding region of a
flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM, or ytfN gene
hybridizes under at least medium stringency conditions to the
non-coding strand of said gene, even more preferably under high
stringency conditions to the non-coding strand of said gene.
[0173] For the purposes of defining the level of stringency, a low
stringency is defined herein as being a hybridisation and/or a wash
carried out in 6.times.SSC buffer, 0.1% (w/v) SDS at 28.degree. C.
A moderate stringency is defined herein as being a hybridisation
and/or washing carried out in 2.times.SSC buffer, 0.1% (w/v) SDS at
a temperature in the range 45.degree. C. to 65.degree. C. A high
stringency is defined herein as being a hybridisation and/or wash
carried out in 0.1.times.SSC buffer, 0.1% (w/v) SDS, or lower salt
concentration, and at a temperature of at least 65.degree. C.
Reference herein to a particular level of stringency encompasses
equivalent conditions using wash/hybridization solutions other than
SSC known to those skilled in the art.
[0174] Generally, the stringency is increased by reducing the
concentration of SSC buffer, and/or increasing the concentration of
SDS and/or increasing the temperature of the hybridisation and/or
wash. Those skilled in the art will be aware that the conditions
for hybridisation and/or wash may vary depending upon the nature of
the hybridisation membrane or the type of hybridisation probe used.
Conditions for hybridisations and washes are well understood by one
normally skilled in the art. For the purposes of clarification of
the parameters affecting hybridisation between nucleic acid
molecules, reference is found in pages 2.10.8 to 2.10.16. of
Ausubel et al. (1987), which is herein incorporated by
reference.
[0175] As used herein, a "nucleic acid molecule in an expressible
format" is a protein-encoding region of a nucleic acid molecule
placed in operable connection with a promoter or other regulatory
sequence capable of regulating expression in the vaccine vector
system.
[0176] Reference herein to a "promoter" Is to be taken in its
broadest context and includes the transcriptional regulatory
sequences of a classical genomic gene, including the TATA box which
Is required for accurate transcription initiation, with or without
a CCAAT box sequence and additional regulatory elements (i.e.,
upstream activating sequences, enhancers and silencers) which alter
gene expression in response to developmental and/or external
stimuli, or in a tissue-specific manner. In the present context,
the term "promoter" is also used to describe a recombinant,
synthetic or fusion molecule, or derivative which confers,
activates or enhances the expression of a nucleic acid molecule to
which it is operably connected, and which encodes the immunogenic
polypeptide. Preferred promoters can contain additional copies of
one or more specific regulatory elements to further enhance
expression and/or to alter the spatial expression and/or temporal
expression of the said nucleic acid molecule.
[0177] Placing a nucleic acid molecule under the regulatory control
of, i.e., "in operable connection with", a promoter sequence means
positioning said molecule such that expression is controlled by the
promoter sequence. Promoters are generally, but not necessarily,
positioned 5' (upstream) to the genes that they control. In the
construction of heterologous promoter/structural gene combinations
it is generally preferred to position the promoter at a distance
from the gene transcription start site that is approximately the
same as the distance between that promoter and the gene it controls
in its natural setting, i.e., the gene from which the promoter is
derived. Furthermore, the regulatory elements comprising a promoter
are usually positioned within 2 kb of the start site of
transcription of the gene. As is known in the art, some variation
in this distance can be accommodated without loss of promoter
function. Similarly, the preferred positioning of a regulatory
sequence element with respect to a heterologous gene to be placed
under its control is defined by the positioning of the element in
its natural setting, i.e., the genes from which it is derived.
Again, as is known in the art, some variation in this distance can
also occur.
[0178] The prerequisite for producing intact polypeptides in
bacteria such as E. coli is the use of a strong promoter with an
effective ribosome binding site. Typical promoters suitable for
expression in bacterial cells such as E. coli include, but are not
limited to, the lacz promoter, temperature-sensitive .lambda..sub.L
or .lambda..sub.R promoters, T7 promoter or the IPTG-inducible tac
promoter. A number of other vector systems for expressing the
nucleic acid molecule of the invention in E. coli are well-known in
the art and are described, for example, in Ausubel et al (1987) or
Sambrook et al (1989). Numerous plasmids with suitable promoter
sequences for expression in bacteria and efficient ribosome binding
sites have been described, such as for example, pKC30
(.lambda..sub.L: Shimatake and Rosenberg, 1981); pKK173-3 (tac:
Amann and Brosius, 1985), pET-3 (T7: Studier and Moffat, 1986); the
pBAD/TOPO or pBAD/Thio-TOPO series of vectors containing an
arabinose-inducible promoter (Invitrogen, Carlsbad, Calif.), the
latter of which is designed to also produce fusion proteins with
thioredoxin to enhance solubility of the expressed protein; the
pFLEX series of expression vectors (Pfizer Inc., CT, USA); or the
pQE series of expression vectors (Qiagen, Calif.), amongst others.
Typical promoters suitable for expression in viruses of eukaryotic
cells and eukaryotic cells include the SV40 late promoter, SV40
early promoter and cytomegalovirus (CMV) promoter, CMV IE
(cytomegalovirus immediate early) promoter amongst others.
[0179] Means for introducing the isolated nucleic acid molecule or
a genetic construct comprising same into a cell for expression of
the immunogenic component of the vaccine composition are well-known
to those skilled in the art. The technique used for a given
organism depends on the known successful techniques. Means for
introducing recombinant DNA into animal cells include
microinjection, transfection mediated by DEAE-dextran, transfection
mediated by liposomes such as by using lipofectamine (Gibco, Md.,
USA) and/or cellfectin (Gibco, Md., USA), PEG-mediated DNA uptake,
electroporation and microparticle bombardment such as by using
DNA-coated tungsten or gold particles (Agracetus Inc., WI, USA)
amongst others.
[0180] The immunogenic component of a vaccine composition as
contemplated herein exhibits excellent therapeutic activity, for
example, in the treatment and/or prophylaxis of PPE when
administered in an amount which depends on the particular case. For
example, for recombinant polypeptide molecules, from about 0.5
.mu.g to about 20 mg may be administered, preferably from about 1
.mu.g to about 10 mg, more preferably from about 10 .mu.g to about
5 mg, and most preferably from about 50 .mu.g to about 1 mg
equivalent of the immunogenic component in a volume of about 1 ml
to about 5 ml. For DNA vaccines, a preferred amount is from about
0.1 .mu.g/ml to about 5 mg/ml in a volume of about 1 to about 5 ml.
The DNA can be present in "naked" form or it can be administered
together with an agent facilitating cellular uptake (e.g., in
liposomes or cationic lipids). The important feature is to
administer sufficient immunogen to induce a protective immune
response. The above amounts can be administered as stated or
calculated per kilogram of body weight. Dosage regime can be
adjusted to provide the optimum therapeutic response. For example,
several divided doses can be administered or the dose can be
proportionally reduced as indicated by the exigencies of the
therapeutic situation. Booster administration may also be
required.
[0181] The vaccine of the present invention can further comprise
one or more additional immunomodulatory components such as, for
example, an adjuvant or cytokine molecule, amongst others, that is
capable of increasing the immune response against the immunogenic
component. Non-limiting examples of adjuvants that can be used in
the vaccine of the present invention include the RIBI adjuvant
system (Ribi Inc., Hamilton, Mont., USA), alum, mineral gels such
as aluminium hydroxide gel, oil-in-water emulsions, water-in-oil
emulsions such as, for example, Block co-polymer (CytRx, Atlanta
Ga., USA), QS-21 (Cambridge Biotech Inc., Cambridge Mass., USA),
SAF-M (Chiron, Emeryville Calif., USA), AMPHIGEN.RTM. adjuvant,
Freund's complete adjuvant; Freund's incomplete adjuvant; and
Saponin, QuilA or other saponin fraction, monophosphoryl lipid A,
and Avridine lipid-amine adjuvant. Other immunomodulatory agents
that can be included in the vaccine include, for example, one or
more cytokines, such as interferon and/or interleukin, or other
known cytokines. Non-ionic surfactants such as, for example,
polyoxyethylene oleyl ether and n-hexadecyl polyethylene ether may
also be included in the vaccines of the present invention. The
vaccine composition can be administered in a convenient manner such
as by oral, intravenous (where water soluble), intramuscular,
subcutaneous, intranasal, intradermal or suppository routes or by
implantation (eg., using slow release technology), provided that a
sufficient degree of the immunogenicity of the immunizing antigen
is retained for the purposes of eliciting an immune response in the
animal being treated. Depending on the route of administration, the
immunogenic component may be required to be coated in a material to
protect it from the action of enzymes, acids and other natural
conditions which may inactivate it, such as those in the digestive
tract.
[0182] The vaccine composition may also be administered
parenterally or intraperitoneally. Dispersions can also be prepared
in glycerol, liquid polyethylene glycols, and mixtures thereof, or
in oils. Under ordinary conditions of storage and use, these
preparations can contain a preservative to prevent the growth of
microorganisms. Alternatively, the vaccine composition can be
stored in lyophilised form to be rehydrated with an appropriate
vehicle or carrier prior to use.
[0183] Pharmaceutical forms suitable for injectable use include
sterile aqueous solutions (where water soluble) or dispersions and
sterile powders for the extemporaneous preparation of sterile
injectable solutions or dispersion. In all cases the form must be
fluid to the extent that easy syringability exists, unless the
pharmaceutical form is a solid or semi-solid such as when slow
release technology is employed. In any event, it must be stable
under the conditions of manufacture and storage and must be
preserved against the contaminating action of microorganisms.
[0184] The carrier may be a solvent or dispersion medium
containing, for example, water, ethanol, polyol (for example,
glycerol, propylene glycol and liquid polyethylene glycol, and the
like), suitable mixtures thereof and vegetable oils. The proper
fluidity can be maintained, for example, by the use of a coating
such as lecithin, by the maintenance of the required particle size
in the case of dispersion and by the use of surfactants. The
prevention of the action of microorganisms can be brought about by
various antibacterial and antifungal agents such as, for example,
parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the
like. In many cases, it will be preferable to include isotonic
agents such as, for example, sugars or sodium chloride. Prolonged
absorption of the injectable compositions can be brought about by
the use in the compositions of agents delaying absorption such as,
for example, aluminum monostearate and gelatin.
[0185] Sterile injectable solutions are prepared by incorporating
the active compound in the required amount in the appropriate
solvent with various of the other ingredients enumerated above, as
required, followed by filter-sterilization. Generally, dispersions
are prepared by incorporating the sterilized active ingredient into
a sterile vehicle which contains the basic dispersion medium and
the required other ingredients selected from those enumerated
above. In the case of sterile powders for the preparation of
sterile injectable solutions, the preferred methods of preparation
are vacuum drying and the freeze-drying technique which yield a
powder of the active ingredient plus any additional desired
ingredient from previously sterile-filtered solution thereof.
[0186] The present invention extends to vaccine compositions which
confer protection against infection by one or more isolates or
sub-types of L. intracellularis including those that belong to the
same serovar or serogroup as L. intracellularis. The vaccine
composition preferably also confers protection against infection by
other species of the genus Lawsonia or other microorganisms related
thereto, as determined at the nucleotide, biochemical, structural,
physiological and/or immunointeractive level; the only requirement
being that said other species or other microorganism expresses a
polypeptide which is immunologically cross-reactive to a
polypeptide selected from the group consisting of flhB, fliR, ntrC,
glnH, motA, motB, tlyC, ytfM, and ytfN polypeptides, or a
homologue, analogue or derivative of any one or more of said
polypeptides as described herein. For example, such related
microorganisms may comprise genomic DNA which is at least about 70%
identical overall to the genomic DNA of L. intracellularis as
determined using standard genomic DNA hybridisation and analysis
techniques.
[0187] The terms "serogroup" and "serovar" relate to a
classification of microorganisms which is based upon serological
typing data, in particular data obtained using agglutination assays
such as the microscopic agglutination test (MAT). Those skilled in
the art will be aware that serovar and serogroup antigens are a
mosaic on the cell surface and, as a consequence there will be no
strict delineation between bacteria belonging to a serovar and/or
serogroup. Moreover, organisms which belong to different species
may be classified into the same serovar or serogroup because they
are indistinguishable by antigenic determination. As used herein,
the term "serovar" means one or more Lawsonia strains which are
antigenically-identical with respect to antigenic determinants
produced by one or more loci. Quantitatively, serovars may be
differentiated from one another by cross-agglutination absorption
techniques. As used herein, the term "serogroup" refers to a group
of Lawsonia spp. whose members cross-agglutinate with shared group
antigens and do not cross-agglutinate with the members of other
groups and, as a consequence, the members of a serogroup have more
or less close antigenic relations with one another by simple
cross-agglutination.
[0188] The present invention thus clearly extends to vaccine
compositions for the treatment and/or prophylaxis of animals, in
particular, vaccine compositions for the treatment and/or
prophylaxis of porcine and/or avian species, against any bacterium
belonging to the same serovar or serogroup as L. intracellularis.
Preferably, such organisms will express a polypeptide homologue,
analogue or derivative of a polypeptide selected from the group
consisting of flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM, and
ytfN polypeptides.
[0189] The present invention extends further to vaccine
compositions capable of conferring protection against a "genetic
variant" of L. intracellularis, the only requirement being that
said variant expresses a polypeptide which is immunologically
cross-reactive to a polypeptide selected from the group consisting
of flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM, and ytfN
polypeptides. Genetic variants of L. intracellularis can be
developed by mutation, recombination, conjugation or transformation
of L. intracellularis or may occur naturally. It will be known to a
person skilled in the art how to produce such derivatives.
[0190] In a particularly preferred embodiment, the vaccine
composition of the invention is intended for or suitable for the
prophylaxis and/or treatment of infection in a porcine or avian
animal and more preferably, for prophylaxis and/or treatment of a
porcine animal for infection by L. intracellularis.
[0191] Accordingly, the present invention clearly extends to the
use of the immunogenic polypeptide of the invention or a DNA or RNA
molecule encoding the same, according to any one of the preceding
embodiments or as exemplified herein in the preparation of a
medicament for the treatment and/or prophylaxis of PPE in animals,
particularly porcine or avian animals.
[0192] The invention further extends to a method of treatment
and/or prophylaxis of PPE in an animal such as an avian or porcine
animal, said method comprising administering the vaccine
composition or the immunogenic polypeptide of the invention or a
DNA or RNA molecule encoding the same, as described or exemplified
herein to said animal for a time and under conditions sufficient
for an immune response to occur thereto. Preferably, in the case of
administration of a vaccine composition, the immune response to the
immunogen is a protective immune response.
[0193] Those skilled in the art will recognise the general
applicability of the invention in vaccinating animals other than
porcine and avian animals against L. intracellularis and/or related
microorganisms. In the general application of the vaccine of the
present invention, the only prerequisite is that the animal on
which protection is conferred is capable of being infected with L.
intracellularis and/or a related microorganism thereto and that, in
the case of a related microorganism to L. intracellularis, said
related microorganism expresses a B-cell or T-cell epitope which
mimics or cross-reacts with the polypeptide component of the
vaccine composition described herein. Animals which may be
protected by the vaccine of the present invention include, but are
not limited to, humans, primates, companion animals (e.g., cats,
dogs), livestock animals (e.g., pigs, sheep, cattle, horses,
donkeys, goats), laboratory test animals (e.g., mice, rats, guinea
pigs, rabbits) and captive wild animals (e.g., kangaroos, foxes,
deer). The present invention also extends to the vaccination of
birds such as poultry birds, game birds and caged birds.
[0194] The present invention further extends to combination
vaccines comprising an effective amount of a first immunogenic
component comprising a polypeptide selected from the group
consisting of flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM, and
ytfN polypeptides, or a homologue, analogue or derivative thereof
as described herein, or a DNA or RNA molecule encoding the same,
combined with an effective amount of a second immunogenic component
comprising one or more other antigens capable of protecting a
porcine animal, or bird, against either Lawsonia spp. or another
pathogen that infects and causes disease in said animal. The second
immunogenic component is different from the first immunogenic
component and is preferably selected from the group consisting of
the L. intracellularis FlgE, hemolysin, OmpH, SodC, flhB, fliR,
ntrC, glnH, motA, motB, tlyC, ytfM, and ytfN polypeptides and
homologues, analogues or derivatives thereof. The present invention
clearly extends to DNA vaccines and vaccine vectors which express
said first immunogenic component and said second immunogenic
component.
[0195] It is within the scope of the invention to encompass vaccine
compositions comprising multimeric and polymeric forms of any one
or more of the immunogenic polypeptides described herein, such as
tandem arrays of homologous amino acid sequences, or,
alternatively, tandem arrays of heterologous immunogenic repeats of
amino acid sequences. The present invention extends further to
nucleic acid molecules encoding such polymeric forms.
[0196] The isolated or recombinant polypeptide of the invention, or
an immunologically-equivalent homologue, analogue or derivative
thereof is also useful for the preparation of immunologically
interactive molecules which are useful in the diagnosis of
infection of an animal by Lawsonia spp., in particular by L.
intracellularis or a related organism thereto.
[0197] As used herein, the term "immunologically interactive
molecule" includes antibodies and antibody derivatives and
functional equivalents, such as a Fab, or a SCAB (single-chain
antibody), any of which optionally can be conjugated to an enzyme,
radioactive or fluorescent tag, amongst others. The only
requirement of such immunologically interactive molecules is that
they are capable of binding specifically to the immunogenic
polypeptide of the present invention as hereinbefore described.
[0198] Accordingly, a further aspect of the invention extends to an
immunologically interactive molecule which is capable of binding to
a polypeptide selected from the group consisting of:
[0199] (i) a polypeptide which comprises an amino acid sequence
which has at least about 60% sequence identity overall to an amino
acid sequence selected from the group consisting of SEQ ID NOs: 2,
4, 6, 8, 10, 12, 14, 16, and 18;
[0200] (ii) a polypeptide which comprises an amino acid sequence
which has at least about 60% sequence identity overall to an amino
acid sequence encoded by L. intracellularis DNA contained within a
plasmid selected from the group consisting of AGAL Accession Nos:
NM00/16476 (plasmid pGTE#1 glnH); NM00/16477 (plasmid pGTE#2 flhB);
NM00/16478 (plasmid pGTE#3 fliR); NM00/16479 (plasmid pGTE#4
motA/B); NM00/16480 (plasmid pGTE#5 tlyC); NM00/16481 (plasmid
pGTE#6 ntrC); NM00/16482 (plasmid pGTE#7 ytfM); and NM01/23286
(plasmid pGTE#8 ytfN);
[0201] (iii) a polypeptide which comprises at least about 5
contiguous amino acids of an amino acid sequence selected from the
group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, and
18;
[0202] (iv) a polypeptide which comprises at least about 5
contiguous amino acids of an amino acid sequence encoded by L.
intracellularis DNA contained within a plasmid selected from the
group consisting of AGAL Accession Nos: NM00/16476 (plasmid pGTE#1
glnH); NM00/16477 (plasmid pGTE#2 flhB); NM00/16478 (plasmid pGTE#3
fliR); NM00/16479 (plasmid pGTE#4 motA/B); NM00/16480 (plasmid
pGTE#5 tlyC); NM00/16481 (plasmid pGTE#6 ntrC); NM00/16482 (plasmid
pGTE#7 ytfM); and NM01/23286 (plasmid pGTE#8 ytfN);
[0203] (v) a polypeptide which comprises an amino acid sequence
encoded by a nucleotide sequence having at least about 60% sequence
identity overall to a nucleotide sequence selected from the group
consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, and 17;
[0204] (vi) a polypeptide which comprises an amino acid sequence
encoded by a nucleotide sequence having at least about 60% sequence
identity overall to the nucleotide sequence of L. intracellularis
DNA contained within a plasmid selected from the group consisting
of AGAL Accession Nos: NM00/16476 (plasmid pGTE#1 glnH); NM00/16477
(plasmid pGTE#2 flhB); NM00/16478 (plasmid pGTE#3 fliR); NM00/16479
(plasmid pGTE#4 motA/B); NM00/16480 (plasmid pGTE#5 tlyC);
NM00/16481 (plasmid pGTE#6 ntrC); NM00/16482 (plasmid pGTE#7 ytfM);
and NM01/23286 (plasmid pGTE#8 ytfN);
[0205] (vii) a polypeptide encoded by at least about 15 contiguous
nucleotides of a nucleotide sequence selected from the group
consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, and 17;
[0206] (viii) a polypeptide encoded by at least about 15 contiguous
nucleotides of a nucleotide sequence of L. intracellularis DNA
contained within a plasmid selected from the group consisting of
AGAL Accession Nos: NM00/16476 (plasmid pGTE#1 glnH); NM00/16477
(plasmid pGTE#2 flhB); NM00/16478 (plasmid pGTE#3 fliR); NM00/16479
(plasmid pGTE#4 mot/B); NM00/16480 (plasmid pGTE#5 tlyC);
NM00/16481 (plasmid pGTE#6 ntrC); NM00/16482 (plasmid pGTE#7 ytfM);
and NM01/23286 (plasmid pGTE#8 ytfN); and
[0207] (ix) a homologue, analogue or derivative of any one of (i)
to (viii) which mimics a B-cell or T-cell epitope of Lawsonia
spp.
[0208] In a preferred embodiment, the immunologically interactive
molecule is an antibody that binds specifically to one or more
epitopes of a polypeptide selected from the group consisting of
flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM, and ytfN
polypeptides. More preferably, the immunologically interactive
molecule binds specifically to one or more epitopes of a
polypeptide from a causative agent of PPE, such as, for example, L.
intracellularis.
[0209] Conventional methods can be used to prepare the
immunologically interactive molecules. For example, by using a
polypeptide immunogen of the present invention, polyclonal antisera
or monoclonal antibodies can be made using standard methods. For
example, a mammal, (e.g., a mouse, hamster, or rabbit) can be
immunized with an immunogenic form of the polypeptide of the
present invention which elicits an antibody response in the mammal.
Techniques for conferring immunogenicity on a polypeptide include
conjugation to carriers, or other techniques well known in the art.
For example, the polypeptide can be administered in the presence of
adjuvant or can be coupled to a carrier molecule, as known in the
art, that enhances the immunogenicity of the polypeptide. The
progress of immunization can be monitored by detection of antibody
titres in plasma or serum. Standard ELISA or other immunoassay can
be used with the immunogen as antigen to assess the levels of
antibodies. Following immunization, antisera can be obtained and,
for example, IgG molecules corresponding to the polyclonal
antibodies can be isolated from the antisera.
[0210] To produce monoclonal antibodies, antibody producing cells
(lymphocytes) can be harvested from an animal immunised with a
polypeptide of the present invention and fused with myeloma cells
by standard somatic cell fusion procedures, thus immortalizing
these cells and yielding hybridoma cells. Such techniques are well
known in the art, for example, the hybridoma technique originally
developed by Kohler and Milstein (1975), as well as other
techniques such as the human B-cell hybridoma technique (Kozbor et
al., 1983), the EBV-hybridoma technique to produce human monoclonal
antibodies (Cole et al., 1985), and screening of combinatorial
antibody libraries (Huse et al., 1989). Hybridoma cells can be
isolated and screened immunochemically for production of antibodies
that are specifically reactive with the polypeptide and monoclonal
antibodies isolated therefrom.
[0211] As with all immunogenic compositions for eliciting
antibodies, the immunogenically effective amounts of the peptides
of the invention must be determined empirically. Factors to be
considered include the immunogenicity of the native polypeptide,
whether or not the polypeptide will be complexed with or covalently
attached to an adjuvant or carrier protein or other carrier, the
route of administration for the composition, i.e., intravenous,
intramuscular, subcutaneous, etc., and the number of immunizing
doses to be administered. Such factors are known in the vaccine art
and it is well within the skill of immunologists to make such
determinations without undue experimentation.
[0212] The term "antibody" as used herein, is intended to include
fragments thereof which are also specifically reactive with a
polypeptide that mimics or cross-reacts with a B-cell or T-cell
epitope of the L. intracellularis polypeptide selected from the
group consisting of flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM,
and ytfN polypeptides. Antibodies can be fragmented using
conventional techniques and the fragments screened for utility in
the same manner as described above for whole antibodies. For
example, F(ab')2 fragments can be generated by treating antibody
with pepsin. The resulting F(ab')2 fragment can be treated to
reduce disulfide bridges to produce Fab' fragments.
[0213] It is within the scope of this invention to include any
secondary antibodies (monoclonal, polyclonal or fragments of
antibodies), including anti-idiotypic antibodies, directed to the
first mentioned antibodies discussed above. Both the first and
second antibodies can be used in detection assays or a first
antibody can be used with a commercially available
anti-immunoglobulin antibody. An antibody as contemplated herein
includes any antibody specific to any region of a polypeptide which
mimics, or cross-reacts with a B-cell or T-cell epitope of a L.
intracellularis polypeptide selected from the group consisting of
flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM, and ytfN
polypeptides.
[0214] The antibodies described herein are useful for determining
B-cell or T-cell epitopes of a polypeptide selected from the group
consisting of flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM, and
ytfN polypeptides, such as, for example, by testing the ability of
synthetic peptides to cross-react immunologically with said
polypeptide or to elicit the production of antibodies which
cross-react with said polypeptide. Using methods described herein,
polyclonal antibodies, monoclonal antibodies or chimeric monoclonal
antibodies can also be raised to peptides which mimic or
cross-react with a B-cell or T-cell epitope of a L. intracellularis
polypeptide selected from the group consisting of flhB, fliR, ntrC,
glnH, motA, motB, tlyC, ytfM, and ytfN polypeptides.
[0215] More particularly, the polyclonal, monoclonal or chimeric
monoclonal antibodies can be used to detect the polypeptide of the
invention and/or any homologues, analogues or derivatives thereof,
in various biological materials. For example, they can be used in
an ELISA, radioimmunoassay, or histochemical test. In other words,
the antibodies can be used to test for binding to a polypeptide of
the invention or to a homologue, analogue or derivative thereof, in
a biological sample to diagnose the presence of L. intracellularis
therein.
[0216] Accordingly, a further aspect of the invention provides a
method of diagnosing infection of an animal by L. intracellularis
or a related microorganism thereto, said method comprising the
steps of contacting a biological sample derived from said animal
with an immunologically interactive molecule which is capable of
binding to a polypeptide selected from the group consisting of
flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM, and ytfN
polypeptides, or a homologue, analogue or derivative thereof, for a
time and under conditions sufficient for an antigen:antibody
complex to form, and detecting said complex formation.
[0217] According to this embodiment of the present invention, the
immunologically interactive molecule is preferably an antibody
molecule prepared against a L. intracellularis polypeptide selected
from the group consisting of flhB, fliR, ntrC, glnH, motA, motB,
tlyC, ytfM, and ytfN polypeptides, or an analogue or derivative
thereof.
[0218] If the biological sample being tested contains one or more
epitopes of a polypeptide selected from the group consisting of
flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM, and ytfN
polypeptides, or an immunologically cross-reactive homologue,
analogue or derivative thereof, it will give a positive binding
result to the immunologically interactive molecule.
[0219] Preferably, the biological sample is derived from a porcine
or avian host of the pathogen L. intracellularis or a related
microorganism thereto, and includes an appropriate tissue or fluid
sample from the animal.
[0220] Preferred biological samples are derived from the ileum,
caecum, small intestine, large intestine, whole serum or lymph
nodes of the porcine or avian host animal being tested.
Alternatively or in addition the biological test sample may
comprise faeces or a rectal swab derived from the animal.
[0221] To distinguish L. intracellularis from other microorganisms
resident in the gut or other organ of an animal, the antibodies
should not be prepared against highly-conserved epitopes of the L.
intracellularis polypeptide, such as, for example, those amino acid
sequences of at least 5 amino acids in length which are conserved
between L. intracellularis and a microorganism which is present in
the gut or other organ of an animal in respect of which diagnosis
is sought such as, for example, E. coli.
[0222] Conventional immunoassays can be used to perform this
embodiment of the invention. A wide range of immunoassay techniques
are available as can be seen by reference to U.S. Pat. Nos.
4,016,043, 4,424,279 and 4,018,653. These, of course, include both
single-site and two-site or "sandwich" assays of the
non-competitive types, as well as the traditional competitive
binding assays. These assays also include direct binding of a
labelled antibody to a target. It will be readily apparent to the
skilled technician how to modify or optimise such assays to perform
this embodiment of the present invention, and all such
modifications and optimisations are encompassed by the present
invention.
[0223] In one alternative embodiment, the present invention
contemplates a method of identifying whether or not an animal has
suffered from a past infection, or is currently infected with L.
intracellularis or a related microorganism thereto, said method
comprising contacting blood or serum derived from said animal with
the immunogenic polypeptide of the invention for a time and under
conditions sufficient for an antigen:antibody complex to form, and
detecting said complex formation. This embodiment differs from the
embodiment described supra in that it relies upon the detection of
circulating antibodies against L. intracellularis or related
organism in the animals blood or serum which are present as a
consequence of a past or present infection by this pathogen.
However, it will be apparent to those skilled in the art that the
principle of the assay format is the same. As with other
embodiments of the invention referred to supra, conventional
immunoassays can be used. Persons skilled in the art will readily
be capable of varying known immunoassay formats to perform the
present embodiment. This embodiment of the invention can also
utilise derivatives of blood and serum which comprise
immunologically interactive molecules such as, for example,
partially-purified IgG or IgM fractions and buffy coat samples,
amongst others. The preparation of such fractions will also be
known to those skilled in the art.
[0224] A further aspect of the present invention provides an
isolated nucleic acid molecule which comprises a sequence of
nucleotides that encodes, or is complementary to a nucleic acid
molecule that encodes a polypeptide selected from the group
consisting of flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM, and
ytfN polypeptides, including any and all genes selected from the
group consisting of flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM,
and ytfN genes as defined hereinabove.
[0225] In a preferred embodiment, the isolated nucleic acid
molecule comprises a nucleotide sequence encoding a polypeptide
that is immunologically cross-reactive with L. intracellularis or
other causative agent of PPE, wherein said nucleotide sequence is
selected from the group consisting of:
[0226] (i) a nucleotide sequence having at least about 60% sequence
identity overall to a nucleotide sequence selected from the group
consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, and 17;
[0227] (ii) a nucleotide sequence having at least about 60%
sequence identity overall to L. intracellularis DNA contained
within a plasmid selected from the group consisting of AGAL
Accession Nos: NM00/16476 (plasmid pGTE#1 glnH); NM00/16477
(plasmid pGTE#2 flhB); NM00/16478 (plasmid pGTE#3 fliR); NM00/16479
(plasmid pGTE#4 motA/B); NM00/16480 (plasmid pGTE#5 tlyC);
NM00/16481 (plasmid pGTE#6 ntrC); NM00/16482 (plasmid pGTE#7 ytfM);
and NM01/23286 (plasmid pGTE#8 ytfN);
[0228] a (iii) a nucleotide sequence which comprises at least about
15 contiguous nucleotides of a sequence selected from the group
consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, and 17;
[0229] (iv) a nucleotide sequence which comprises at least about 15
contiguous nucleotides of L. intracellularis DNA contained within a
plasmid selected from the group consisting of AGAL Accession Nos:
NM00/16476 (plasmid pGTE#1 glnH); NM00/16477 (plasmid pGTE#2 flhB);
NM00/16478 (plasmid pGTE#3 fliR); NM00/16479 (plasmid pGTE#4
motA/B); NM00/16480 (plasmid pGTE#5 tlyC); NM00/16481 (plasmid
pGTE#6 ntrC); NM00/16482 (plasmid pGTE#7 ytfM); and NM01/23286
(plasmid pGTE#8 ytfN);
[0230] (v) a nucleotide sequence which hybridizes under at least
low stringency conditions to a nucleotide sequence selected from
the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, and
17 or a complementary nucleotide sequence thereto;
[0231] (vi) a nucleotide sequence which hybridizes under at least
low stringency conditions to L. intracellularis DNA contained
within a plasmid selected from the group consisting of AGAL
Accession Nos: NM00/16476 (plasmid pGTE#1 glnH); NM00/16477
(plasmid pGTE#2 flhB); NM00/16478 (plasmid pGTE#3 fliR); NM00/16479
(plasmid pGTE#4 motA/B); NM00/16480 (plasmid pGTE#5 tlyC);
NM00/16481 (plasmid pGTE#6 ntrC); NM00/16482 (plasmid pGTE#7 ytfM);
and NM01/23286 (plasmid pGTE#8 ytfN); and
[0232] (vii) a homologue, analogue or derivative of any one of (i)
to (vi) which encodes a polypeptide which mimics a B-cell or T-cell
epitope of Lawsonia spp.
[0233] For the present purpose, a "homologue" of a nucleotide
sequence shall be taken to refer to an isolated nucleic acid
molecule which encodes a polypeptide that is immunologically
cross-reactive to a polypeptide selected from the group consisting
of flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM, and ytfN
polypeptides, but which includes one or more nucleotide
substitutions, insertions, deletions, or rearrangements.
[0234] An "analogue" of a nucleotide sequence set forth herein
shall be taken to refer to an isolated nucleic acid molecule which
encodes a polypeptide which is immunologically cross-reactive to a
polypeptide selected from the group consisting of flhB, fliR, ntrC,
glnH, motA, motB, tlyC, ytfM, and ytfN polypeptides, but which
includes one or more non-nucleotide constituents not normally
present in said isolated nucleic acid molecule, such as, for
example, carbohydrates, radiochemicals including radio nucleotides,
reporter molecules such as, but not limited to biotin, DIG,
alkaline phosphatase or horseradish peroxidase, amongst others.
[0235] A "derivative" of a nucleotide sequence set forth herein
shall be taken to refer to any isolated nucleic acid molecule which
contains at least about 60% nucleotide sequence identity to 15 or
more contiguous nucleotides present in the nucleotide sequence of a
gene selected from the group consisting of flhB, fliR, ntrC, glnH,
motA, motB, tlyC, ytfM, and ytfN genes.
[0236] Generally, a flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM,
or ytfN gene may be subjected to mutagenesis to produce single or
multiple nucleotide substitutions, deletions and/or insertions.
Nucleotide insertional derivatives include 5' and 3' terminal
fusions as well as intra-sequence insertions of single or multiple
nucleotides or nucleotide analogues. Insertional nucleotide
sequence variants are those in which one or more nucleotides or
nucleotide analogues are introduced into a predetermined site in
the nucleotide sequence of the gene, although random insertion is
also possible with suitable screening of the resulting product
being performed. Deletional nucleotide sequence variants are
characterised by the removal of one or more nucleotides from the
gene. Substitutional nucleotide sequence variants are those in
which at least one nucleotide in the gene sequence has been removed
and a different nucleotide or nucleotide analogue inserted in its
place. In a preferred embodiment, such substitutions are selected
based on the degeneracy of the genetic code, as known in the art,
with the resulting substitutional variant encoding the amino acid
sequence of a flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM, or
ytfN polypeptide.
[0237] Preferred homologues, analogues and derivatives of a flhB,
fliR, ntrC, glnH, motA, motB, tlyC, ytfM, or ytfN gene comprise a
sequence of nucleotides which has at least about 80% identity, even
more preferably at least about 90% identity, and yet still more
preferably at least about 95% identity to said gene.
[0238] In determining whether or not two nucleotide sequences fall
within these percentage limits, reference is made to the
description supra of methods for conducting a side-by-side
comparison or multiple alignment of nucleotide sequences.
[0239] Alternatively or in addition, preferred homologues,
analogues and derivatives of a flhB, fliR, ntrC, glnH, motA, motB,
tlyC, ytfM, or ytfN gene comprise a sequence of nucleotides which
hybridizes under at least moderate stringency conditions and to the
nucleotide sequence of said gene, or to a nucleic acid fragment
comprising at least about 20 contiguous nucleotides in length
derived therefrom, and even more preferably, under high stringency
conditions to said gene, or to said nucleic acid fragment. For the
purposes of defining the level of stringency, reference is made to
the description hereinabove of hybridization stringencies.
[0240] In a more preferred embodiment, such a nucleotide sequence
encodes a polypeptide that is immunologically cross-reactive with
L. intracellularis or other causative agent of PPE.
[0241] In a particularly preferred embodiment, the isolated nucleic
acid molecule of the present invention comprises or consists of a
nucleotide sequence selected from the group consisting of:
[0242] (i) a nucleotide sequence selected from the group consisting
of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, and 17;
[0243] (ii) a nucleotide sequence of the L. intracellularis DNA
contained within a deposited plasmid selected from the group
consisting of AGAL Accession Nos: NM00/16476 (plasmid pGTE#1 glnH);
NM00/16477 (plasmid pGTE#2 flhB); NM00/16478 (plasmid pGTE#3 fliR);
NM00/16479 (plasmid pGTE#4 motB); NM00/16480 (plasmid pGTE#5 tlyC);
NM00/16481 (plasmid pGTE#6 ntrC); NM00/16482 (plasmid pGTE#7 ytfM);
and NM01/23286 (plasmid pGTE#8 ytfN);
[0244] (iii) a nucleotide sequence that encodes the same
polypeptide as (i) or (ii), wherein said polypeptide is selected
from the group consisting of flhB, fliR, ntrC, glnH, motA, motB,
tlyC, ytfM, and ytfN polypeptides; and
[0245] (iv) a nucleotide sequence that is complementary to (i) or
(ii) or (iii).
[0246] The present invention clearly encompasses genetic constructs
comprising the subject nucleic acid molecule in an expressible
format suitable for the preparation of a recombinant immunogenic
polypeptide selected from the group consisting of flhB, fliR, ntrC,
glnH, motA, motB, tlyC, ytfM, and ytfN polypeptides, such as for
use in recombinant univalent or polyvalent recombinant
vaccines.
[0247] In such cases, the nucleic acid molecule will be operably
connected to a promoter sequence which can thereby regulate
expression of said nucleic acid molecule in a prokaryotic or
eukaryotic cell as described supra. The genetic construct
optionally further comprises a terminator sequence. The term
"terminator" refers to a DNA sequence at the end of a
transcriptional unit which signals termination of transcription. A
"terminator" is a nucleotide sequence, generally located within the
3'-non-translated region of a gene or mRNA, comprising a
polyadenylation signal to facilitate the post-transcriptional
addition of a polyadenylate sequence to the 3'-end of a primary
mRNA transcript. Terminator sequences may be isolated from the
genetic sequences of bacteria, fungi, viruses, animals and/or
plants. Terminators active in animal cells are known and described
in the literature.
[0248] In a preferred embodiment, the genetic construct can be a
cloning or expression vector, as known in the art, such as a
plasmid, cosmid, or phage, comprising a nucleic acid molecule of
the present invention, and host cells transformed or transfected
therewith. In a non-limiting embodiment, the vector is a plasmid
selected from the group consisting of AGAL Accession Nos:
NM00/16476 (plasmid pGTE#1 glnH); NM00/16477 (plasmid pGTE#2 flhB);
NM00/16478 (plasmid pGTE#3 fliR); NM00/16479 (plasmid pGTE#4 motB);
NM00/16480 (plasmid pGTE#5 tlyC); NM00/16481 (plasmid pGTE#6 ntrC);
NM00/16482 (plasmid pGTE#7 ytfM); and NM01/23286 (plasmid pGTE#8
ytfN).
[0249] The genetic constructs of the present invention are
particularly useful for producing the immunogenic component of the
vaccine composition described herein or for use in a DNA
vaccine.
[0250] A range of genetic diagnostic assays to detect infection of
an animal by L. intracellularis or a related microorganism can be
employed using the nucleic acid molecule described herein such as,
for example, assays based upon the polymerase chain reaction (PCR)
and nucleic acid hybridisation. All such assays are contemplated in
the present invention.
[0251] Accordingly, a still further aspect of the invention
provides a diagnostic method of detecting L. intracellularis or
related microorganism in a biological sample derived from an animal
subject, said method comprising the steps of hybridising one or
more probes or primers derived from a nucleotide sequence of a
flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM, or ytfN gene as
defined hereinabove, or a homologue, analogue or derivative
thereof, to a DNA or RNA molecule present in said sample and then
detecting said hybridisation using a detection means.
[0252] As used herein, the term "probe" refers to a nucleic acid
molecule which is capable of being used in the detection of a gene
selected from the group consisting of flhB, fliR, ntrC, glnH, motA,
motB, tlyC, ytfM, and ytfN genes. Probes may comprise DNA
(single-stranded or double-stranded) or RNA (i.e., riboprobes) or
analogues thereof.
[0253] The term "primer" refers to a probe as hereinbefore defined
which is further capable of being used to amplify a nucleotide
sequence from L. intracellularis or a related microorganism thereto
in a PCR.
[0254] Preferred probes and primers include fragments of a gene
selected from the group consisting of flhB, fliR, ntrC, glnH, motA,
motB, tlyC, ytfM, and ytfN genes, including synthetic
single-stranded DNA or RNA molecules of at least about 15
nucleotides in length.
[0255] Preferably, probes and primers according to this embodiment
will comprise at least about 20 contiguous nucleotides in length
from a gene selected from the group consisting of flhB, fliR, ntrC,
glnH, motA, motB, tlyC, ytfM, and ytfN genes, even more preferably
at least about 25 contiguous nucleotides, still even more
preferably at least about 50 contiguous nucleotides, and even more
preferably at least about 100 nucleotides to about 500 nucleotides
in length from said gene. Probes and primers comprising the
full-length gene or a complementary nucleotide sequence thereto are
also encompassed by the present invention.
[0256] Probes or primers can comprise inosine, adenine, guanine,
thymidine, cytidine or uracil residues or functional analogues or
derivatives thereof that are capable of being incorporated into a
polynucleotide molecule, provided that the resulting probe or
primer is capable of hybridising under at least low stringency
conditions to a gene selected from the group consisting of flhB,
fliR, ntrC, glnH, motA, motB, tlyC, ytfM, and ytfN genes, or is at
least about 60% identical to one strand of said gene.
[0257] The biological sample according to this aspect of the
invention includes any organ, tissue, cell or exudate which
contains or is likely to contain L. intracellularis or a nucleic
acid derived therefrom. A biological sample can be prepared in a
suitable solution such as, for example, an extraction buffer or
suspension buffer. The present invention extends to the testing of
biological solutions thus prepared, the only requirement being that
said solution at least comprises a biological sample as described
herein.
[0258] The diagnostic assay of the present invention is useful for
the detection of L. intracellularis or a microorganism which is
related thereto which expresses a polypeptide selected from the
group consisting of flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM,
and ytfN polypeptides.
[0259] The present invention clearly contemplates diagnostic assays
which are capable of both genus-specific and species-specific
detection. Accordingly, in one embodiment, the probe or primer, or
a homologue, analogue or derivative thereof, comprises DNA capable
of being used to detect multiple Lawsonia spp. In an alternative
embodiment, the probe or primer or a homologue, analogue or
derivative thereof comprises DNA capable of being used to
distinguish L. intracellularis from related microorganisms.
[0260] Less-highly conserved regions within the flhB, fliR, ntrC,
glnH, motA, motB, tlyC, ytfM, or ytfN genes are particularly useful
as species-specific probes and/or primers for the detection of L.
intracellularis and very closely related species.
[0261] Furthermore, the diagnostic assays described herein can be
adapted to a genus-specific or species-specific assay by varying
the stringency of the hybridisation step. Accordingly, a low
stringency hybridisation can be used to detect several different
species of Lawsonia in one or more biological samples being
assayed, while a high stringency hybridisation can be used to
distinguish L. intracellularis from such other species.
[0262] The detection means according to this aspect of the
invention may be any nucleic acid-based detection means such as,
for example, nucleic acid hybridisation techniques or paper
chromatography hybridisation assay (PACHA), or an amplification
reaction such as PCR, or nucleic acid sequence-based amplification
(NASBA) system. The invention further encompasses the use of
different assay formats of said nucleic acid-based detection means,
including restriction fragment length polymorphism (RFLP),
amplified fragment length polymorphism (AFLP), single-strand chain
polymorphism (SSCP), amplification and mismatch detection (AMD),
interspersed repetitive sequence polymerase chain reaction
(IRS-PCR), inverse polymerase chain reaction (iPCR), in situ
polymerase chain reaction and reverse transcription polymerase
chain reaction (RT-PCR), amongst others.
[0263] Where the detection means is a nucleic acid hybridisation
technique, the probe can be labelled with a reporter molecule
capable of producing an identifiable signal (e.g., a radioisotope
such as .sup.32P or .sup.35S, or a biotinylated molecule).
According to this embodiment, those skilled in the art will be
aware that the detection of said reporter molecule provides for
identification of the probe and that, following the hybridisation
reaction, the detection of the corresponding nucleotide sequences
in the biological sample is facilitated. Additional probes can be
used to confirm the assay results obtained using a single
probe.
[0264] A variation of the nucleic acid hybridisation technique
contemplated by the present invention is the paper chromatography
hybridisation assay (PACHA) described by Reinhartz et al. (1993)
and equivalents thereof, wherein a target nucleic acid molecule is
labelled with a reporter molecule such as biotin, applied to one
end of a nitrocellulose or nylon membrane filter strip and
subjected to chromatography under the action of capillary or other
forces (e.g., an electric field) for a time and under conditions
sufficient to promote migration of said target nucleic acid along
the length of said membrane to a zone at which a DNA probe is
immobilised thereto such as, for example, in the middle region.
According to this detection format, labelled target nucleic acid
comprising the Lawsonia spp. nucleotide sequences complementary to
the probe will hybridise thereto and become immobilised in that
region of the membrane to which the probe is bound.
Non-complementary sequences to the probe will diffuse past the site
at which the probe is bound. The target nucleic acid may comprise a
crude or partially-pure extract of DNA or RNA or, alternatively, an
amplified or purified DNA. Additional variations of this detection
means which utilise the nucleotide sequences described herein are
clearly encompassed by the present invention.
[0265] Wherein the detection means is a RFLP, nucleic acid derived
from the biological sample, in particular DNA, is digested with one
or more restriction endonuclease enzymes and the digested DNA is
subjected to electrophoresis, transferred to a solid support such
as, for example, a nylon or nitrocellulose membrane, and hybridised
to a probe optionally labelled with a reporter molecule as
hereinbefore defined. According to this embodiment, a specific
pattern of DNA fragments is displayed on the support, wherein said
pattern is preferably specific for a particular Lawsonia spp., to
enable the user to distinguish between different species of the
bacterium.
[0266] Wherein the detection means is an amplification reaction
such as, for example, a polymerase chain reaction or a nucleic acid
sequence-based amplification (NASBA) system or a variant thereof,
one or more nucleic acid primer molecules of at least 15 contiguous
nucleotides in length derivable from a gene selected from the group
consisting of flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM, and
ytfN genes is hybridised to nucleic acid derived from a biological
sample, and nucleic acid copies of the FlgE-encoding genetic
sequences in said sample, or a part or fragment thereof, are
enzymically-amplified.
[0267] Those skilled in the art will be aware that there must be a
sufficiently high percentage of nucleotide sequence identity
between the primers and the sequences in the biological sample
template molecule to which they hybridise (i.e., the "template
molecule"). As stated previously, the stringency conditions can be
selected to promote hybridisation.
[0268] Preferably, each primer is at least about 95% identical to a
region of a gene selected from the group consisting of flhB, fliR,
ntrC, glnH, motA, motB, tlyC, ytfM, and ytfN genes in the template
molecule to which it hybridises.
[0269] Those skilled in the art will also be aware that, in one
format, PCR provides for the hybridisation of non-complementary
primers to different strands of the template molecule, such that
the hybridised primers are positioned to facilitate the 5'-3'
synthesis of nucleic acid in the intervening region, under the
control of a thermostable DNA polymerase enzyme. As a consequence,
PCR provides an advantage over other detection means in so far as
the nucleotide sequence in the region between the hybridised
primers may be unknown and unrelated to any known nucleotide
sequence.
[0270] In an alternative embodiment, wherein the detection means is
AFLP, the primers are selected such that, when nucleic acid derived
from the biological sample, in particular DNA, is amplified,
different length amplification products are produced from different
Lawsonia spp. The amplification products can be subjected to
electrophoresis, transferred to a solid support such as, for
example, a nylon or nitrocellulose membrane, and hybridised to a
probe optionally labelled with a reporter molecule as hereinbefore
described. According to this embodiment, a specific pattern of
amplified DNA fragments is displayed on the support, said pattern
optionally specific for a particular Lawsonia ssp., to enable the
user to distinguish between different species of the bacterium in
much the same way as for RFLP analysis.
[0271] The technique of AMD facilitates, not only the detection of
Lawsonia spp. DNA in a biological sample, but also the
determination of nucleotide sequence variants which differ from the
primers and probes used in the assay format. Wherein the detection
means is AMD, the probe is end-labelled with a suitable reporter
molecule and mixed with an excess of the amplified template
molecule. The mixtures are subsequently denatured and allowed to
renature to form nucleic acid "probe:template hybrid molecules" or
"hybrids", such that any nucleotide sequence variation between the
probe and the temple molecule to which it is hybridised will
disrupt base-pairing in the hybrids. These regions of mismatch are
sensitive to specific chemical modification using hydroxylamine
(mismatched cytosine residues) or osmium tetroxide (mismatched
thymidine residues), allowing subsequent cleavage of the modified
site using piperidine. The cleaved nucleic acid may be analysed
using denaturing polyacrylamide gel electrophoresis, followed by
standard nucleic acid hybridisation as described supra, to detect
the Lawsonia-derived nucleotide sequences. Those skilled in the art
will be aware of the means of end-labelling a genetic probe
according to the performance of the invention described in this
embodiment.
[0272] According to this embodiment, the use of a single
end-labelled probe allows unequivocal localisation of the sequence
variation. The distance between the point(s) of sequence variation
and the end-label is represented by the size of the cleavage
product.
[0273] In an alternative embodiment of AMD, the probe is labelled
at both ends with a reporter molecule, to facilitate the
simultaneous analysis of both DNA strands.
[0274] Wherein the detection means is RT-PCR, the nucleic acid
sample comprises an RNA molecule which is a transcription product
of Lawsonia-derived DNA or a homologue, analogue or derivative
thereof. As a consequence, this assay format is particularly useful
when it is desirable to determine expression of one or more
Lawsonia genes. According to this embodiment, the RNA sample is
reverse-transcribed to produce the complementary single-stranded
DNA which is subsequently amplified using standard procedures.
[0275] Variations of the embodiments described herein are described
in detail by McPherson et al. (1991).
[0276] The present invention clearly extends to the use of any and
all detection means referred to supra for the purposes of
diagnosing Lawsonia spp. and in particular L. intracellularis
infection in animals.
[0277] The amplification reaction detection means described supra
can be further coupled to a classical hybridisation reaction
detection means to further enhance sensitivity and specificity of
the inventive method, such as by hybridising the amplified DNA with
a probe which is different from any of the primers used in the
amplification reaction.
[0278] Similarly, the hybridisation reaction detection means
described supra can be further coupled to a second hybridisation
step employing a probe which is different from the probe used in
the first hybridisation reaction.
[0279] A further aspect of the invention provides an isolated probe
or primer derived from a gene selected from the group consisting of
flhB, fliR, ntrC, glnH, motA, motB, tlyC, ytfM, and ytfN genes.
Preferably, the probe or primer of the invention comprises a
nucleotide sequence selected from the group consisting of SEQ ID
NO: 19 to SEQ ID NO: 68 or a complementary nucleotide sequence
thereto.
[0280] The present invention does not extend to any nucleic acid or
polypeptide of Camplylobacter or Helicobacter that was disclosed
publicly before the filing date or priority date of this
application, or otherwise takes priority over the instant
application, and which is homologous to a nucleotide sequence or
amino acid sequence of Lawsonia spp. disclosed herein.
[0281] The present invention is further described with reference to
the following non-limiting Examples.
EXAMPLE 1
Molecular Cloning of Lawsonia intracellularis Genes
[0282] Isolation of DNA and Construction of DNA Libraries
[0283] L. intracellularis DNA was purified from pig intestinal
mucosa isolated from the ileum of pigs experimentally infected with
L. intracellularis. DNA purification from homogenized intestinal
mucosa was performed according to the method of Nollau et al.
(1996); or alternatively, by phenol extraction and sodium
acetate-ethanol precipitation of DNA.
[0284] To facilitate cloning of L. intracellularis gene sequences,
several genomic libraries were constructed. These libraries were
specifically modified by ligation of a known sequence (Vectorette
II.TM., Genosys Biotechnologies, Inc., The Woodlands, Tex.) to the
5'- and 3'-ends of restricted DNA fragments. Vectorette.TM.
libraries were constructed by separately digesting aliquots of L.
intracellularis-infected pig mucosal DNA extract with restriction
endonucleases 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.TM. tailing was carried out by addition
of T.sub.4 DNA Ligase (1 unit) plus 3 pMol of the appropriate
compatible Vectorette.TM. linker (HindIII Vectorette.TM.:
HindIII-digested DNA; EcoRI: EcoRI digested DNA; Blunt: DraI-,
HpaI-digested DNA). The mixture was incubated for three cycles,
each cycle consisting of 20.degree. C. for 60 min; followed by
37.degree. C. for 30 min, to complete the tailing reaction.
Reaction volumes were then adjusted to 200 .mu.l with water, and
reactions were stored at -20.degree. C.
EXAMPLE 2
Expression of the YtfN and YtfM genes of L. intracellularis
[0285] i) Isolation of a C-Terminal Fragment of ytfN Gene by Genome
Walking
[0286] The complete sequence of the L. intracellularis YffN gene
was determined from genomic DNA and is set forth herein as SEQ ID
NO: 17. Based upon the 2,035 bp sequence obtained for the amino
terminal portion of the ytfN gene fragment, oligonucleotide primer
KWK-Li-YtfN-4C (SEQ ID NO:29) was designed and synthesized (Life
Technologies; Rockville, Md.). This oligonucleotide binds within
the 3'-region of the YtfN gene in the L. intracellularis chromosome
to allow amplification of DNA downstream of the existing gene
fragment. For polymerase chain amplification, primer KWK-Li-YtfN-4C
(SEQ ID NO:29) was used in combination with a Vectorette.TM.
specific oligonucleotide primer (ER70; SEQ ID NO:108) in 50 .mu.l
reactions containing 1.times.PCR Buffer II (Perkin Elmer; Foster
City, Calif.), 2.0 mM MgCl.sub.2, 250 .mu.M each deoxy-NTP, 50 pMol
each primer, and 2.5 U AmpliTaq.TM. Gold (Perkin Elmer)
thermostable polymerase. Reactions were performed with 1 .mu.l of
the Vectorette.TM. libraries as DNA template. Amplification was
carried out as follows: denaturation (94.degree. C., 9 min); 40
cycles of denaturation (94.degree. C., 30 sec), annealing
(60.degree. C., 30 sec), and polymerization (72.degree. C., 4.0
min); this was followed by a final extension at 72.degree. C. for 7
minutes.
[0287] The amplified products were visualized by separation on a
1.0% agarose gel (Sigma; St. Louis, Mo.). Screening of the HpaI
library by PCR resulted in amplification of a fragment
approximately 1.5 kb in length. The PCR product was purified using
a QIAquick.TM. PCR Purification kit (Qiagen; Valencia, Calif.) and
cloned into the TA cloning site of pCR2.1-TOPO (Invitrogen;
Carlsbad, Calif.); the ligated product was transformed into Max
Efficiency E. coli DH5.alpha. cells (Life Technologies; Rockville,
Md.). Sequence analysis of the cloned fragment failed to identify a
termination codon for ytfN. Therefore, oligonucleotide primer
KWK-Li-YtfN-12C (SEQ ID NO:21) was designed and synthesized to be
used in a second round of PCR amplification using the
Vectorette.TM. libraries. The above-mentioned conditions for
amplification were used, and products were visualized by agarose
gel electrophoresis. A fragment approximately 1.6 kb in length was
amplified from the DraI library. The PCR product was purified using
a QIAquick PCR Purification kit, cloned into pCR2.1-TOPO, and
subsequently transformed into Max Efficiency E. coli DH5.alpha.
cells. Sequence analysis of the fragment identified a terminal TAA
codon indicating the end of the ytfN gene.
[0288] ii) Determination of Genomic Sequence of Complete ytfN
Gene
[0289] Results from the preliminary sequencing described above were
used to design oligonucleotide primers for the specific
amplification of two overlapping ytfN gene fragments directly from
L. intracellularis chromosomal DNA. These products encompass the
entire ytfN gene and were sequenced directly in an attempt to avoid
introduction of sequence artifacts due to mutations which might
arise during PCR amplification and subsequent cloning steps. To
obtain the first of the two fragments, PCR amplifications were
carried out in triplicate and contained 100 pMol of primers YtfN-D
(SEQ. ID NO:45) and YtfN-U (SEQ. ID NO:46), 100 ng purified
chromosomal DNA, 1.times.PC2 buffer (Ab Peptides; St. Louis, Mo.),
200 .mu.M each dNTP, 15 U KlenTaq1 (Ab Peptides) and 0.3 U cloned
Pfu (Stratagene; La Jolla, Calif.) thermostable polymerases in a
100 .mu.l final sample volume. Conditions for amplification
consisted of denaturation (94.degree. C., 9 min), followed by 40
cycles of denaturation (94.degree. C., 30 sec), annealing
(60.degree. C., 30 sec), and polymerization (72.degree. C., 4.0
min), and a final extension at 72.degree. C. for 7 min. To obtain
the second (overlapping) fragment, PCR amplifications were carried
out in triplicate as described above, except that primers
KWK-Li-YtfN-12C (SEQ ID NO:21) and KWK-LI-YtfN-15N (SEQ ID NO:24)
were used. Conditions for amplification consisted of denaturation
(94.degree. C., 9 min), followed by 40 cycles of denaturation
(94.degree. C., 30 sec), annealing (55.degree. C., 30 sec), and
polymerization (72.degree. C., 2.5 min), and a final extension at
72.degree. C. for 7 min.
[0290] Following amplification, each set of triplicate samples was
pooled and the specific product from each was purified
(QIAquick.TM. PCR Purification kit). Both purified DNA fragments
were then subjected to direct sequence analysis using DyeDeoxy
termination reactions on an ABI automated DNA sequencer (Lark
Technologies Inc., Houston, Tex.).
[0291] Synthetic oligonucleotide primers (SEQ ID NOs:21, 24, 26-38,
43-46, 51-53, and 55-60) were used to sequence both DNA strands of
the amplified products.
[0292] The ytfN ORF extends from nucleotides 1-4149 of SEQ ID NO:17
and encodes a 1382 amino acid protein (SEQ ID NO:18), having a
theoretical molecular weight of 150,887 Daltons. The sequence of
the amino terminus of the encoded protein resembles a prokaryotic
signal sequence (von Heijne, 1985; Nielsen, et al., 1997), although
the precise site of cleavage is not presently known. The ytfN ORF
was compared against existing nucleotide and protein databases
using the Basic Local Alignment Search Tool (BLAST) programs
(Altschul, et al., 1990). The entry with which it shared the
greatest homology was a hypothetical 40.5 kDa protein from
Zymomonas mobilis. The second-most-significant homologous sequence
identified was a YtfN homolog from Neissena meningitidis.
[0293] iii) Cloning of Recombinant ytfM Gene into Expression
Vectors
[0294] For the purpose of recombinant protein expression, both the
ytfM and ytfN genes or fragments thereof were cloned without the
sequences encoding their respective signal peptides.
[0295] The ytfM gene was amplified from L. intracellularis
chromosomal DNA using oligonucleotide primers RA202-b (SEQ ID NO:
50) and RA201-b (SEQ ID NO: 49). For polymerase chain
amplification, triplicate 50 .mu.l reactions were set up with
eachcontaining 100 ng of chromosomal DNA as template, 1.times.PC2
buffer, 200 .mu.M each dNTP, 50 pMol each primer, 7.5 U KIenTaq1
and 0.15 U cloned Pfu thermostable polymerases. Amplification was
carried out as follows: denaturation (94.degree. C., 9 min); 40
cycles of denaturation (94.degree. C., 30 sec), annealing
(60.degree. C., 30 sec), and polymerization (72.degree. C., 2.5
min), followed by a final extension at 72.degree. C. for 7 minutes.
Following amplification, the samples were purified (QIAquick.TM.
PCR Purification kit) and pooled. The purified PCR product was
cloned directly into the TA cloning site of both pBAD-TOPO and
pBAD-TOPO (Invitrogen). The ligated products were transformed into
Max Efficiency E. coli DH5.alpha. cells. The predicted amino
terminal sequence of the encoded protein expressed from
pBAD-TOPO:YtfM would consist of the vector-encoded sequence
MGSGSGDDDDKLALLTM (SEQ ID NO: 61) followed immediately by the
sequence ATSITTS (SEQ ID NO: 62) beginning at Alanine-24 of the
YtfM ORF (SEQ ID NO:16). A clone containing the appropriate plasmid
was identified, and purified plasmid was isolated from a
small-scale broth culture using a QIAprep Spin Miniprep kit
(Qiagen). This plasmid was transformed into E. coli BL21 (Novagen;
Madison, Wis.) and BL21-CodonPlus-RIL cells (Stratagene); a clone
was identified in each strain that contained the appropriate
plasmid.
[0296] The predicted amino terminal sequence of the encoded fusion
protein expressed from pBAD/Thio-TOPO:YtfM would consist of the
thioredoxin protein and a 15 amino acid residue linker followed
immediately by the sequence ATSITTS (SEQ ID NO: 62) beginning at
Alanine-24 of the YtfM ORF (SEQ ID NO:16). A clone containing the
appropriate plasmid was identified, and purified plasmid was
isolated from a small-scale broth culture using a QIAprep Spin
Miniprep kit. This plasmid was transformed into E. coli BL21 and
BL21-CodonPlus-RIL cells; a clone was identified in each strain
that contained the appropriate plasmid.
[0297] For cloning into pET-30a, the purified PCR product encoding
YtfM was digested with BamHI and NcoI, then purified using a
QIAquick.TM. PCR Purification kit. pET-30a was also digested with
BamHI and NcoI; the linearized plasmid was purified using a
JETsorb.TM. kit (Genomed; Frederick, Md.) prior to ligation. The
ligated product was transformed into Max Efficiency E. coli
DH5.alpha. cells. The predicted amino terminal sequence of the
encoded fusion protein expressed from pET-30a:YtfM would consist of
MHHHHHHSSGLVPRGSGMKETAAAKFERQHMDSPDLGTDDDDK- AM (SEQ ID NO: 63)
encoded by the vector, followed by the sequence ATSITTS (SEQ ID NO:
62) beginning at Alanine-24 of the YtfM ORF (SEQ ID NO:16). A clone
containing the appropriate plasmid was identified, and purified
plasmid was isolated from a small-scale broth culture using a
QIAprep Spin Miniprep kit. This plasmid was transformed into E.
coli BL21(DE3) (Novagen) and BL21-CodonPlus(DE3)-RIL cells
(Stratagene); a clone was identified in each strain that contained
the appropriate plasmid.
[0298] The ytfM gene was also amplified from L. intracellularis
chromosomal DNA by PCR amplification using oligonucleotide primers
RA200 (SEQ ID NO: 47) and RA201 (SEQ ID NO: 48). Duplicate 50 pI
reactions were set up each containing 100 ng of chromosomal DNA as
template, 1.times.PC2 buffer, 200 .mu.M each dNTP, 50 pMol each
primer, 7.5 U KlenTaq1 and 0.15 U cloned Pfu thermostable
polymerases. Amplification was carried out as follows: denaturation
(94.degree. C., 9 min); 30 cycles of denaturation (94.degree. C.,
30 sec), annealing (60.degree. C., 30 sec), and polymerization
(72.degree. C., 2 min), followed by a final extension at 72.degree.
C., for 7 minutes. Following amplification, the samples were
purified (QIAquick.TM. PCR Purification kit) and pooled. The
purified PCR product was cloned directly into the TA cloning site
of pCR2.1-TOPO. The ligated product was transformed into Max
Efficiency E. coli DH5.alpha. cells. A clone containing the
appropriate plasmid was identified, propagated, and plasmid DNA was
isolated using a QIAprep Spin Miniprep kit. Following digestion of
the plasmid with EcoRI, a fragment corresponding to bp 437 of SEQ
ID NO:15 to the EcoRI site in the MCS of pCR2.1-TOPO was purified
using a JETsorb.TM. kit. pET-30a was also digested with EcoRI, and
purified using a QIAquick PCR.TM. Purification kit. The two
fragments were ligated and transformed into Max Efficiency E. coli
DH5.alpha. cells. The predicted amino terminal sequence of the
encoded fusion protein would consist of
MHHHHHHSSGLVPRGSGMKETAAAKFERQHMDS- PDLGTDDDDKAMADIGS (SEQ ID NO:
64) encoded by the vector followed by the sequence EFNLSKG (SEQ ID
NO: 65) beginning at Aspartate-146 of the YtfM ORF (SEQ ID NO:16).
A clone containing the plasmid with the gene fragment inserted in
the proper orientation was identified, and purified plasmid was
isolated from a small-scale broth culture using a QIAprep Spin
Miniprep kit. This plasmid was transformed into E. coli
BL21-CodonPlus(DE3)-RIL cells; a clone was identified that
contained the appropriate plasmid.
[0299] iv) Cloning of Recombinant ytfN Gene into Expression
Vectors
[0300] The 5' half of the ytfN gene, excluding that encoding the
signal sequence, was amplified from L. intracellularis chromosomal
DNA using oligonucleotide primers RA205-b (SEQ ID NO: 53) and
RA204-b (SEQ ID NO: 52). For polymerase chain amplification,
triplicate 100 .mu.l reactions were set up each containing 100 ng
of chromosomal DNA as template, 1.times.PC2 buffer, 200 .mu.M each
dNTP, 100 pMol each primer, 15 U KlenTaq1 and 0.3 U cloned Pfu
thermostable polymerases. Amplification was carried out as follows:
denaturation (94.degree. C., 9 min); 40 cycles of denaturation
(94.degree. C., 30 sec), annealing (60.degree. C., 30 sec), and
polymerization (72.degree. C., 4 min), followed by a final
extension at 72.degree. C. for 7 minutes. Following amplification,
the samples were purified (QIAquick.TM. PCR Purification kit) and
pooled. The purified PCR product was cloned directly into the TA
cloning site of both pBAD-TOPO and pBAD/Thio-TOPO (Invitrogen). The
ligated products were transformed into Max Efficiency E. coli
DH5.alpha. cells. The predicted amino terminal sequence of the
encoded protein expressed from pBAD-TOPO:YtfN would consist of the
vector-encoded sequence MGSGSGDDDDKLALGHM (SEQ ID NO: 66) followed
immediately by the sequence RTSTGIA (SEQ ID NO: 67) beginning at
Arginine-33 of the YtfN ORF (SEQ ID NO:18). A clone containing the
appropriate plasmid was identified, and purified plasmid was
isolated from a small-scale broth culture using a QIAprep Spin
Miniprep kit. This plasmid was transformed into E. coli
BL21-CodonPlus-RIL cells; a clone was identified that contained the
appropriate plasmid.
[0301] The predicted amino terminal sequence of the encoded protein
expressed from pBAD/Thio-TOPO:YtfN would consist of the thioredoxin
protein and a 15 amino acid linker followed immediately by the
sequence RTSTGIA (SEQ ID NO: 67) beginning at Arginine-33 of the
YtfN ORF (SEQ ID NO:18). A clone containing the appropriate plasmid
was identified, and purified plasmid was isolated from a
small-scale broth culture using a QIAprep Spin Miniprep kit. This
plasmid was transformed into E. coli BL21-CodonPlus-RIL cells; a
clone was identified that contained the appropriate plasmid.
[0302] For cloning into pET-30a, the purified PCR product was
digested with BamHI and NdeI and extracted using a QIAquick PCR
Purification kit. The linearized plasmid was purified using a
JETsorb.TM. kit prior to ligation. The ligated product was
transformed into Max Efficiency E. coli DH5.alpha. cells. The
predicted amino terminal sequence of the protein expressed from
pET-30a:YtfN would consist of Met encoded by RA205-b (SEQ ID NO:
53) followed by the sequence RTSTGIA (SEQ ID NO: 67) beginning at
Arginine-33 of the YtfN ORF (SEQ ID NO:18). A clone containing the
appropriate plasmid was identified, and purified plasmid was
isolated from a small-scale broth culture using a QIAprep Spin
Miniprep kit. This plasmid was transformed into E. coli
BL21-CodonPlus(DE3)-RIL and BL21-CodonPlus(DE3)-RP cells
(Stratagene); a clone was identified in each strain that contained
the appropriate plasmid.
[0303] Utilizing oligonucleotide primers RA205-b (SEQ ID NO: 53)
and KWK-Li-YtfN-BgIII-3' (SEQ ID NO:39), an approximate 1 kb
fragment encoding the N-terminal portion of YtfN, excluding the
signal sequence, was amplified by PCR. Triplicate 50 .mu.l
reactions were set up each containing 100 ng of chromosomal DNA as
template, 1.times.PC2 buffer, 200 .mu.M each dNTP, 50 pMol each
primer, 7.5 U KlenTaq1 and 0.15 U cloned Pfu thermostable
polymerases. Amplification was carried out as follows: denaturation
(94.degree. C., 9 min); 40 cycles of denaturation (94.degree. C.,
30 sec), annealing (55.degree. C., 30 sec), and polymerization
(72.degree. C., 1 min); followed by a final extension at 72.degree.
C. for 7 minutes. Following amplification, the samples were
purified (QIAquick.TM. PCR Purification kit) and pooled. The
purified PCR product was cloned directly into the TA cloning site
of both pBAD-TOPO and pBAD/Thio-TOPO. The ligated products were
transformed into Max Efficiency E. coli DH5.alpha. cells. The
predicted amino terminal sequence of the protein expressed from
pBAD-TOPO would consist of the vector-encoded sequence
MGSGSGDDDDKLALGHM (SEQ ID NO: 66) followed immediately by the
sequence RTSTGIA (SEQ ID NO: 67) beginning at Arginine-33 of the
YtfN ORF (SEQ ID NO:18); the protein would terminate with
Isoleucine-332 of ytfN (SEQ ID NO:18). A clone containing the
appropriate plasmid was identified, and purified plasmid was
isolated from a small-scale broth culture using a QIAprep Spin
Miniprep kit. This plasmid was transformed into E. coli
BL21-CodonPlus-RIL cells; a clone was identified that contained the
appropriate plasmid.
[0304] The predicted amino terminal sequence of the encoded fusion
protein expressed from pBAD/Thio-TOPO:YtfN would consist of the
thioredoxin protein and a 15 amino acid linker followed immediately
by the sequence RTSTGIA (SEQ ID NO: 67) beginning at Arginine-33 of
the YtfN ORF (SEQ ID NO:18); again, the polypeptide would terminate
with Isoleucine-332 of ytfN (SEQ ID NO:18). A clone containing the
appropriate plasmid was identified, and purified plasmid was
isolated from a small-scale broth culture using a QIAprep Spin
Miniprep kit. This plasmid was transformed into E. coli
BL21-CodonPlus-RIL cells; a clone was identified that contained the
appropriate plasmid.
[0305] In order to generate a construct consisting of the
above-described 1 kb (5' to Bgll site) ytfN fragment in pET30a, the
plasmid pET-30a:YtfN containing the 5' half of ytfN amplified using
RA205-b (SEQ ID NO: 53) and RA204-b (SEQ ID NO: 52) was digested
with BgIII and BamHI, thus excising the gene sequence downstream of
the BgIII site within ytfN. The fragment containing the vector and
ytfN sequence up to the BgIII site was purified using a JETsorb.TM.
kit, and the remaining fragment was religated and transformed into
E. coli Max Efficiency DH5.alpha. cells. The predicted amino
terminal sequence of the protein expressed would consist of Met
encoded by RA205-b (SEQ ID NO: 53) followed by the sequence RTSTGIA
(SEQ ID NO: 67) beginning at Arginine-33 of the YtfN ORF (SEQ ID
NO:18). The polypeptide would terminate with Isoleucine-332 of SEQ
ID NO:18, followed by a C-terminal extension consisting of
DPNSSSVDKLAAALEHHHHHH (SEQ ID NO: 68) encoded by the vector. A
clone containing the appropriate plasmid was identified, and
purified plasmid was isolated from a small-scale broth culture
using a QIAprep Spin Miniprep kit (Qiagen). This plasmid was
transformed into E. coli BL21(DE3) and BL21-CodonPlus(DE3)-RIL
cells; a clone was identified in each strain that contained the
appropriate plasmid. Stocks of the clone containing this plasmid in
E. coli BL21 (DE3) were frozen at -80.degree. C.
[0306] v) Expression of Recombinant YtfN Polypeptide
[0307] Frozen working stock of the E. coli BL21 (DE3) transformant
harboring pET-30a containing the 5' portion of ytfN up to the BgIII
site (eg. corresponding to amino acids 33-332 of the encoded YtfN
protein) was thawed and seeded at a 1:5000 dilution in RWLDM/G vi
defined medium [K.sub.2HPO.sub.4 (6 g/L), KH.sub.2PO.sub.4 (3 g/L),
(NH.sub.4).sub.2SO.sub.4 (5 g/L), NaCl (2 g/L), 0.2 mL CaCl.sub.2
(15 g/L), 0.4 mL FeCl.sub.3.6H.sub.2O (5 g/L), 0.4 ml
MgSO.sub.4.7H.sub.2O (480 g/L), ZnCl.sub.2 (6.5 g/L),
MnSO.sub.4.H.sub.2O (12 g/L), Na.sub.2MoO.sub.4.2H.sub.2O (5 g/L),
CuSO.sub.4 (1.5 g/L), CoCl.sub.2.6H.sub.2O (2 g/L), H.sub.3BO.sub.3
(0.5 g/L), and 37% HCl (5 ml/L)]. Kanamycin was also added to a
concentration of 25 .mu.g/ml to maintain the expression plasmid.
The culture was grown under fed-batch (50% glycerol) in a 5 liter
working volume BioFlow 3000 fermentor (New Brunswick Scientific;
Edison, N.J.) at 37.degree. C. until A.sub.625 was 2.9. At this
time, IPTG was added to 0.1 mM, and culture samples were collected
at 0, 2.25, and 3 hours post induction to monitor expression of
recombinant YtfN (see Figure). The primary culture was maintained
28 hours post inoculation then immediately chilled, and wet cells
were collected by centrifugation and stored at -20.degree. C. Data
showing expression of ytfN protein are presented in FIG. 1.
REFERENCES
[0308] 1. Altuvia, Y., Schueler, O., and Margalit, H. (1995) J.
Mol. Biol. 249:244-250.
[0309] 2. Amann and Brosius (1985). Gene 40: 183.
[0310] 3. Anderson, B. J., M. M. Bills, J. R. Egerton, and J. S.
Mattick. (1984) Journal of Bacteriology 160:748-754.
[0311] 4. Altschul, S. F., Gish, W., Miller, W., Myers, E. W.,
Lipman, D. J., 1990, J. Mol. Biol. 215:403-410.
[0312] 5. Ausubel, F. M., Brent, R., Kingston, R E, Moore, D. D.,
Seidman, J. G., Smith, J. A., and Struhl, K. (1987). In: Current
Protocols in Molecular Biology. Wiley Interscience (ISBN
047150338).
[0313] 6. Barker, I. K. and Van Dreumel, A. A. (1985) In
.quadrature.Pathology of Domestic Animals,.quadrature. 3rd Edition,
Vol. 2 p. 1-237, eds K. V. F. Jubb, P. C. Kennedy and N. Palmer.
(Academic Press: Orlando).
[0314] 7. Cole et al. (1985) In: Monoclonal antibodies in cancer
therapy, Alan R. Bliss Inc., pp 77-96.
[0315] 8. Dayhof, M. D. (1978) In: Nat. Biomed. Res. Found.
Washington D.C. Vol5, Suppl. 3.
[0316] 9. De Groot, A. S., Carter, E. J., Roberts, C. G. P.,
Edelson, B. T., Jesdale, B. M., Meister, G. E., Houghten, R. A.,
Montoya, J., Romulo, R. C., Berzofsky, J. A., and Ramirezm, B. D.
L. L. (1995) Vaccines 96, Cold Spring Harbor Laboratory, Cold
Spring Harbor N.Y.
[0317] 10. Devereux, J., Haeberli, P. and Smithies, O. (1984).
Nucl. Acids Res. 12: 387-395.
[0318] 11. Elwell, M R, Chapman, A L and Frenkel, J K (1981)
Veterinary Pathology 18: 136-139.
[0319] 12. Fox, J G, Murphy, J C, Otto, G Pecquet-Goad, M E,
Larson, Q H K and Scott J A (1989) Veterinary Pathology 26:
515-517.
[0320] 13. Gabriel, E. Meister, G. E., Caroline, G. P., Roberts, C.
G. P., Berzofsky, J. A., and De Groot, A. S. (1995) Vaccines 95,
Cold Spring Harbor Laboratory, Cold Spring Harbor N.Y.
[0321] 14. Gebhart, C. J., Ward, G. E., Chang, K. And Kurtz, H. J.
(1983). American Journal of Veterinary Research 44:361-367.
[0322] 15. Gish, W and States, D. J. (1993) Nature Genetics 3:
266-272.
[0323] 16. Goodman et al. (1987) Biopolymers 26: 525-532.
[0324] 17. Huse et al. (1989) Science 246: 1275-1281.
[0325] 18. Jones, L. A., Nibbelink, S., and Glock, R. D. (1997) Am.
J. Vet. Res. 58: 1125-1131.
[0326] 19. Jonsson, L. and Martinsson, K. (1976) Acta Veterinaria
Scandinavica 17:223-232.
[0327] 20. Kohler and Milstein (1975) Nature 256: 495499
[0328] 21. Kozbor et al. (1983) Immunol. Today 4: 72.
[0329] 22. Lawson, G. H. K., McOrist, S., Jansi, S. and Mackie, R.
A. (1993) Journal of Clinical Microbiology 31:1136-1142.
[0330] 23. Love, R. J. and Love, D. M. (1977) Veterinary Record
100:473
[0331] 24. Margalit, H., Spouge, J. L., Cornette, J. L., Cease, K.
B., DeLisi, C., and Berzofsky, J. A. (1987) J. Immunol.
138:2213-2229.
[0332] 25. Mason, R W, Monkton, P and Hasse D (1998) Australian
Veterinary Journal (submitted for publication).
[0333] 26. McOrist, S., Boid, R., Lawson, G. H. K. and McConnell,
I. (1987) The Veterinary Record 121:421422.
[0334] 27. McOrist, S, Jasni, S, Mackie, R A, MacIntyre, N, Neef,
N. and Lawson G H K (1993) Infection and Immunity 61: 42864292.
[0335] 28. McOrist, S et al (1995) International Journal of
Systematic Bacteriology 45: 820-825.
[0336] 29. McPherson, M. J., Quirke, P., and Taylor, G. R.
(1991)/n: PCR: A Practical Approach. (series editors, D. Rickwood
and B. D. Hames) IRL Press Limited, Oxford. pp1-253.
[0337] 30. Meister, G. E., Roberts, C. G. P., Berzofsky, J. A., and
De Groot, A. S. (1995) Vaccine 13: 581-591.
[0338] 31. Mierke et al. (1990) Int. J. Peptide Protein Research
35:3545.
[0339] 32. Mohapatra, S. S., Cao, Y., Ni, H., and Salo, D. (1995)
Allergy 50:37-44.
[0340] 33. Needleman and Wunsch (1970) J. Mol. Biol.
48:443-453.
[0341] 34. Nielsen, H., Engelbrecht, J., Brunak, S., and von
Heijne, G., (1997) Protein Engineering, 10: 1-6
[0342] 35. Nollau, P., Moser, C. and C. Wagener (1996)
BioTechniques 20:784-788.
[0343] 36. O'Neil, I. P. A. (1970) Veterinary Record
87:742-747.
[0344] 37. Parker, K. C., Bednarek, M. A., and Coligan, J. E.
(1994) J. Immunol. 152:163-175.
[0345] 38. Portoghese et al. (1990) J. Med. Chem. 33:1714-1720.
[0346] 39. Reinhartz, A., Alajem, S., Samson, A. and Herzberg, M.
(1993). Gene 136: 221-226.
[0347] 40. Rowland, A. C. and Lawson, G. H. K. (1976) Veterinary
Record 97:178-180.
[0348] 41. Sambrook, J., E. F. Fritsch, and T. Maniatis. (1989)
Molecular cloning. A laboratory manual. Second edition. Cold Spring
Harbour Laboratory, Cold Spring Harbour, N.Y.
[0349] 42. Schodeb, T R and Fox J G (1990) Veterinary Pathology 27:
73-80.
[0350] 43. Shimatake and Rosenberg (1981) Nature 292: 128.
[0351] 44. Stills, H. F. (1991). Infection and immunology 59:
3227-3236.
[0352] 45. Straw, B. E. (1990). Journal of American Veterinary
Medical Association 197: 355-357.
[0353] 46. Studier and Moffat (1986) J. Mol. Biol. 189: 113.
[0354] 47. Thompson, J. D., Higgins, D. G., and Gibson, T. J.
(1994) Nucl. Acids Res. 22: 4673-4680.
[0355] 48. Vajda, S. and DeLisi, C. (1990) Biopolymers
29:1755-1772.
[0356] 49. van Regenmortel, M. (1992) Molecular dissection of
protein antigens. In: Structure of antigens, (van Regenmortel M.
ed.) CRC Press, London, pp1-27.
[0357] 50. von Heijne, (1985), J. Mol. Biol. 184: 99-105.
Sequence CWU 1
1
68 1 622 DNA Lawsonia intracellularis CDS (1)...(621) 1 atg tct gat
gat ccc agt aaa aca gag aaa gca acc ccg aaa cga cgt 48 Met Ser Asp
Asp Pro Ser Lys Thr Glu Lys Ala Thr Pro Lys Arg Arg 1 5 10 15 cag
gaa gct cgt tct gaa ggg agt gtc cct aaa tca gaa gag gtt act 96 Gln
Glu Ala Arg Ser Glu Gly Ser Val Pro Lys Ser Glu Glu Val Thr 20 25
30 aaa gca ttg act act gca gca ggg atg ctg ggg ctt gct att tat tca
144 Lys Ala Leu Thr Thr Ala Ala Gly Met Leu Gly Leu Ala Ile Tyr Ser
35 40 45 ggc gta atg gga cgt cat ttt gaa aca att ttc tac tat att
ttt aca 192 Gly Val Met Gly Arg His Phe Glu Thr Ile Phe Tyr Tyr Ile
Phe Thr 50 55 60 gaa tca ttt cgg ttt gag gtt aca gca cag tca gta
tat gct tta ttt 240 Glu Ser Phe Arg Phe Glu Val Thr Ala Gln Ser Val
Tyr Ala Leu Phe 65 70 75 80 att tat gtt gct caa gag ata gct att tta
ttg atg cca ata tta ctt 288 Ile Tyr Val Ala Gln Glu Ile Ala Ile Leu
Leu Met Pro Ile Leu Leu 85 90 95 ttt att gct gtt acg gca tgg att
tca tta cgt gta caa gtt ggt gca 336 Phe Ile Ala Val Thr Ala Trp Ile
Ser Leu Arg Val Gln Val Gly Ala 100 105 110 tta tgg act aca aag gtt
ttt aaa ttt aaa tgg agt aaa ttt aat ata 384 Leu Trp Thr Thr Lys Val
Phe Lys Phe Lys Trp Ser Lys Phe Asn Ile 115 120 125 ata aaa ggg ttg
aaa gga atg ttt gct tct caa caa aca ctt gtt cga 432 Ile Lys Gly Leu
Lys Gly Met Phe Ala Ser Gln Gln Thr Leu Val Arg 130 135 140 ctt tta
cgt agt tta gtt caa gta att gtt ata ggt att gtt cca tat 480 Leu Leu
Arg Ser Leu Val Gln Val Ile Val Ile Gly Ile Val Pro Tyr 145 150 155
160 atg att ata aaa gga gag ttt tca aac ttt tta cca tta tat tat gca
528 Met Ile Ile Lys Gly Glu Phe Ser Asn Phe Leu Pro Leu Tyr Tyr Ala
165 170 175 agt cct tca ggt gtg gca gat tat atg ctt aat aca gga ata
gta ctt 576 Ser Pro Ser Gly Val Ala Asp Tyr Met Leu Asn Thr Gly Ile
Val Leu 180 185 190 gtt tta tat acg cta att cct atg aca att att gca
gtc gca gat 621 Val Leu Tyr Thr Leu Ile Pro Met Thr Ile Ile Ala Val
Ala Asp 195 200 205 c 622 2 207 PRT Lawsonia intracellularis 2 Met
Ser Asp Asp Pro Ser Lys Thr Glu Lys Ala Thr Pro Lys Arg Arg 1 5 10
15 Gln Glu Ala Arg Ser Glu Gly Ser Val Pro Lys Ser Glu Glu Val Thr
20 25 30 Lys Ala Leu Thr Thr Ala Ala Gly Met Leu Gly Leu Ala Ile
Tyr Ser 35 40 45 Gly Val Met Gly Arg His Phe Glu Thr Ile Phe Tyr
Tyr Ile Phe Thr 50 55 60 Glu Ser Phe Arg Phe Glu Val Thr Ala Gln
Ser Val Tyr Ala Leu Phe 65 70 75 80 Ile Tyr Val Ala Gln Glu Ile Ala
Ile Leu Leu Met Pro Ile Leu Leu 85 90 95 Phe Ile Ala Val Thr Ala
Trp Ile Ser Leu Arg Val Gln Val Gly Ala 100 105 110 Leu Trp Thr Thr
Lys Val Phe Lys Phe Lys Trp Ser Lys Phe Asn Ile 115 120 125 Ile Lys
Gly Leu Lys Gly Met Phe Ala Ser Gln Gln Thr Leu Val Arg 130 135 140
Leu Leu Arg Ser Leu Val Gln Val Ile Val Ile Gly Ile Val Pro Tyr 145
150 155 160 Met Ile Ile Lys Gly Glu Phe Ser Asn Phe Leu Pro Leu Tyr
Tyr Ala 165 170 175 Ser Pro Ser Gly Val Ala Asp Tyr Met Leu Asn Thr
Gly Ile Val Leu 180 185 190 Val Leu Tyr Thr Leu Ile Pro Met Thr Ile
Ile Ala Val Ala Asp 195 200 205 3 789 DNA Lawsonia intracellularis
CDS (1)...(786) 3 atg aat tta ttt aat ttt gat cct agt atg ttt ctt
agt ttt tta ctt 48 Met Asn Leu Phe Asn Phe Asp Pro Ser Met Phe Leu
Ser Phe Leu Leu 1 5 10 15 aca ttt tta cgt att agt gtt gtc tta ttt
atg ctt cct ttt ttt tct 96 Thr Phe Leu Arg Ile Ser Val Val Leu Phe
Met Leu Pro Phe Phe Ser 20 25 30 att gat ggt ttt cct aat atg tta
aaa gca tca ata gct ctt att cta 144 Ile Asp Gly Phe Pro Asn Met Leu
Lys Ala Ser Ile Ala Leu Ile Leu 35 40 45 act ata gtt ctt tgg ggg
cgt ctt tct ctt tca gga aca caa atg cca 192 Thr Ile Val Leu Trp Gly
Arg Leu Ser Leu Ser Gly Thr Gln Met Pro 50 55 60 gcg cat cct ttc
gat cta gta ttg tta atc ata agc gag gtt ttt ctt 240 Ala His Pro Phe
Asp Leu Val Leu Leu Ile Ile Ser Glu Val Phe Leu 65 70 75 80 ggt att
gta ttg ggg ctt gcg gta aac ttt ttc ttt gca gga att caa 288 Gly Ile
Val Leu Gly Leu Ala Val Asn Phe Phe Phe Ala Gly Ile Gln 85 90 95
gct ggg gga gaa att ctt gct aca caa atg ggg ttt aca atg att acg 336
Ala Gly Gly Glu Ile Leu Ala Thr Gln Met Gly Phe Thr Met Ile Thr 100
105 110 ctt gca gac cca tta act ggt aac acc aca ggt ttt att gca cat
ttt 384 Leu Ala Asp Pro Leu Thr Gly Asn Thr Thr Gly Phe Ile Ala His
Phe 115 120 125 ctt tat atg gtt gct aca tta gtt ttt ctt gct ctt aat
ggc cat ttg 432 Leu Tyr Met Val Ala Thr Leu Val Phe Leu Ala Leu Asn
Gly His Leu 130 135 140 ttt ctt ata aaa gct ttt aca tat act ttt aaa
atg gtt cca gca gga 480 Phe Leu Ile Lys Ala Phe Thr Tyr Thr Phe Lys
Met Val Pro Ala Gly 145 150 155 160 gga ctt gtt gta aga gaa att tta
ttg agt gaa ctt ctt aat atg gca 528 Gly Leu Val Val Arg Glu Ile Leu
Leu Ser Glu Leu Leu Asn Met Ala 165 170 175 ggg atg att ttt gtt ttt
gcc tta cat gtt gcg gca cca gtt atg tca 576 Gly Met Ile Phe Val Phe
Ala Leu His Val Ala Ala Pro Val Met Ser 180 185 190 gct ctt ttt tta
gta gag atc tct tta gga ctt atg gca aga gct gct 624 Ala Leu Phe Leu
Val Glu Ile Ser Leu Gly Leu Met Ala Arg Ala Ala 195 200 205 cct cag
att cat att atg gaa gtt gga ttt cct gta aaa att ggt gta 672 Pro Gln
Ile His Ile Met Glu Val Gly Phe Pro Val Lys Ile Gly Val 210 215 220
gga ttt ttt ttc att gga cta tta ttt act atc tta tca aaa gaa acc 720
Gly Phe Phe Phe Ile Gly Leu Leu Phe Thr Ile Leu Ser Lys Glu Thr 225
230 235 240 tat cga ttt att gca ggc cta gag gga cta ttt ttt aac tta
ctt act 768 Tyr Arg Phe Ile Ala Gly Leu Glu Gly Leu Phe Phe Asn Leu
Leu Thr 245 250 255 gta atg ggt agt gga aaa tag 789 Val Met Gly Ser
Gly Lys 260 4 262 PRT Lawsonia intracellularis 4 Met Asn Leu Phe
Asn Phe Asp Pro Ser Met Phe Leu Ser Phe Leu Leu 1 5 10 15 Thr Phe
Leu Arg Ile Ser Val Val Leu Phe Met Leu Pro Phe Phe Ser 20 25 30
Ile Asp Gly Phe Pro Asn Met Leu Lys Ala Ser Ile Ala Leu Ile Leu 35
40 45 Thr Ile Val Leu Trp Gly Arg Leu Ser Leu Ser Gly Thr Gln Met
Pro 50 55 60 Ala His Pro Phe Asp Leu Val Leu Leu Ile Ile Ser Glu
Val Phe Leu 65 70 75 80 Gly Ile Val Leu Gly Leu Ala Val Asn Phe Phe
Phe Ala Gly Ile Gln 85 90 95 Ala Gly Gly Glu Ile Leu Ala Thr Gln
Met Gly Phe Thr Met Ile Thr 100 105 110 Leu Ala Asp Pro Leu Thr Gly
Asn Thr Thr Gly Phe Ile Ala His Phe 115 120 125 Leu Tyr Met Val Ala
Thr Leu Val Phe Leu Ala Leu Asn Gly His Leu 130 135 140 Phe Leu Ile
Lys Ala Phe Thr Tyr Thr Phe Lys Met Val Pro Ala Gly 145 150 155 160
Gly Leu Val Val Arg Glu Ile Leu Leu Ser Glu Leu Leu Asn Met Ala 165
170 175 Gly Met Ile Phe Val Phe Ala Leu His Val Ala Ala Pro Val Met
Ser 180 185 190 Ala Leu Phe Leu Val Glu Ile Ser Leu Gly Leu Met Ala
Arg Ala Ala 195 200 205 Pro Gln Ile His Ile Met Glu Val Gly Phe Pro
Val Lys Ile Gly Val 210 215 220 Gly Phe Phe Phe Ile Gly Leu Leu Phe
Thr Ile Leu Ser Lys Glu Thr 225 230 235 240 Tyr Arg Phe Ile Ala Gly
Leu Glu Gly Leu Phe Phe Asn Leu Leu Thr 245 250 255 Val Met Gly Ser
Gly Lys 260 5 1371 DNA Lawsonia intracellularis CDS (1)...(1368) 5
atg tca gca cgt ata ctt att ata gat gat gaa gac tct att aga ttt 48
Met Ser Ala Arg Ile Leu Ile Ile Asp Asp Glu Asp Ser Ile Arg Phe 1 5
10 15 tca ttg aaa gga att ttt gaa gat gag ggc cat gaa gtt tta gaa
aga 96 Ser Leu Lys Gly Ile Phe Glu Asp Glu Gly His Glu Val Leu Glu
Arg 20 25 30 gct tca gca gaa gaa gga ctt aag tgt gtt gat gta gag
tct cca gat 144 Ala Ser Ala Glu Glu Gly Leu Lys Cys Val Asp Val Glu
Ser Pro Asp 35 40 45 ctt gtt ttt ctt gat att tgg ctt cct ggg atg
gat ggt ctt atg gct 192 Leu Val Phe Leu Asp Ile Trp Leu Pro Gly Met
Asp Gly Leu Met Ala 50 55 60 tta gac cat att cag gct ctt cat cag
gaa tta cct gtt att atg att 240 Leu Asp His Ile Gln Ala Leu His Gln
Glu Leu Pro Val Ile Met Ile 65 70 75 80 tca ggt cat gcc aca att gaa
act gct gta aca gct atc cgt caa ggt 288 Ser Gly His Ala Thr Ile Glu
Thr Ala Val Thr Ala Ile Arg Gln Gly 85 90 95 gct tat gat ttt att
gaa aag cct ctt tct ttg gaa aaa gtc ctt att 336 Ala Tyr Asp Phe Ile
Glu Lys Pro Leu Ser Leu Glu Lys Val Leu Ile 100 105 110 aca gct aat
aga gct ata gaa aca gta aga tta aga agg gaa aac aaa 384 Thr Ala Asn
Arg Ala Ile Glu Thr Val Arg Leu Arg Arg Glu Asn Lys 115 120 125 tta
cta cgt act gta tta cct gag gag agt gag ttt ata gga cag tct 432 Leu
Leu Arg Thr Val Leu Pro Glu Glu Ser Glu Phe Ile Gly Gln Ser 130 135
140 cct gtt atc tta aaa ttt aaa agt tta tta tca cag gtc gct cca aca
480 Pro Val Ile Leu Lys Phe Lys Ser Leu Leu Ser Gln Val Ala Pro Thr
145 150 155 160 gat gct tgg gta cta ctt aca gga gag aat ggt aca ggt
aaa gag tta 528 Asp Ala Trp Val Leu Leu Thr Gly Glu Asn Gly Thr Gly
Lys Glu Leu 165 170 175 gct gca caa gca ttg cac aaa gga agc tca cga
tat caa aaa cca ttt 576 Ala Ala Gln Ala Leu His Lys Gly Ser Ser Arg
Tyr Gln Lys Pro Phe 180 185 190 ata gct gtt aat tgt gct gct atc cct
gaa gaa ttg att gaa agc gaa 624 Ile Ala Val Asn Cys Ala Ala Ile Pro
Glu Glu Leu Ile Glu Ser Glu 195 200 205 cta ttt ggt cat gaa aaa ggg
gcc ttt act ggt gcc gat gct tct cgt 672 Leu Phe Gly His Glu Lys Gly
Ala Phe Thr Gly Ala Asp Ala Ser Arg 210 215 220 gca ggt cgt ttt gag
ttg gca cat aaa gga aca tta ttt ctt gat gaa 720 Ala Gly Arg Phe Glu
Leu Ala His Lys Gly Thr Leu Phe Leu Asp Glu 225 230 235 240 ata gga
gat atg agt tta aaa aca caa gca aaa att ttg cgt att ttg 768 Ile Gly
Asp Met Ser Leu Lys Thr Gln Ala Lys Ile Leu Arg Ile Leu 245 250 255
caa gaa caa tgt ttt gaa aaa att ggt agt gtt aga act att aaa gtt 816
Gln Glu Gln Cys Phe Glu Lys Ile Gly Ser Val Arg Thr Ile Lys Val 260
265 270 gat gta aga gtt att gca gca aca aat aag aat ctt gaa gac gct
att 864 Asp Val Arg Val Ile Ala Ala Thr Asn Lys Asn Leu Glu Asp Ala
Ile 275 280 285 agc gat gga aca ttt cgt caa gat ttg tat tat cgc tta
cga gtt gtt 912 Ser Asp Gly Thr Phe Arg Gln Asp Leu Tyr Tyr Arg Leu
Arg Val Val 290 295 300 cca ttg cat ctt ccc cct ctt cgt gaa cgt gat
tct gat att gag cta 960 Pro Leu His Leu Pro Pro Leu Arg Glu Arg Asp
Ser Asp Ile Glu Leu 305 310 315 320 tta tta aat agg ttt gtg att cag
ttg agt aaa cgt tat aga cgt gag 1008 Leu Leu Asn Arg Phe Val Ile
Gln Leu Ser Lys Arg Tyr Arg Arg Glu 325 330 335 ccg cct att ttt tta
gat gag gtc ttc cct gta ttg aaa caa tat tgt 1056 Pro Pro Ile Phe
Leu Asp Glu Val Phe Pro Val Leu Lys Gln Tyr Cys 340 345 350 tgg cca
ggg aat gta aga gaa tta ctt aat ttt gta gaa cga atg gtt 1104 Trp
Pro Gly Asn Val Arg Glu Leu Leu Asn Phe Val Glu Arg Met Val 355 360
365 att ctt tat tca ggg aag aaa gta tgt ttg aca gat cct aag gta aaa
1152 Ile Leu Tyr Ser Gly Lys Lys Val Cys Leu Thr Asp Pro Lys Val
Lys 370 375 380 agc aat tta aaa tat tta ccc aag aaa ttt tct tcc cat
tat aac ttt 1200 Ser Asn Leu Lys Tyr Leu Pro Lys Lys Phe Ser Ser
His Tyr Asn Phe 385 390 395 400 ctt ccc gat ata gat ttt aac cag gct
aaa ata gct ttt gaa cca aaa 1248 Leu Pro Asp Ile Asp Phe Asn Gln
Ala Lys Ile Ala Phe Glu Pro Lys 405 410 415 ttt tta act gaa aaa tta
cat gct tat caa gga aat att acc cga tta 1296 Phe Leu Thr Glu Lys
Leu His Ala Tyr Gln Gly Asn Ile Thr Arg Leu 420 425 430 gca gaa gct
att gga ctt gaa aga agt tat tta tat aga aag cta aaa 1344 Ala Glu
Ala Ile Gly Leu Glu Arg Ser Tyr Leu Tyr Arg Lys Leu Lys 435 440 445
agc tat ggt att tat ctg tct gag tga 1371 Ser Tyr Gly Ile Tyr Leu
Ser Glu 450 455 6 456 PRT Lawsonia intracellularis 6 Met Ser Ala
Arg Ile Leu Ile Ile Asp Asp Glu Asp Ser Ile Arg Phe 1 5 10 15 Ser
Leu Lys Gly Ile Phe Glu Asp Glu Gly His Glu Val Leu Glu Arg 20 25
30 Ala Ser Ala Glu Glu Gly Leu Lys Cys Val Asp Val Glu Ser Pro Asp
35 40 45 Leu Val Phe Leu Asp Ile Trp Leu Pro Gly Met Asp Gly Leu
Met Ala 50 55 60 Leu Asp His Ile Gln Ala Leu His Gln Glu Leu Pro
Val Ile Met Ile 65 70 75 80 Ser Gly His Ala Thr Ile Glu Thr Ala Val
Thr Ala Ile Arg Gln Gly 85 90 95 Ala Tyr Asp Phe Ile Glu Lys Pro
Leu Ser Leu Glu Lys Val Leu Ile 100 105 110 Thr Ala Asn Arg Ala Ile
Glu Thr Val Arg Leu Arg Arg Glu Asn Lys 115 120 125 Leu Leu Arg Thr
Val Leu Pro Glu Glu Ser Glu Phe Ile Gly Gln Ser 130 135 140 Pro Val
Ile Leu Lys Phe Lys Ser Leu Leu Ser Gln Val Ala Pro Thr 145 150 155
160 Asp Ala Trp Val Leu Leu Thr Gly Glu Asn Gly Thr Gly Lys Glu Leu
165 170 175 Ala Ala Gln Ala Leu His Lys Gly Ser Ser Arg Tyr Gln Lys
Pro Phe 180 185 190 Ile Ala Val Asn Cys Ala Ala Ile Pro Glu Glu Leu
Ile Glu Ser Glu 195 200 205 Leu Phe Gly His Glu Lys Gly Ala Phe Thr
Gly Ala Asp Ala Ser Arg 210 215 220 Ala Gly Arg Phe Glu Leu Ala His
Lys Gly Thr Leu Phe Leu Asp Glu 225 230 235 240 Ile Gly Asp Met Ser
Leu Lys Thr Gln Ala Lys Ile Leu Arg Ile Leu 245 250 255 Gln Glu Gln
Cys Phe Glu Lys Ile Gly Ser Val Arg Thr Ile Lys Val 260 265 270 Asp
Val Arg Val Ile Ala Ala Thr Asn Lys Asn Leu Glu Asp Ala Ile 275 280
285 Ser Asp Gly Thr Phe Arg Gln Asp Leu Tyr Tyr Arg Leu Arg Val Val
290 295 300 Pro Leu His Leu Pro Pro Leu Arg Glu Arg Asp Ser Asp Ile
Glu Leu 305 310 315 320 Leu Leu Asn Arg Phe Val Ile Gln Leu Ser Lys
Arg Tyr Arg Arg Glu 325 330 335 Pro Pro Ile Phe Leu Asp Glu Val Phe
Pro Val Leu Lys Gln Tyr Cys 340 345 350 Trp Pro Gly Asn Val Arg Glu
Leu Leu Asn Phe Val Glu Arg Met Val 355 360 365 Ile Leu Tyr Ser Gly
Lys Lys Val Cys Leu Thr Asp Pro Lys Val Lys 370 375 380 Ser Asn Leu
Lys Tyr Leu Pro Lys Lys Phe Ser Ser His Tyr Asn Phe 385 390 395 400
Leu Pro Asp Ile Asp Phe Asn Gln Ala Lys Ile Ala Phe Glu Pro Lys 405
410 415 Phe Leu Thr Glu Lys Leu His Ala Tyr Gln Gly Asn Ile Thr Arg
Leu 420 425 430
Ala Glu Ala Ile Gly Leu Glu Arg Ser Tyr Leu Tyr Arg Lys Leu Lys 435
440 445 Ser Tyr Gly Ile Tyr Leu Ser Glu 450 455 7 412 DNA Lawsonia
intracellularis CDS (1)...(411) 7 aaa caa att gat ata atc att agt
ggg gct acg ata act ctt gaa cgt 48 Lys Gln Ile Asp Ile Ile Ile Ser
Gly Ala Thr Ile Thr Leu Glu Arg 1 5 10 15 aat ctt caa gtc aat ttt
tct aac cca tac cat caa aca gat att gaa 96 Asn Leu Gln Val Asn Phe
Ser Asn Pro Tyr His Gln Thr Asp Ile Glu 20 25 30 gtc ctg gct aat
gca aaa aaa gtt aaa ggg atg aag ttt cca caa gac 144 Val Leu Ala Asn
Ala Lys Lys Val Lys Gly Met Lys Phe Pro Gln Asp 35 40 45 ttt aat
aaa cct gaa gtt ata gtt gct ata cgt aat ggt agt aca gtt 192 Phe Asn
Lys Pro Glu Val Ile Val Ala Ile Arg Asn Gly Ser Thr Val 50 55 60
att act cct gca aag caa ctt ctt cct aaa gca tct ttt aga ctc ttt 240
Ile Thr Pro Ala Lys Gln Leu Leu Pro Lys Ala Ser Phe Arg Leu Phe 65
70 75 80 gat gat gaa gtt gca tct ata aaa gat gta gaa tct gga caa
tca cat 288 Asp Asp Glu Val Ala Ser Ile Lys Asp Val Glu Ser Gly Gln
Ser His 85 90 95 ata tta tta gct tca gca cca tta cca gcg att caa
gct ata aac tca 336 Ile Leu Leu Ala Ser Ala Pro Leu Pro Ala Ile Gln
Ala Ile Asn Ser 100 105 110 aat ggc aac ctt att cgt tta gat aca ctc
ccc att act cat caa tct 384 Asn Gly Asn Leu Ile Arg Leu Asp Thr Leu
Pro Ile Thr His Gln Ser 115 120 125 gta gga ttt gca ata aag aag gga
gat c 412 Val Gly Phe Ala Ile Lys Lys Gly Asp 130 135 8 137 PRT
Lawsonia intracellularis 8 Lys Gln Ile Asp Ile Ile Ile Ser Gly Ala
Thr Ile Thr Leu Glu Arg 1 5 10 15 Asn Leu Gln Val Asn Phe Ser Asn
Pro Tyr His Gln Thr Asp Ile Glu 20 25 30 Val Leu Ala Asn Ala Lys
Lys Val Lys Gly Met Lys Phe Pro Gln Asp 35 40 45 Phe Asn Lys Pro
Glu Val Ile Val Ala Ile Arg Asn Gly Ser Thr Val 50 55 60 Ile Thr
Pro Ala Lys Gln Leu Leu Pro Lys Ala Ser Phe Arg Leu Phe 65 70 75 80
Asp Asp Glu Val Ala Ser Ile Lys Asp Val Glu Ser Gly Gln Ser His 85
90 95 Ile Leu Leu Ala Ser Ala Pro Leu Pro Ala Ile Gln Ala Ile Asn
Ser 100 105 110 Asn Gly Asn Leu Ile Arg Leu Asp Thr Leu Pro Ile Thr
His Gln Ser 115 120 125 Val Gly Phe Ala Ile Lys Lys Gly Asp 130 135
9 849 DNA Lawsonia intracellularis CDS (1)...(846) 9 atg tat att
att att gga tac ttt att gtt att gct tcc att att gga 48 Met Tyr Ile
Ile Ile Gly Tyr Phe Ile Val Ile Ala Ser Ile Ile Gly 1 5 10 15 ggc
tac ctt atg gct aaa ggg aat ctt gct tta ctc ttt caa cct gca 96 Gly
Tyr Leu Met Ala Lys Gly Asn Leu Ala Leu Leu Phe Gln Pro Ala 20 25
30 gaa ctt gtt atc att att ggg gca gca tta ggt gct ttt ttt gct tca
144 Glu Leu Val Ile Ile Ile Gly Ala Ala Leu Gly Ala Phe Phe Ala Ser
35 40 45 cag acg aaa tat tca ttt act ctg gtc att aaa aat tta tca
cac att 192 Gln Thr Lys Tyr Ser Phe Thr Leu Val Ile Lys Asn Leu Ser
His Ile 50 55 60 ttt ggc gat cca aac agt aca aaa ata aaa tac ctt
gaa aca ctt gcc 240 Phe Gly Asp Pro Asn Ser Thr Lys Ile Lys Tyr Leu
Glu Thr Leu Ala 65 70 75 80 ctt ctc tat gga ctt ttc tta aaa atg aat
aga gaa ggt gtc att agt 288 Leu Leu Tyr Gly Leu Phe Leu Lys Met Asn
Arg Glu Gly Val Ile Ser 85 90 95 ata gaa agt gat ata gaa aaa cct
gaa tca agt cct atc ttt agt aaa 336 Ile Glu Ser Asp Ile Glu Lys Pro
Glu Ser Ser Pro Ile Phe Ser Lys 100 105 110 tac cct aca att gta aaa
gat act aaa gtt gtt gcc ttt att gca gat 384 Tyr Pro Thr Ile Val Lys
Asp Thr Lys Val Val Ala Phe Ile Ala Asp 115 120 125 aca tta cga gtt
tat ctg aca aca ggt gca cca gaa gat ata gat aac 432 Thr Leu Arg Val
Tyr Leu Thr Thr Gly Ala Pro Glu Asp Ile Asp Asn 130 135 140 ctc atg
gaa tct gac atg aaa att aca cac gaa gaa gaa tta tta cct 480 Leu Met
Glu Ser Asp Met Lys Ile Thr His Glu Glu Glu Leu Leu Pro 145 150 155
160 gca cat tcc atc agc cat atg gca gag tcg cta cca gga atg ggt att
528 Ala His Ser Ile Ser His Met Ala Glu Ser Leu Pro Gly Met Gly Ile
165 170 175 gtt gct gca gta tta ggt gtt gtt att acc atg gga aaa att
aat gag 576 Val Ala Ala Val Leu Gly Val Val Ile Thr Met Gly Lys Ile
Asn Glu 180 185 190 cct cca gaa gtc ctt ggg cat tat att gga gca gct
ttg gtt ggt aca 624 Pro Pro Glu Val Leu Gly His Tyr Ile Gly Ala Ala
Leu Val Gly Thr 195 200 205 ttt ata ggt att ctt ttc tgt tat ggt ttt
ttt gga cct atg ggt tca 672 Phe Ile Gly Ile Leu Phe Cys Tyr Gly Phe
Phe Gly Pro Met Gly Ser 210 215 220 aag ctt gaa acc tct gca gaa gaa
gca cat ttt tat tat aat tcc att 720 Lys Leu Glu Thr Ser Ala Glu Glu
Ala His Phe Tyr Tyr Asn Ser Ile 225 230 235 240 aaa gaa gct gtt gca
gct gct atc cga ggt tct aca cca atg ata gca 768 Lys Glu Ala Val Ala
Ala Ala Ile Arg Gly Ser Thr Pro Met Ile Ala 245 250 255 gta gaa tat
gga aga cgt gcc ata cct aat aca ttt cgt cca tca ttt 816 Val Glu Tyr
Gly Arg Arg Ala Ile Pro Asn Thr Phe Arg Pro Ser Phe 260 265 270 tcg
gaa atg gaa gaa cgt cta aaa aca gga taa 849 Ser Glu Met Glu Glu Arg
Leu Lys Thr Gly 275 280 10 282 PRT Lawsonia intracellularis 10 Met
Tyr Ile Ile Ile Gly Tyr Phe Ile Val Ile Ala Ser Ile Ile Gly 1 5 10
15 Gly Tyr Leu Met Ala Lys Gly Asn Leu Ala Leu Leu Phe Gln Pro Ala
20 25 30 Glu Leu Val Ile Ile Ile Gly Ala Ala Leu Gly Ala Phe Phe
Ala Ser 35 40 45 Gln Thr Lys Tyr Ser Phe Thr Leu Val Ile Lys Asn
Leu Ser His Ile 50 55 60 Phe Gly Asp Pro Asn Ser Thr Lys Ile Lys
Tyr Leu Glu Thr Leu Ala 65 70 75 80 Leu Leu Tyr Gly Leu Phe Leu Lys
Met Asn Arg Glu Gly Val Ile Ser 85 90 95 Ile Glu Ser Asp Ile Glu
Lys Pro Glu Ser Ser Pro Ile Phe Ser Lys 100 105 110 Tyr Pro Thr Ile
Val Lys Asp Thr Lys Val Val Ala Phe Ile Ala Asp 115 120 125 Thr Leu
Arg Val Tyr Leu Thr Thr Gly Ala Pro Glu Asp Ile Asp Asn 130 135 140
Leu Met Glu Ser Asp Met Lys Ile Thr His Glu Glu Glu Leu Leu Pro 145
150 155 160 Ala His Ser Ile Ser His Met Ala Glu Ser Leu Pro Gly Met
Gly Ile 165 170 175 Val Ala Ala Val Leu Gly Val Val Ile Thr Met Gly
Lys Ile Asn Glu 180 185 190 Pro Pro Glu Val Leu Gly His Tyr Ile Gly
Ala Ala Leu Val Gly Thr 195 200 205 Phe Ile Gly Ile Leu Phe Cys Tyr
Gly Phe Phe Gly Pro Met Gly Ser 210 215 220 Lys Leu Glu Thr Ser Ala
Glu Glu Ala His Phe Tyr Tyr Asn Ser Ile 225 230 235 240 Lys Glu Ala
Val Ala Ala Ala Ile Arg Gly Ser Thr Pro Met Ile Ala 245 250 255 Val
Glu Tyr Gly Arg Arg Ala Ile Pro Asn Thr Phe Arg Pro Ser Phe 260 265
270 Ser Glu Met Glu Glu Arg Leu Lys Thr Gly 275 280 11 717 DNA
Lawsonia intracellularis CDS (1)...(714) 11 atg tct ggc tca tgg aaa
gtg gct tat gca gac ttt gtt aca gct atg 48 Met Ser Gly Ser Trp Lys
Val Ala Tyr Ala Asp Phe Val Thr Ala Met 1 5 10 15 atg gct ttc ttt
cta ctg atg tgg att ctt gca atg aca ccc cct gag 96 Met Ala Phe Phe
Leu Leu Met Trp Ile Leu Ala Met Thr Pro Pro Glu 20 25 30 gtt aaa
gaa ggt ctt gct gca tat ttt tct tca tct gat gct aca ttt 144 Val Lys
Glu Gly Leu Ala Ala Tyr Phe Ser Ser Ser Asp Ala Thr Phe 35 40 45
aaa aca cct gat agt tcg cca atc tct aac aat cct ctt atc aac caa 192
Lys Thr Pro Asp Ser Ser Pro Ile Ser Asn Asn Pro Leu Ile Asn Gln 50
55 60 ata gat aaa ctt gat act cga caa tta aaa att aat gaa aca gaa
caa 240 Ile Asp Lys Leu Asp Thr Arg Gln Leu Lys Ile Asn Glu Thr Glu
Gln 65 70 75 80 tct cat tat gct ctt gct aat aaa tta aaa aaa atg tta
atg gct gat 288 Ser His Tyr Ala Leu Ala Asn Lys Leu Lys Lys Met Leu
Met Ala Asp 85 90 95 gct atc cca cag tca gca aca gga ata agt gct
gac gat gtt ggt gta 336 Ala Ile Pro Gln Ser Ala Thr Gly Ile Ser Ala
Asp Asp Val Gly Val 100 105 110 tta tta cgt gta aat tct aat tcc acg
ttt ttt cct ggt aca gca act 384 Leu Leu Arg Val Asn Ser Asn Ser Thr
Phe Phe Pro Gly Thr Ala Thr 115 120 125 ctt aca ccc gaa ggg aaa aaa
gtt atg gga act gtt tta gcc gtt ctc 432 Leu Thr Pro Glu Gly Lys Lys
Val Met Gly Thr Val Leu Ala Val Leu 130 135 140 cgt gaa tat aat ctt
tac ctt gtg ata cgt ggc cat gct gat att ggt 480 Arg Glu Tyr Asn Leu
Tyr Leu Val Ile Arg Gly His Ala Asp Ile Gly 145 150 155 160 gaa ata
aca aaa ggc agc cct ttt gct tct aac tgg gaa ctt tca gga 528 Glu Ile
Thr Lys Gly Ser Pro Phe Ala Ser Asn Trp Glu Leu Ser Gly 165 170 175
gct cgt gca gct gca gct gca cag tat ctt gta gag cac ggg ata aag 576
Ala Arg Ala Ala Ala Ala Ala Gln Tyr Leu Val Glu His Gly Ile Lys 180
185 190 gct tca cga att cgc tct gta gga tat gca gat aca aga cct cta
gaa 624 Ala Ser Arg Ile Arg Ser Val Gly Tyr Ala Asp Thr Arg Pro Leu
Glu 195 200 205 cct agt tct cct gaa gga agt aca aaa aat cgt cgt ata
gaa ttc tat 672 Pro Ser Ser Pro Glu Gly Ser Thr Lys Asn Arg Arg Ile
Glu Phe Tyr 210 215 220 ttt cat cgg cca gaa gtt atg tct tat ggc gtt
gta tat taa 714 Phe His Arg Pro Glu Val Met Ser Tyr Gly Val Val Tyr
* 225 230 235 tag 717 12 237 PRT Lawsonia intracellularis 12 Met
Ser Gly Ser Trp Lys Val Ala Tyr Ala Asp Phe Val Thr Ala Met 1 5 10
15 Met Ala Phe Phe Leu Leu Met Trp Ile Leu Ala Met Thr Pro Pro Glu
20 25 30 Val Lys Glu Gly Leu Ala Ala Tyr Phe Ser Ser Ser Asp Ala
Thr Phe 35 40 45 Lys Thr Pro Asp Ser Ser Pro Ile Ser Asn Asn Pro
Leu Ile Asn Gln 50 55 60 Ile Asp Lys Leu Asp Thr Arg Gln Leu Lys
Ile Asn Glu Thr Glu Gln 65 70 75 80 Ser His Tyr Ala Leu Ala Asn Lys
Leu Lys Lys Met Leu Met Ala Asp 85 90 95 Ala Ile Pro Gln Ser Ala
Thr Gly Ile Ser Ala Asp Asp Val Gly Val 100 105 110 Leu Leu Arg Val
Asn Ser Asn Ser Thr Phe Phe Pro Gly Thr Ala Thr 115 120 125 Leu Thr
Pro Glu Gly Lys Lys Val Met Gly Thr Val Leu Ala Val Leu 130 135 140
Arg Glu Tyr Asn Leu Tyr Leu Val Ile Arg Gly His Ala Asp Ile Gly 145
150 155 160 Glu Ile Thr Lys Gly Ser Pro Phe Ala Ser Asn Trp Glu Leu
Ser Gly 165 170 175 Ala Arg Ala Ala Ala Ala Ala Gln Tyr Leu Val Glu
His Gly Ile Lys 180 185 190 Ala Ser Arg Ile Arg Ser Val Gly Tyr Ala
Asp Thr Arg Pro Leu Glu 195 200 205 Pro Ser Ser Pro Glu Gly Ser Thr
Lys Asn Arg Arg Ile Glu Phe Tyr 210 215 220 Phe His Arg Pro Glu Val
Met Ser Tyr Gly Val Val Tyr 225 230 235 13 1047 DNA Lawsonia
intracellularis CDS (1)...(1044) 13 atg ata atc ctt tta gga act gtt
ttt ctt att gtt ctt atc tct gca 48 Met Ile Ile Leu Leu Gly Thr Val
Phe Leu Ile Val Leu Ile Ser Ala 1 5 10 15 tta tgc tca atg atg gaa
gct gct ata tac tct atc cct att act tat 96 Leu Cys Ser Met Met Glu
Ala Ala Ile Tyr Ser Ile Pro Ile Thr Tyr 20 25 30 att gaa cac ctt
cgt gaa cag gga agc aaa aaa gga gaa aaa ctt tat 144 Ile Glu His Leu
Arg Glu Gln Gly Ser Lys Lys Gly Glu Lys Leu Tyr 35 40 45 tat tta
cat agt aat att gat cag cct att aca gcc gta tta ata ttg 192 Tyr Leu
His Ser Asn Ile Asp Gln Pro Ile Thr Ala Val Leu Ile Leu 50 55 60
aat act ata gca aat act gct gga gct gcc ctt gct gga gca att gct 240
Asn Thr Ile Ala Asn Thr Ala Gly Ala Ala Leu Ala Gly Ala Ile Ala 65
70 75 80 aca aca aca ctt cat gaa tct act aag cct ttc ttt gca gca
atc ctc 288 Thr Thr Thr Leu His Glu Ser Thr Lys Pro Phe Phe Ala Ala
Ile Leu 85 90 95 acc ttg ctt att tta gct ttt ggg gaa att ata cct
aaa aca cta ggt 336 Thr Leu Leu Ile Leu Ala Phe Gly Glu Ile Ile Pro
Lys Thr Leu Gly 100 105 110 gtt gct tac tct aaa cgt att gct ata att
ctc ctt aat cct ctc tct 384 Val Ala Tyr Ser Lys Arg Ile Ala Ile Ile
Leu Leu Asn Pro Leu Ser 115 120 125 att ctt ata gtt act tta aaa ccc
ctt att atg ctt tca agc tac tta 432 Ile Leu Ile Val Thr Leu Lys Pro
Leu Ile Met Leu Ser Ser Tyr Leu 130 135 140 aca cga ctt gtt tca cct
cga aaa cgt cct aca gtt aca gaa gat gac 480 Thr Arg Leu Val Ser Pro
Arg Lys Arg Pro Thr Val Thr Glu Asp Asp 145 150 155 160 atc cgt gca
ctt aca agt ctt tcc aga gag tct ggt cgt att aag cca 528 Ile Arg Ala
Leu Thr Ser Leu Ser Arg Glu Ser Gly Arg Ile Lys Pro 165 170 175 tat
gaa gaa cat gtc ata aaa aat atc ctt agt ctt gat tta aaa tat 576 Tyr
Glu Glu His Val Ile Lys Asn Ile Leu Ser Leu Asp Leu Lys Tyr 180 185
190 gct cat gaa att atg act ccc aga act atg gtc ttt tca ctt cat gaa
624 Ala His Glu Ile Met Thr Pro Arg Thr Met Val Phe Ser Leu His Glu
195 200 205 aac ctt act gtc tct gaa gct tat agc aac ccc aaa ata tgg
aac tat 672 Asn Leu Thr Val Ser Glu Ala Tyr Ser Asn Pro Lys Ile Trp
Asn Tyr 210 215 220 agt cgc atc cct act tat gga gaa aat aac gaa gac
att act ggc att 720 Ser Arg Ile Pro Thr Tyr Gly Glu Asn Asn Glu Asp
Ile Thr Gly Ile 225 230 235 240 atc caa cga tat gaa att gga cga tat
atg acc aat gga gaa aca gaa 768 Ile Gln Arg Tyr Glu Ile Gly Arg Tyr
Met Thr Asn Gly Glu Thr Glu 245 250 255 aaa aaa ctt tta gaa att atg
caa cca gca aaa ttt gtc ctt gaa agt 816 Lys Lys Leu Leu Glu Ile Met
Gln Pro Ala Lys Phe Val Leu Glu Ser 260 265 270 caa act gta gat cat
tta ctt ctt gca ttt tta gaa gaa aga caa cat 864 Gln Thr Val Asp His
Leu Leu Leu Ala Phe Leu Glu Glu Arg Gln His 275 280 285 ctt ttt att
gta ctt gat gag tat ggg gga tta tct ggt gtt gtt tcc 912 Leu Phe Ile
Val Leu Asp Glu Tyr Gly Gly Leu Ser Gly Val Val Ser 290 295 300 tta
gaa gat gta tta gaa act atg ctt gga aga gaa att gtt gat gaa 960 Leu
Glu Asp Val Leu Glu Thr Met Leu Gly Arg Glu Ile Val Asp Glu 305 310
315 320 agt gat aca aca cct gat ctt aga gca ctt gca aaa aaa aga cat
agt 1008 Ser Asp Thr Thr Pro Asp Leu Arg Ala Leu Ala Lys Lys Arg
His Ser 325 330 335 gca tta atc caa aat aat aaa aat act ctt tta aaa
taa 1047 Ala Leu Ile Gln Asn Asn Lys Asn Thr Leu Leu Lys 340 345 14
348 PRT Lawsonia intracellularis 14 Met Ile Ile Leu Leu Gly Thr Val
Phe Leu Ile Val Leu Ile Ser Ala 1 5 10 15 Leu Cys Ser Met Met Glu
Ala Ala Ile Tyr Ser Ile Pro Ile Thr Tyr 20 25 30 Ile Glu His Leu
Arg Glu Gln Gly Ser Lys Lys Gly Glu Lys Leu Tyr 35 40 45 Tyr Leu
His Ser Asn Ile Asp Gln Pro Ile Thr Ala Val Leu Ile Leu 50 55 60
Asn Thr Ile Ala Asn Thr Ala Gly Ala Ala Leu Ala Gly Ala Ile Ala 65
70 75 80 Thr Thr Thr Leu His Glu
Ser Thr Lys Pro Phe Phe Ala Ala Ile Leu 85 90 95 Thr Leu Leu Ile
Leu Ala Phe Gly Glu Ile Ile Pro Lys Thr Leu Gly 100 105 110 Val Ala
Tyr Ser Lys Arg Ile Ala Ile Ile Leu Leu Asn Pro Leu Ser 115 120 125
Ile Leu Ile Val Thr Leu Lys Pro Leu Ile Met Leu Ser Ser Tyr Leu 130
135 140 Thr Arg Leu Val Ser Pro Arg Lys Arg Pro Thr Val Thr Glu Asp
Asp 145 150 155 160 Ile Arg Ala Leu Thr Ser Leu Ser Arg Glu Ser Gly
Arg Ile Lys Pro 165 170 175 Tyr Glu Glu His Val Ile Lys Asn Ile Leu
Ser Leu Asp Leu Lys Tyr 180 185 190 Ala His Glu Ile Met Thr Pro Arg
Thr Met Val Phe Ser Leu His Glu 195 200 205 Asn Leu Thr Val Ser Glu
Ala Tyr Ser Asn Pro Lys Ile Trp Asn Tyr 210 215 220 Ser Arg Ile Pro
Thr Tyr Gly Glu Asn Asn Glu Asp Ile Thr Gly Ile 225 230 235 240 Ile
Gln Arg Tyr Glu Ile Gly Arg Tyr Met Thr Asn Gly Glu Thr Glu 245 250
255 Lys Lys Leu Leu Glu Ile Met Gln Pro Ala Lys Phe Val Leu Glu Ser
260 265 270 Gln Thr Val Asp His Leu Leu Leu Ala Phe Leu Glu Glu Arg
Gln His 275 280 285 Leu Phe Ile Val Leu Asp Glu Tyr Gly Gly Leu Ser
Gly Val Val Ser 290 295 300 Leu Glu Asp Val Leu Glu Thr Met Leu Gly
Arg Glu Ile Val Asp Glu 305 310 315 320 Ser Asp Thr Thr Pro Asp Leu
Arg Ala Leu Ala Lys Lys Arg His Ser 325 330 335 Ala Leu Ile Gln Asn
Asn Lys Asn Thr Leu Leu Lys 340 345 15 1812 DNA Lawsonia
intracellularis CDS (1)...(1809) 15 atg caa aaa gta tgt tat ttt ttt
ctt ata acc ttt ttc tac ttt ttc 48 Met Gln Lys Val Cys Tyr Phe Phe
Leu Ile Thr Phe Phe Tyr Phe Phe 1 5 10 15 ata aca gaa aat tat ctc
ttt gct aca tca att acc act tcc aca att 96 Ile Thr Glu Asn Tyr Leu
Phe Ala Thr Ser Ile Thr Thr Ser Thr Ile 20 25 30 aac caa caa cat
ata gca tat aca gtt act ttt acc tct cca gaa aat 144 Asn Gln Gln His
Ile Ala Tyr Thr Val Thr Phe Thr Ser Pro Glu Asn 35 40 45 cct aat
ctt gca aca gag atg gaa aca cat agt gaa tta gta aag ctt 192 Pro Asn
Leu Ala Thr Glu Met Glu Thr His Ser Glu Leu Val Lys Leu 50 55 60
gca aat caa tct tta gat agt aaa ata ggt tta aat tta cgt gtt aaa 240
Ala Asn Gln Ser Leu Asp Ser Lys Ile Gly Leu Asn Leu Arg Val Lys 65
70 75 80 gaa gat ata agt aca gca caa aaa att ctt gac tcg aat ggt
tat tat 288 Glu Asp Ile Ser Thr Ala Gln Lys Ile Leu Asp Ser Asn Gly
Tyr Tyr 85 90 95 agt gga agt gtc gag gga aag att gac tgg cag acg
aac cct att agt 336 Ser Gly Ser Val Glu Gly Lys Ile Asp Trp Gln Thr
Asn Pro Ile Ser 100 105 110 atc caa atc caa ttt aaa cca aat gta caa
tat aaa ata aat aca ata 384 Ile Gln Ile Gln Phe Lys Pro Asn Val Gln
Tyr Lys Ile Asn Thr Ile 115 120 125 cat atc caa tac ctt gat agt gaa
ctt gca tat ctc cct ctt tcc tta 432 His Ile Gln Tyr Leu Asp Ser Glu
Leu Ala Tyr Leu Pro Leu Ser Leu 130 135 140 gaa gaa ttc aat ctc tct
aaa ggt aat cct gct ctt gct gtt aat atc 480 Glu Glu Phe Asn Leu Ser
Lys Gly Asn Pro Ala Leu Ala Val Asn Ile 145 150 155 160 cta tcc tct
gta agt agc ctc atg caa tat ata cat aat aat gga tat 528 Leu Ser Ser
Val Ser Ser Leu Met Gln Tyr Ile His Asn Asn Gly Tyr 165 170 175 cca
tta gcc aaa ata aaa aaa act caa tac ata att aat cgg atg gat 576 Pro
Leu Ala Lys Ile Lys Lys Thr Gln Tyr Ile Ile Asn Arg Met Asp 180 185
190 tat aca ttt gat att gat tta gta ata aga caa gga ccg tta ctc cat
624 Tyr Thr Phe Asp Ile Asp Leu Val Ile Arg Gln Gly Pro Leu Leu His
195 200 205 atg ggt aaa gta caa cct caa cat aat ctc aat att tca aca
ata ttc 672 Met Gly Lys Val Gln Pro Gln His Asn Leu Asn Ile Ser Thr
Ile Phe 210 215 220 cta aat aaa att gct aca tgg aag gaa gga agg gta
tgg aac aat gca 720 Leu Asn Lys Ile Ala Thr Trp Lys Glu Gly Arg Val
Trp Asn Asn Ala 225 230 235 240 ctc ctt gat tct tat cga aca cgg ctt
caa caa aca ggc ctt ttc agt 768 Leu Leu Asp Ser Tyr Arg Thr Arg Leu
Gln Gln Thr Gly Leu Phe Ser 245 250 255 tct ata act ctc aat cca agg
aat caa aaa gaa caa aat ggt aac acc 816 Ser Ile Thr Leu Asn Pro Arg
Asn Gln Lys Glu Gln Asn Gly Asn Thr 260 265 270 tct ata gaa ctt gtt
gca aca gaa gcc cct cca agg act att agt ggt 864 Ser Ile Glu Leu Val
Ala Thr Glu Ala Pro Pro Arg Thr Ile Ser Gly 275 280 285 ggc tta caa
tac tct tct gat caa ggt att ggt gca cgt ggg act tgg 912 Gly Leu Gln
Tyr Ser Ser Asp Gln Gly Ile Gly Ala Arg Gly Thr Trp 290 295 300 gaa
cat cga aat gtt ttt ggt aat gga gaa ctt ttt cgt ata aca gca 960 Glu
His Arg Asn Val Phe Gly Asn Gly Glu Leu Phe Arg Ile Thr Ala 305 310
315 320 cca ata agt cga gat gat caa aaa att atg gca aac ttc caa aaa
cca 1008 Pro Ile Ser Arg Asp Asp Gln Lys Ile Met Ala Asn Phe Gln
Lys Pro 325 330 335 gcc ttt ggc cgt cca aat caa tca tta att agt gaa
gca caa ctt aaa 1056 Ala Phe Gly Arg Pro Asn Gln Ser Leu Ile Ser
Glu Ala Gln Leu Lys 340 345 350 aaa gaa aat aca aaa agt tac aaa caa
caa ctt gca tct att gct tta 1104 Lys Glu Asn Thr Lys Ser Tyr Lys
Gln Gln Leu Ala Ser Ile Ala Leu 355 360 365 gga att gaa cga caa ttt
aat aga cgt tgg ttt ggt agt agc agt ctt 1152 Gly Ile Glu Arg Gln
Phe Asn Arg Arg Trp Phe Gly Ser Ser Ser Leu 370 375 380 tca gtt gat
aca gga ttt atg gat gat cga gat tct ata aaa aaa ata 1200 Ser Val
Asp Thr Gly Phe Met Asp Asp Arg Asp Ser Ile Lys Lys Ile 385 390 395
400 ttt act ctt ttt ggc atc ccc tta tca ata aca agg gat agt tct aaa
1248 Phe Thr Leu Phe Gly Ile Pro Leu Ser Ile Thr Arg Asp Ser Ser
Lys 405 410 415 gat cct ctt aat cct atc caa gga aca aaa gct acc tta
aat gtt act 1296 Asp Pro Leu Asn Pro Ile Gln Gly Thr Lys Ala Thr
Leu Asn Val Thr 420 425 430 cct tat att ggt aaa tat aaa aaa aag att
ttg act tta cgt agt cgg 1344 Pro Tyr Ile Gly Lys Tyr Lys Lys Lys
Ile Leu Thr Leu Arg Ser Arg 435 440 445 ttt gat ttt agc ttt tac ata
gac gtt ctt aaa aca ggg aaa ctt atc 1392 Phe Asp Phe Ser Phe Tyr
Ile Asp Val Leu Lys Thr Gly Lys Leu Ile 450 455 460 ttg gct aac aaa
ata gca ata ggt tcc ctc cta ggg aaa gat ata gaa 1440 Leu Ala Asn
Lys Ile Ala Ile Gly Ser Leu Leu Gly Lys Asp Ile Glu 465 470 475 480
aac tat cct gca ata cta agg ttt tat gct ggg ggt ggt ggt agt gta
1488 Asn Tyr Pro Ala Ile Leu Arg Phe Tyr Ala Gly Gly Gly Gly Ser
Val 485 490 495 aga ggg tat gac tat caa tca ttg gga cca aaa aat aaa
tat ggg gat 1536 Arg Gly Tyr Asp Tyr Gln Ser Leu Gly Pro Lys Asn
Lys Tyr Gly Asp 500 505 510 gct att gga gga ctt tct ttt tca act att
agt ttt gaa tta cga tta 1584 Ala Ile Gly Gly Leu Ser Phe Ser Thr
Ile Ser Phe Glu Leu Arg Leu 515 520 525 aaa ata aca gaa tcc att ggc
att gtg cca att tat tgg atg ggg gaa 1632 Lys Ile Thr Glu Ser Ile
Gly Ile Val Pro Ile Tyr Trp Met Gly Glu 530 535 540 tat tta cga aaa
aaa aat ttc ctg act tta aaa aaa tca ata tat tgg 1680 Tyr Leu Arg
Lys Lys Asn Phe Leu Thr Leu Lys Lys Ser Ile Tyr Trp 545 550 555 560
ggg gta ggc ctg ggg cta cga tat tat aca agt ttt gcc ccc ata cgt
1728 Gly Val Gly Leu Gly Leu Arg Tyr Tyr Thr Ser Phe Ala Pro Ile
Arg 565 570 575 tta gat ata gca act cca ctt caa gat aga agc cat aat
aaa cac ttt 1776 Leu Asp Ile Ala Thr Pro Leu Gln Asp Arg Ser His
Asn Lys His Phe 580 585 590 caa ctt tat att agt att ggg caa gca ttc
taa tga 1812 Gln Leu Tyr Ile Ser Ile Gly Gln Ala Phe * 595 600 16
602 PRT Lawsonia intracellularis 16 Met Gln Lys Val Cys Tyr Phe Phe
Leu Ile Thr Phe Phe Tyr Phe Phe 1 5 10 15 Ile Thr Glu Asn Tyr Leu
Phe Ala Thr Ser Ile Thr Thr Ser Thr Ile 20 25 30 Asn Gln Gln His
Ile Ala Tyr Thr Val Thr Phe Thr Ser Pro Glu Asn 35 40 45 Pro Asn
Leu Ala Thr Glu Met Glu Thr His Ser Glu Leu Val Lys Leu 50 55 60
Ala Asn Gln Ser Leu Asp Ser Lys Ile Gly Leu Asn Leu Arg Val Lys 65
70 75 80 Glu Asp Ile Ser Thr Ala Gln Lys Ile Leu Asp Ser Asn Gly
Tyr Tyr 85 90 95 Ser Gly Ser Val Glu Gly Lys Ile Asp Trp Gln Thr
Asn Pro Ile Ser 100 105 110 Ile Gln Ile Gln Phe Lys Pro Asn Val Gln
Tyr Lys Ile Asn Thr Ile 115 120 125 His Ile Gln Tyr Leu Asp Ser Glu
Leu Ala Tyr Leu Pro Leu Ser Leu 130 135 140 Glu Glu Phe Asn Leu Ser
Lys Gly Asn Pro Ala Leu Ala Val Asn Ile 145 150 155 160 Leu Ser Ser
Val Ser Ser Leu Met Gln Tyr Ile His Asn Asn Gly Tyr 165 170 175 Pro
Leu Ala Lys Ile Lys Lys Thr Gln Tyr Ile Ile Asn Arg Met Asp 180 185
190 Tyr Thr Phe Asp Ile Asp Leu Val Ile Arg Gln Gly Pro Leu Leu His
195 200 205 Met Gly Lys Val Gln Pro Gln His Asn Leu Asn Ile Ser Thr
Ile Phe 210 215 220 Leu Asn Lys Ile Ala Thr Trp Lys Glu Gly Arg Val
Trp Asn Asn Ala 225 230 235 240 Leu Leu Asp Ser Tyr Arg Thr Arg Leu
Gln Gln Thr Gly Leu Phe Ser 245 250 255 Ser Ile Thr Leu Asn Pro Arg
Asn Gln Lys Glu Gln Asn Gly Asn Thr 260 265 270 Ser Ile Glu Leu Val
Ala Thr Glu Ala Pro Pro Arg Thr Ile Ser Gly 275 280 285 Gly Leu Gln
Tyr Ser Ser Asp Gln Gly Ile Gly Ala Arg Gly Thr Trp 290 295 300 Glu
His Arg Asn Val Phe Gly Asn Gly Glu Leu Phe Arg Ile Thr Ala 305 310
315 320 Pro Ile Ser Arg Asp Asp Gln Lys Ile Met Ala Asn Phe Gln Lys
Pro 325 330 335 Ala Phe Gly Arg Pro Asn Gln Ser Leu Ile Ser Glu Ala
Gln Leu Lys 340 345 350 Lys Glu Asn Thr Lys Ser Tyr Lys Gln Gln Leu
Ala Ser Ile Ala Leu 355 360 365 Gly Ile Glu Arg Gln Phe Asn Arg Arg
Trp Phe Gly Ser Ser Ser Leu 370 375 380 Ser Val Asp Thr Gly Phe Met
Asp Asp Arg Asp Ser Ile Lys Lys Ile 385 390 395 400 Phe Thr Leu Phe
Gly Ile Pro Leu Ser Ile Thr Arg Asp Ser Ser Lys 405 410 415 Asp Pro
Leu Asn Pro Ile Gln Gly Thr Lys Ala Thr Leu Asn Val Thr 420 425 430
Pro Tyr Ile Gly Lys Tyr Lys Lys Lys Ile Leu Thr Leu Arg Ser Arg 435
440 445 Phe Asp Phe Ser Phe Tyr Ile Asp Val Leu Lys Thr Gly Lys Leu
Ile 450 455 460 Leu Ala Asn Lys Ile Ala Ile Gly Ser Leu Leu Gly Lys
Asp Ile Glu 465 470 475 480 Asn Tyr Pro Ala Ile Leu Arg Phe Tyr Ala
Gly Gly Gly Gly Ser Val 485 490 495 Arg Gly Tyr Asp Tyr Gln Ser Leu
Gly Pro Lys Asn Lys Tyr Gly Asp 500 505 510 Ala Ile Gly Gly Leu Ser
Phe Ser Thr Ile Ser Phe Glu Leu Arg Leu 515 520 525 Lys Ile Thr Glu
Ser Ile Gly Ile Val Pro Ile Tyr Trp Met Gly Glu 530 535 540 Tyr Leu
Arg Lys Lys Asn Phe Leu Thr Leu Lys Lys Ser Ile Tyr Trp 545 550 555
560 Gly Val Gly Leu Gly Leu Arg Tyr Tyr Thr Ser Phe Ala Pro Ile Arg
565 570 575 Leu Asp Ile Ala Thr Pro Leu Gln Asp Arg Ser His Asn Lys
His Phe 580 585 590 Gln Leu Tyr Ile Ser Ile Gly Gln Ala Phe 595 600
17 4149 DNA Lawsonia intracellularis CDS (1)...(4146) 17 atg aat
aac aca aaa ata ctt tct aag tta ctc tat acc ctc tta gga 48 Met Asn
Asn Thr Lys Ile Leu Ser Lys Leu Leu Tyr Thr Leu Leu Gly 1 5 10 15
gca ttt acg tta ttt tta gga ctt att att aca ggc att ctt ttt ata 96
Ala Phe Thr Leu Phe Leu Gly Leu Ile Ile Thr Gly Ile Leu Phe Ile 20
25 30 cgg acc tct aca ggc att gct tgg att aaa aat aca gtt tct tct
tta 144 Arg Thr Ser Thr Gly Ile Ala Trp Ile Lys Asn Thr Val Ser Ser
Leu 35 40 45 ctt caa caa caa gga att ata cta caa gta tct tca att
att gga cca 192 Leu Gln Gln Gln Gly Ile Ile Leu Gln Val Ser Ser Ile
Ile Gly Pro 50 55 60 ttc cca gaa caa att act att aat gaa ctt agc
ctt agt gat gtg aat 240 Phe Pro Glu Gln Ile Thr Ile Asn Glu Leu Ser
Leu Ser Asp Val Asn 65 70 75 80 gga act tac ctt aca ata tct aac tta
gaa atc caa tca aac tta tgg 288 Gly Thr Tyr Leu Thr Ile Ser Asn Leu
Glu Ile Gln Ser Asn Leu Trp 85 90 95 gct tta ttc aaa ggt caa ctt
gaa att ctg tct ttt gaa ctt aat gat 336 Ala Leu Phe Lys Gly Gln Leu
Glu Ile Leu Ser Phe Glu Leu Asn Asp 100 105 110 ctt gta tta tat cgc
tta ccc tca aat aat aat cta aaa aaa tca tct 384 Leu Val Leu Tyr Arg
Leu Pro Ser Asn Asn Asn Leu Lys Lys Ser Ser 115 120 125 aca agt ttt
gtg tta cct cac ata tca ttt gat tta act cca tgg tgg 432 Thr Ser Phe
Val Leu Pro His Ile Ser Phe Asp Leu Thr Pro Trp Trp 130 135 140 act
gaa cat att cgt att caa aac atc cat att aac aat aca caa ctt 480 Thr
Glu His Ile Arg Ile Gln Asn Ile His Ile Asn Asn Thr Gln Leu 145 150
155 160 tcc tct gat att ata ggt att cca ttg gta tta tcc ctt gag ggt
gat 528 Ser Ser Asp Ile Ile Gly Ile Pro Leu Val Leu Ser Leu Glu Gly
Asp 165 170 175 ggt aca tta aca aat tgg aat gga aca ttt caa cta tcc
tct tct aac 576 Gly Thr Leu Thr Asn Trp Asn Gly Thr Phe Gln Leu Ser
Ser Ser Asn 180 185 190 aaa aca aaa att ata gga acg ctt cgt tac caa
ggg aat aag aca caa 624 Lys Thr Lys Ile Ile Gly Thr Leu Arg Tyr Gln
Gly Asn Lys Thr Gln 195 200 205 ttt ttt gaa tat gtt cat cct aca cgg
ata gta aca cta gag ata gac 672 Phe Phe Glu Tyr Val His Pro Thr Arg
Ile Val Thr Leu Glu Ile Asp 210 215 220 agc gta gct gat aaa aag tca
tat aat aat agt atc ctt gaa caa cct 720 Ser Val Ala Asp Lys Lys Ser
Tyr Asn Asn Ser Ile Leu Glu Gln Pro 225 230 235 240 cta cat tta cac
ctt tct att tat cct gaa cat aat aga att atc tta 768 Leu His Leu His
Leu Ser Ile Tyr Pro Glu His Asn Arg Ile Ile Leu 245 250 255 cac tca
tta cta gct gaa tat ggt agc tgg tta ctt aca tca gaa agt 816 His Ser
Leu Leu Ala Glu Tyr Gly Ser Trp Leu Leu Thr Ser Glu Ser 260 265 270
att gaa gta tct aat gag caa tta aaa gga aat att tta tta aaa tat 864
Ile Glu Val Ser Asn Glu Gln Leu Lys Gly Asn Ile Leu Leu Lys Tyr 275
280 285 aat gga gaa gct act cat caa ctt cct ata aaa aaa ctt aac tca
tca 912 Asn Gly Glu Ala Thr His Gln Leu Pro Ile Lys Lys Leu Asn Ser
Ser 290 295 300 att acc ctc agt ggc tca cta aat aaa cct aat ttt agt
ata caa atg 960 Ile Thr Leu Ser Gly Ser Leu Asn Lys Pro Asn Phe Ser
Ile Gln Met 305 310 315 320 aca tta cct gaa att aac att aca aaa aac
ata ata gat ctt caa aca 1008 Thr Leu Pro Glu Ile Asn Ile Thr Lys
Asn Ile Ile Asp Leu Gln Thr 325 330 335 gaa ctt gtt att aat cta gga
ctt ttc tct act cac tct gat att ctt 1056 Glu Leu Val Ile Asn Leu
Gly Leu Phe Ser Thr His Ser Asp Ile Leu 340 345 350 aca tct ggg aca
att aca gta cag gga gaa act ata ccc aat agt att 1104 Thr Ser Gly
Thr Ile Thr Val Gln Gly Glu Thr Ile Pro Asn Ser Ile 355
360 365 ctt tcc agt gca gtt gat ata ata gcc tct aca aca aca cat aca
att 1152 Leu Ser Ser Ala Val Asp Ile Ile Ala Ser Thr Thr Thr His
Thr Ile 370 375 380 acc tta gag cat gca acc tta aca tct cca gaa atg
cat ttt tcc cta 1200 Thr Leu Glu His Ala Thr Leu Thr Ser Pro Glu
Met His Phe Ser Leu 385 390 395 400 tct gga gaa ttt aat agt ctt cta
gga aat atc gat gca aac cta aaa 1248 Ser Gly Glu Phe Asn Ser Leu
Leu Gly Asn Ile Asp Ala Asn Leu Lys 405 410 415 ggt aat act cca act
ctt agt ata ttt tct tct ctt ctt gga cta cct 1296 Gly Asn Thr Pro
Thr Leu Ser Ile Phe Ser Ser Leu Leu Gly Leu Pro 420 425 430 gat ctt
act ggg caa agt aac att act ata gga tta cac cgt caa ggg 1344 Asp
Leu Thr Gly Gln Ser Asn Ile Thr Ile Gly Leu His Arg Gln Gly 435 440
445 tct tcc tct tca ata gaa gga aca gca act gtc tca ctt aat aat atg
1392 Ser Ser Ser Ser Ile Glu Gly Thr Ala Thr Val Ser Leu Asn Asn
Met 450 455 460 aac tgg gga gta caa gca tta cag ggg aca tta ggt gat
aat gca act 1440 Asn Trp Gly Val Gln Ala Leu Gln Gly Thr Leu Gly
Asp Asn Ala Thr 465 470 475 480 cta agt gga ata tat aat tta act ccc
ata gac tgg tct att tct tta 1488 Leu Ser Gly Ile Tyr Asn Leu Thr
Pro Ile Asp Trp Ser Ile Ser Leu 485 490 495 aac aaa ttg aaa tta aca
gca aag aat gtt tat gct gaa ggc ctt att 1536 Asn Lys Leu Lys Leu
Thr Ala Lys Asn Val Tyr Ala Glu Gly Leu Ile 500 505 510 aat ttt caa
aaa aaa tac ata gat agc tct ata aat ctt ata att cct 1584 Asn Phe
Gln Lys Lys Tyr Ile Asp Ser Ser Ile Asn Leu Ile Ile Pro 515 520 525
aac ctt cag cta ata gct cct cct ata tct gga gag tta caa tcc tta
1632 Asn Leu Gln Leu Ile Ala Pro Pro Ile Ser Gly Glu Leu Gln Ser
Leu 530 535 540 att aca gtg tct gga aaa ctt gac gca cct tct ata gaa
agc aaa att 1680 Ile Thr Val Ser Gly Lys Leu Asp Ala Pro Ser Ile
Glu Ser Lys Ile 545 550 555 560 ttt tca tca caa ctc acc tgg aat gcg
ctc caa ctt aat aat cct caa 1728 Phe Ser Ser Gln Leu Thr Trp Asn
Ala Leu Gln Leu Asn Asn Pro Gln 565 570 575 ctc ata ata act act act
caa tct tct tcc tct gcg att aaa ggt aat 1776 Leu Ile Ile Thr Thr
Thr Gln Ser Ser Ser Ser Ala Ile Lys Gly Asn 580 585 590 ata aca ctc
tcg gct gag cca gct tca tct gag gca tta acc ttt tca 1824 Ile Thr
Leu Ser Ala Glu Pro Ala Ser Ser Glu Ala Leu Thr Phe Ser 595 600 605
agt aat tgg gga atc cta cct acg gaa ata cta gta gaa aaa att ata
1872 Ser Asn Trp Gly Ile Leu Pro Thr Glu Ile Leu Val Glu Lys Ile
Ile 610 615 620 gga aat ata tta gga gta aat ctt gat ggt aat att aaa
ata aca aaa 1920 Gly Asn Ile Leu Gly Val Asn Leu Asp Gly Asn Ile
Lys Ile Thr Lys 625 630 635 640 aaa gat tac ctt ata aat ggt gat att
att gca gaa gtt cag tct tgg 1968 Lys Asp Tyr Leu Ile Asn Gly Asp
Ile Ile Ala Glu Val Gln Ser Trp 645 650 655 aaa gat att gca aac ata
ttg caa ata cct att aga ggt tca gca tca 2016 Lys Asp Ile Ala Asn
Ile Leu Gln Ile Pro Ile Arg Gly Ser Ala Ser 660 665 670 ata aaa ata
cag ttt gat cca aag aat caa caa tgt att tct act caa 2064 Ile Lys
Ile Gln Phe Asp Pro Lys Asn Gln Gln Cys Ile Ser Thr Gln 675 680 685
tgg caa tta aaa aat ttc ata tta ggt aat aat ttt aat gta act act
2112 Trp Gln Leu Lys Asn Phe Ile Leu Gly Asn Asn Phe Asn Val Thr
Thr 690 695 700 ata aaa gga aga gca gat aca ata caa ctt cat aag aat
cct aca att 2160 Ile Lys Gly Arg Ala Asp Thr Ile Gln Leu His Lys
Asn Pro Thr Ile 705 710 715 720 gct ctc tct tca aaa att ggt gct ggt
aca tat gaa gac ttt caa tgg 2208 Ala Leu Ser Ser Lys Ile Gly Ala
Gly Thr Tyr Glu Asp Phe Gln Trp 725 730 735 aca caa ggg acg tta gac
ata aaa ggc aca tta aaa aat ttt aat agt 2256 Thr Gln Gly Thr Leu
Asp Ile Lys Gly Thr Leu Lys Asn Phe Asn Ser 740 745 750 aaa ata aat
ata gca gga caa aca act gta aac gca aac ttt caa aca 2304 Lys Ile
Asn Ile Ala Gly Gln Thr Thr Val Asn Ala Asn Phe Gln Thr 755 760 765
aat ctt ttt gaa aaa aat att aat ata act act ctt aat tta aaa aat
2352 Asn Leu Phe Glu Lys Asn Ile Asn Ile Thr Thr Leu Asn Leu Lys
Asn 770 775 780 att caa aaa aat ata gga att aag ctc ctt cag cca ata
aaa att ata 2400 Ile Gln Lys Asn Ile Gly Ile Lys Leu Leu Gln Pro
Ile Lys Ile Ile 785 790 795 800 gtc tca cct caa caa ttt gtt ctt aat
aac tgt tca cta gca att ctt 2448 Val Ser Pro Gln Gln Phe Val Leu
Asn Asn Cys Ser Leu Ala Ile Leu 805 810 815 cca tct gga aca att aca
act gat ata tat gtt act cct caa cga ctt 2496 Pro Ser Gly Thr Ile
Thr Thr Asp Ile Tyr Val Thr Pro Gln Arg Leu 820 825 830 aat gct aat
gca atc att aaa gaa gtt tca ctt ctc tct ttc caa cca 2544 Asn Ala
Asn Ala Ile Ile Lys Glu Val Ser Leu Leu Ser Phe Gln Pro 835 840 845
ttt agt ata ctt ctt cct caa gga aat ata aat gga cac ata aca ctt
2592 Phe Ser Ile Leu Leu Pro Gln Gly Asn Ile Asn Gly His Ile Thr
Leu 850 855 860 aca gga ata cct agt aaa cct aaa gga aca ctc tca ttt
gat att cta 2640 Thr Gly Ile Pro Ser Lys Pro Lys Gly Thr Leu Ser
Phe Asp Ile Leu 865 870 875 880 aac ata cat tat cca agg cca aat cca
tca ata gca aac tta cat gta 2688 Asn Ile His Tyr Pro Arg Pro Asn
Pro Ser Ile Ala Asn Leu His Val 885 890 895 gaa ggg gaa att ata tct
tct cct aac aat ata tgt aaa ctt aat gca 2736 Glu Gly Glu Ile Ile
Ser Ser Pro Asn Asn Ile Cys Lys Leu Asn Ala 900 905 910 acc cta aca
gaa aaa aaa gag cct ata cct ata tca ata caa gca aca 2784 Thr Leu
Thr Glu Lys Lys Glu Pro Ile Pro Ile Ser Ile Gln Ala Thr 915 920 925
ctc cct ttt gag ttc aca gaa aac aat atc cct atg cta tct aaa atg
2832 Leu Pro Phe Glu Phe Thr Glu Asn Asn Ile Pro Met Leu Ser Lys
Met 930 935 940 agg cct ttt tct gcc cat atc aag tgg act gga ata tta
gat aca ctt 2880 Arg Pro Phe Ser Ala His Ile Lys Trp Thr Gly Ile
Leu Asp Thr Leu 945 950 955 960 tgg aaa ctc att cca ctt act gat tac
att atg gct ggg aat gga tct 2928 Trp Lys Leu Ile Pro Leu Thr Asp
Tyr Ile Met Ala Gly Asn Gly Ser 965 970 975 tta gat gct tct ctt tct
ggg act tta gat agt cca aca tat gca att 2976 Leu Asp Ala Ser Leu
Ser Gly Thr Leu Asp Ser Pro Thr Tyr Ala Ile 980 985 990 ata aca aca
ctt tct aat gct aac ttt caa gat ctc tcc ctt ggt ctt 3024 Ile Thr
Thr Leu Ser Asn Ala Asn Phe Gln Asp Leu Ser Leu Gly Leu 995 1000
1005 tac tta gaa aat atc aat gct aaa tta cag gtc ttt tct aat aga
atc 3072 Tyr Leu Glu Asn Ile Asn Ala Lys Leu Gln Val Phe Ser Asn
Arg Ile 1010 1015 1020 tcc cat att caa gct aca gca tct gat ggt aaa
caa ggt agt ata caa 3120 Ser His Ile Gln Ala Thr Ala Ser Asp Gly
Lys Gln Gly Ser Ile Gln 1025 1030 1035 1040 ctt att ggt aat att ggc
tca tct aaa gaa cac ttt cct ttg tct att 3168 Leu Ile Gly Asn Ile
Gly Ser Ser Lys Glu His Phe Pro Leu Ser Ile 1045 1050 1055 aat ggc
tcc ttt aca aac ctt gct cca tta caa cgt aaa gac cta agt 3216 Asn
Gly Ser Phe Thr Asn Leu Ala Pro Leu Gln Arg Lys Asp Leu Ser 1060
1065 1070 ctt aca ctt tca gga gca gct act ctt gaa gga aca tta aaa
cag tct 3264 Leu Thr Leu Ser Gly Ala Ala Thr Leu Glu Gly Thr Leu
Lys Gln Ser 1075 1080 1085 gaa gtt aaa ggc gat att gtt att aac caa
ggc gaa ttt caa ctt act 3312 Glu Val Lys Gly Asp Ile Val Ile Asn
Gln Gly Glu Phe Gln Leu Thr 1090 1095 1100 gaa ggg tta acc agt aat
att cca act ctt aat gta gtt gat agc act 3360 Glu Gly Leu Thr Ser
Asn Ile Pro Thr Leu Asn Val Val Asp Ser Thr 1105 1110 1115 1120 caa
caa caa aat aca aag acc aaa aaa gct acc tat caa caa cct acc 3408
Gln Gln Gln Asn Thr Lys Thr Lys Lys Ala Thr Tyr Gln Gln Pro Thr
1125 1130 1135 tta tct att gcg tta agt atc ccg aat cgt ttt ttt gtc
cgt agt agt 3456 Leu Ser Ile Ala Leu Ser Ile Pro Asn Arg Phe Phe
Val Arg Ser Ser 1140 1145 1150 atg ttt gaa agt gag tgg gga ggg aac
cta act att aac aaa gtc ata 3504 Met Phe Glu Ser Glu Trp Gly Gly
Asn Leu Thr Ile Asn Lys Val Ile 1155 1160 1165 aca agt cct gtt att
aca gga gca cta act tct ata aga gga aat ttt 3552 Thr Ser Pro Val
Ile Thr Gly Ala Leu Thr Ser Ile Arg Gly Asn Phe 1170 1175 1180 aat
tta cta gga aaa caa ttt tct ctt gct aaa agt aca ata tca ttt 3600
Asn Leu Leu Gly Lys Gln Phe Ser Leu Ala Lys Ser Thr Ile Ser Phe
1185 1190 1195 1200 tca gga tca gtt cca cca aac cca cta ctc aat att
tct tta aca tat 3648 Ser Gly Ser Val Pro Pro Asn Pro Leu Leu Asn
Ile Ser Leu Thr Tyr 1205 1210 1215 tca tca cct tct att aca gct ata
ggc att att aaa ggt aca act agt 3696 Ser Ser Pro Ser Ile Thr Ala
Ile Gly Ile Ile Lys Gly Thr Thr Ser 1220 1225 1230 aat cct aat att
act ttt tca agt aca cca cct tta cct caa gat gaa 3744 Asn Pro Asn
Ile Thr Phe Ser Ser Thr Pro Pro Leu Pro Gln Asp Glu 1235 1240 1245
ata gtt tcc caa gtt ctt ttt ggt aaa agc tca caa agt ctt agc agg
3792 Ile Val Ser Gln Val Leu Phe Gly Lys Ser Ser Gln Ser Leu Ser
Arg 1250 1255 1260 ata caa gcc ata caa ctt gct caa gaa tta gca aac
tta aca gga ttt 3840 Ile Gln Ala Ile Gln Leu Ala Gln Glu Leu Ala
Asn Leu Thr Gly Phe 1265 1270 1275 1280 aat act gga agt atg aat ttc
cta aca aat att cga cag aca tta ggg 3888 Asn Thr Gly Ser Met Asn
Phe Leu Thr Asn Ile Arg Gln Thr Leu Gly 1285 1290 1295 tta gat ata
ctt agc tta ggg aca act tct aat aga aaa gcc aat aca 3936 Leu Asp
Ile Leu Ser Leu Gly Thr Thr Ser Asn Arg Lys Ala Asn Thr 1300 1305
1310 tcc aac tca aac gat caa ata gaa gat atc cct gtt ata gaa cta
ggt 3984 Ser Asn Ser Asn Asp Gln Ile Glu Asp Ile Pro Val Ile Glu
Leu Gly 1315 1320 1325 aaa tat att aca gac act gtt tat gtt ggt gtt
gaa caa agt tat tta 4032 Lys Tyr Ile Thr Asp Thr Val Tyr Val Gly
Val Glu Gln Ser Tyr Leu 1330 1335 1340 gat agt aat gat act ggg gca
aga ata tca gtt gaa ctt gca cct aat 4080 Asp Ser Asn Asp Thr Gly
Ala Arg Ile Ser Val Glu Leu Ala Pro Asn 1345 1350 1355 1360 ttt aat
ctt gaa ggt aga aca ggg act caa tat agt gag ata ggt att 4128 Phe
Asn Leu Glu Gly Arg Thr Gly Thr Gln Tyr Ser Glu Ile Gly Ile 1365
1370 1375 aat tgg aaa aaa gat tat taa 4149 Asn Trp Lys Lys Asp Tyr
1380 18 1382 PRT Lawsonia intracellularis 18 Met Asn Asn Thr Lys
Ile Leu Ser Lys Leu Leu Tyr Thr Leu Leu Gly 1 5 10 15 Ala Phe Thr
Leu Phe Leu Gly Leu Ile Ile Thr Gly Ile Leu Phe Ile 20 25 30 Arg
Thr Ser Thr Gly Ile Ala Trp Ile Lys Asn Thr Val Ser Ser Leu 35 40
45 Leu Gln Gln Gln Gly Ile Ile Leu Gln Val Ser Ser Ile Ile Gly Pro
50 55 60 Phe Pro Glu Gln Ile Thr Ile Asn Glu Leu Ser Leu Ser Asp
Val Asn 65 70 75 80 Gly Thr Tyr Leu Thr Ile Ser Asn Leu Glu Ile Gln
Ser Asn Leu Trp 85 90 95 Ala Leu Phe Lys Gly Gln Leu Glu Ile Leu
Ser Phe Glu Leu Asn Asp 100 105 110 Leu Val Leu Tyr Arg Leu Pro Ser
Asn Asn Asn Leu Lys Lys Ser Ser 115 120 125 Thr Ser Phe Val Leu Pro
His Ile Ser Phe Asp Leu Thr Pro Trp Trp 130 135 140 Thr Glu His Ile
Arg Ile Gln Asn Ile His Ile Asn Asn Thr Gln Leu 145 150 155 160 Ser
Ser Asp Ile Ile Gly Ile Pro Leu Val Leu Ser Leu Glu Gly Asp 165 170
175 Gly Thr Leu Thr Asn Trp Asn Gly Thr Phe Gln Leu Ser Ser Ser Asn
180 185 190 Lys Thr Lys Ile Ile Gly Thr Leu Arg Tyr Gln Gly Asn Lys
Thr Gln 195 200 205 Phe Phe Glu Tyr Val His Pro Thr Arg Ile Val Thr
Leu Glu Ile Asp 210 215 220 Ser Val Ala Asp Lys Lys Ser Tyr Asn Asn
Ser Ile Leu Glu Gln Pro 225 230 235 240 Leu His Leu His Leu Ser Ile
Tyr Pro Glu His Asn Arg Ile Ile Leu 245 250 255 His Ser Leu Leu Ala
Glu Tyr Gly Ser Trp Leu Leu Thr Ser Glu Ser 260 265 270 Ile Glu Val
Ser Asn Glu Gln Leu Lys Gly Asn Ile Leu Leu Lys Tyr 275 280 285 Asn
Gly Glu Ala Thr His Gln Leu Pro Ile Lys Lys Leu Asn Ser Ser 290 295
300 Ile Thr Leu Ser Gly Ser Leu Asn Lys Pro Asn Phe Ser Ile Gln Met
305 310 315 320 Thr Leu Pro Glu Ile Asn Ile Thr Lys Asn Ile Ile Asp
Leu Gln Thr 325 330 335 Glu Leu Val Ile Asn Leu Gly Leu Phe Ser Thr
His Ser Asp Ile Leu 340 345 350 Thr Ser Gly Thr Ile Thr Val Gln Gly
Glu Thr Ile Pro Asn Ser Ile 355 360 365 Leu Ser Ser Ala Val Asp Ile
Ile Ala Ser Thr Thr Thr His Thr Ile 370 375 380 Thr Leu Glu His Ala
Thr Leu Thr Ser Pro Glu Met His Phe Ser Leu 385 390 395 400 Ser Gly
Glu Phe Asn Ser Leu Leu Gly Asn Ile Asp Ala Asn Leu Lys 405 410 415
Gly Asn Thr Pro Thr Leu Ser Ile Phe Ser Ser Leu Leu Gly Leu Pro 420
425 430 Asp Leu Thr Gly Gln Ser Asn Ile Thr Ile Gly Leu His Arg Gln
Gly 435 440 445 Ser Ser Ser Ser Ile Glu Gly Thr Ala Thr Val Ser Leu
Asn Asn Met 450 455 460 Asn Trp Gly Val Gln Ala Leu Gln Gly Thr Leu
Gly Asp Asn Ala Thr 465 470 475 480 Leu Ser Gly Ile Tyr Asn Leu Thr
Pro Ile Asp Trp Ser Ile Ser Leu 485 490 495 Asn Lys Leu Lys Leu Thr
Ala Lys Asn Val Tyr Ala Glu Gly Leu Ile 500 505 510 Asn Phe Gln Lys
Lys Tyr Ile Asp Ser Ser Ile Asn Leu Ile Ile Pro 515 520 525 Asn Leu
Gln Leu Ile Ala Pro Pro Ile Ser Gly Glu Leu Gln Ser Leu 530 535 540
Ile Thr Val Ser Gly Lys Leu Asp Ala Pro Ser Ile Glu Ser Lys Ile 545
550 555 560 Phe Ser Ser Gln Leu Thr Trp Asn Ala Leu Gln Leu Asn Asn
Pro Gln 565 570 575 Leu Ile Ile Thr Thr Thr Gln Ser Ser Ser Ser Ala
Ile Lys Gly Asn 580 585 590 Ile Thr Leu Ser Ala Glu Pro Ala Ser Ser
Glu Ala Leu Thr Phe Ser 595 600 605 Ser Asn Trp Gly Ile Leu Pro Thr
Glu Ile Leu Val Glu Lys Ile Ile 610 615 620 Gly Asn Ile Leu Gly Val
Asn Leu Asp Gly Asn Ile Lys Ile Thr Lys 625 630 635 640 Lys Asp Tyr
Leu Ile Asn Gly Asp Ile Ile Ala Glu Val Gln Ser Trp 645 650 655 Lys
Asp Ile Ala Asn Ile Leu Gln Ile Pro Ile Arg Gly Ser Ala Ser 660 665
670 Ile Lys Ile Gln Phe Asp Pro Lys Asn Gln Gln Cys Ile Ser Thr Gln
675 680 685 Trp Gln Leu Lys Asn Phe Ile Leu Gly Asn Asn Phe Asn Val
Thr Thr 690 695 700 Ile Lys Gly Arg Ala Asp Thr Ile Gln Leu His Lys
Asn Pro Thr Ile 705 710 715 720 Ala Leu Ser Ser Lys Ile Gly Ala Gly
Thr Tyr Glu Asp Phe Gln Trp 725 730 735 Thr Gln Gly Thr Leu Asp Ile
Lys Gly Thr Leu Lys Asn Phe Asn Ser 740 745 750 Lys Ile Asn Ile Ala
Gly Gln Thr Thr Val Asn Ala Asn Phe Gln Thr 755 760 765 Asn Leu Phe
Glu Lys Asn Ile Asn Ile Thr Thr Leu Asn Leu Lys Asn 770 775 780 Ile
Gln Lys Asn Ile Gly Ile Lys Leu Leu Gln Pro Ile Lys Ile Ile 785
790
795 800 Val Ser Pro Gln Gln Phe Val Leu Asn Asn Cys Ser Leu Ala Ile
Leu 805 810 815 Pro Ser Gly Thr Ile Thr Thr Asp Ile Tyr Val Thr Pro
Gln Arg Leu 820 825 830 Asn Ala Asn Ala Ile Ile Lys Glu Val Ser Leu
Leu Ser Phe Gln Pro 835 840 845 Phe Ser Ile Leu Leu Pro Gln Gly Asn
Ile Asn Gly His Ile Thr Leu 850 855 860 Thr Gly Ile Pro Ser Lys Pro
Lys Gly Thr Leu Ser Phe Asp Ile Leu 865 870 875 880 Asn Ile His Tyr
Pro Arg Pro Asn Pro Ser Ile Ala Asn Leu His Val 885 890 895 Glu Gly
Glu Ile Ile Ser Ser Pro Asn Asn Ile Cys Lys Leu Asn Ala 900 905 910
Thr Leu Thr Glu Lys Lys Glu Pro Ile Pro Ile Ser Ile Gln Ala Thr 915
920 925 Leu Pro Phe Glu Phe Thr Glu Asn Asn Ile Pro Met Leu Ser Lys
Met 930 935 940 Arg Pro Phe Ser Ala His Ile Lys Trp Thr Gly Ile Leu
Asp Thr Leu 945 950 955 960 Trp Lys Leu Ile Pro Leu Thr Asp Tyr Ile
Met Ala Gly Asn Gly Ser 965 970 975 Leu Asp Ala Ser Leu Ser Gly Thr
Leu Asp Ser Pro Thr Tyr Ala Ile 980 985 990 Ile Thr Thr Leu Ser Asn
Ala Asn Phe Gln Asp Leu Ser Leu Gly Leu 995 1000 1005 Tyr Leu Glu
Asn Ile Asn Ala Lys Leu Gln Val Phe Ser Asn Arg Ile 1010 1015 1020
Ser His Ile Gln Ala Thr Ala Ser Asp Gly Lys Gln Gly Ser Ile Gln
1025 1030 1035 1040 Leu Ile Gly Asn Ile Gly Ser Ser Lys Glu His Phe
Pro Leu Ser Ile 1045 1050 1055 Asn Gly Ser Phe Thr Asn Leu Ala Pro
Leu Gln Arg Lys Asp Leu Ser 1060 1065 1070 Leu Thr Leu Ser Gly Ala
Ala Thr Leu Glu Gly Thr Leu Lys Gln Ser 1075 1080 1085 Glu Val Lys
Gly Asp Ile Val Ile Asn Gln Gly Glu Phe Gln Leu Thr 1090 1095 1100
Glu Gly Leu Thr Ser Asn Ile Pro Thr Leu Asn Val Val Asp Ser Thr
1105 1110 1115 1120 Gln Gln Gln Asn Thr Lys Thr Lys Lys Ala Thr Tyr
Gln Gln Pro Thr 1125 1130 1135 Leu Ser Ile Ala Leu Ser Ile Pro Asn
Arg Phe Phe Val Arg Ser Ser 1140 1145 1150 Met Phe Glu Ser Glu Trp
Gly Gly Asn Leu Thr Ile Asn Lys Val Ile 1155 1160 1165 Thr Ser Pro
Val Ile Thr Gly Ala Leu Thr Ser Ile Arg Gly Asn Phe 1170 1175 1180
Asn Leu Leu Gly Lys Gln Phe Ser Leu Ala Lys Ser Thr Ile Ser Phe
1185 1190 1195 1200 Ser Gly Ser Val Pro Pro Asn Pro Leu Leu Asn Ile
Ser Leu Thr Tyr 1205 1210 1215 Ser Ser Pro Ser Ile Thr Ala Ile Gly
Ile Ile Lys Gly Thr Thr Ser 1220 1225 1230 Asn Pro Asn Ile Thr Phe
Ser Ser Thr Pro Pro Leu Pro Gln Asp Glu 1235 1240 1245 Ile Val Ser
Gln Val Leu Phe Gly Lys Ser Ser Gln Ser Leu Ser Arg 1250 1255 1260
Ile Gln Ala Ile Gln Leu Ala Gln Glu Leu Ala Asn Leu Thr Gly Phe
1265 1270 1275 1280 Asn Thr Gly Ser Met Asn Phe Leu Thr Asn Ile Arg
Gln Thr Leu Gly 1285 1290 1295 Leu Asp Ile Leu Ser Leu Gly Thr Thr
Ser Asn Arg Lys Ala Asn Thr 1300 1305 1310 Ser Asn Ser Asn Asp Gln
Ile Glu Asp Ile Pro Val Ile Glu Leu Gly 1315 1320 1325 Lys Tyr Ile
Thr Asp Thr Val Tyr Val Gly Val Glu Gln Ser Tyr Leu 1330 1335 1340
Asp Ser Asn Asp Thr Gly Ala Arg Ile Ser Val Glu Leu Ala Pro Asn
1345 1350 1355 1360 Phe Asn Leu Glu Gly Arg Thr Gly Thr Gln Tyr Ser
Glu Ile Gly Ile 1365 1370 1375 Asn Trp Lys Lys Asp Tyr 1380 19 26
DNA Artificial Sequence Oligonucleotide probe/primer 19 catattcaag
gtacagcatc tgatgg 26 20 21 DNA Artificial Sequence Oligonucleotide
probe/primer 20 ctcctttaca aaccttgctc c 21 21 22 DNA Artificial
Sequence Oligonucleotide probe/primer 21 gctcatctaa agaacacttt cc
22 22 23 DNA Artificial Sequence Oligonucleotide probe/primer 22
caaggtagta tacaacttat tgg 23 23 23 DNA Artificial Sequence
Oligonucleotide probe/primer 23 gacctaagtc ttacactttc agg 23 24 24
DNA Artificial Sequence Oligonucleotide probe/primer 24 gtattaatac
tacattagtt gacg 24 25 21 DNA Artificial Sequence Oligonucleotide
probe/primer 25 ggataataat ggaaaaagtg g 21 26 22 DNA Artificial
Sequence Oligonucleotide probe/primer 26 caagcaatgc ctgtagaggt cc
22 27 24 DNA Artificial Sequence Oligonucleotide probe/primer 27
aagaatgcct gtaataataa gtcc 24 28 19 DNA Artificial Sequence
Oligonucleotide probe/primer 28 ttggggaatc ctacctacg 19 29 21 DNA
Artificial Sequence Oligonucleotide probe/primer 29 tattaggagt
aaatcttgat g 21 30 22 DNA Artificial Sequence Oligonucleotide
probe/primer 30 gcaggacaaa caactgtaaa cg 22 31 22 DNA Artificial
Sequence Oligonucleotide probe/primer 31 gaggaagaag tatactaaat gg
22 32 20 DNA Artificial Sequence Oligonucleotide probe/primer 32
tgttggacta tctaaagtcc 20 33 22 DNA Artificial Sequence
Oligonucleotide probe/primer 33 ctattgatgg atttggcctt gg 22 34 20
DNA Artificial Sequence Oligonucleotide probe/primer 34 gtgctggtac
atatgaagac 20 35 20 DNA Artificial Sequence Oligonucleotide
probe/primer 35 ttcatcacct tctattacag 20 36 20 DNA Artificial
Sequence Oligonucleotide probe/primer 36 ggaaactatt tcatcttgag 20
37 20 DNA Artificial Sequence Oligonucleotide probe/primer 37
attaggtgca agttcaactg 20 38 20 DNA Artificial Sequence
Oligonucleotide probe/primer 38 tttagatagt aatgatactg 20 39 36 DNA
Artificial Sequence Oligonucleotide probe/primer 39 ttattatatt
atgttttttg taatgttaat ttcagg 36 40 28 DNA Artificial Sequence
Oligonucleotide probe/primer 40 gacatatgaa taacacaaaa atactttc 28
41 41 DNA Artificial Sequence Oligonucleotide probe/primer 41
gaggatcctc tagagttaat caaactgtat ttttattgat g 41 42 31 DNA
Artificial Sequence Oligonucleotide probe/primer 42 gacatatgcg
gacctctaca ggcattgctt g 31 43 24 DNA Artificial Sequence
Oligonucleotide probe/primer 43 gatcaggtag tccaagaaga gaag 24 44 45
DNA Artificial Sequence Oligonucleotide probe/primer 44 ttggaggatc
ctctagagtt atcaggttgt aattgttcca gatgg 45 45 21 DNA Artificial
Sequence Oligonucleotide probe/primer 45 ccttggttaa taacaatatc g 21
46 23 DNA Artificial Sequence Oligonucleotide probe/primer 46
caactccact tcaagataga agc 23 47 29 DNA Artificial Sequence oligo 47
gaccatggaa aaagtatgtt atttttttc 29 48 36 DNA Artificial Sequence
oligo 48 gaggatcctc tagagttaga atgcttgccc aatact 36 49 39 DNA
Artificial Sequence oligo 49 ttggaggatc ctctagagtt agaatgcttg
cccaatact 39 50 32 DNA Artificial Sequence oligo 50 ttgaccatgg
ctacatcaat taccacttcc ac 32 51 29 DNA Artificial Sequence oligo 51
ggacatatga ataacacaaa aatactttc 29 52 44 DNA Artificial Sequence
oligo 52 ttggaggatc ctctagagtt aatcaaactg tatttttatt gatg 44 53 32
DNA Artificial Sequence oligo 53 ggacatatgc ggacctctac aggcattgct
tg 32 54 20 DNA Artificial Sequence oligo 54 tgaggattat taagttggag
20 55 20 DNA Artificial Sequence oligo 55 gcatgcaacc ttaacatctc 20
56 20 DNA Artificial Sequence oligo 56 tttctgatgt aagtaaccag 20 57
20 DNA Artificial Sequence oligo 57 tctgcccata tcaagtggac 20 58 20
DNA Artificial Sequence oligo 58 ggaacatttc aactatcctc 20 59 20 DNA
Artificial Sequence oligo 59 gtaaggtaag ttccattcac 20 60 25 DNA
Artificial Sequence oligo 60 caacgtggat ccgaattcaa gcttc 25 61 16
PRT Artificial Sequence Peptide 61 Met Gly Ser Gly Ser Gly Asp Asp
Asp Asp Lys Leu Ala Leu Leu Thr 1 5 10 15 62 7 PRT Artificial
Sequence Peptide 62 Ala Thr Ser Ile Thr Thr Ser 1 5 63 45 PRT
Artificial Sequence Peptide 63 Met His His His His His His Ser Ser
Gly Leu Val Pro Arg Gly Ser 1 5 10 15 Gly Met Lys Glu Thr Ala Ala
Ala Lys Phe Glu Arg Gln His Met Asp 20 25 30 Ser Pro Asp Leu Gly
Thr Asp Asp Asp Asp Lys Ala Met 35 40 45 64 50 PRT Artificial
Sequence Peptide 64 Met His His His His His His Ser Ser Gly Leu Val
Pro Arg Gly Ser 1 5 10 15 Gly Met Lys Glu Thr Ala Ala Ala Lys Phe
Glu Arg Gln His Met Asp 20 25 30 Ser Pro Asp Leu Gly Thr Asp Asp
Asp Asp Lys Ala Met Ala Asp Ile 35 40 45 Gly Ser 50 65 7 PRT
Artificial Sequence Peptide 65 Glu Phe Asn Leu Ser Lys Gly 1 5 66
17 PRT Artificial Sequence Peptide 66 Met Gly Ser Gly Ser Gly Asp
Asp Asp Asp Lys Leu Ala Leu Gly His 1 5 10 15 Met 67 7 PRT
Artificial Sequence Peptide 67 Arg Thr Ser Thr Gly Ile Ala 1 5 68
21 PRT Artificial Sequence Peptide 68 Asp Pro Asn Ser Ser Ser Val
Asp Lys Leu Ala Ala Ala Leu Glu His 1 5 10 15 His His His His His
20
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