U.S. patent application number 14/205995 was filed with the patent office on 2014-11-13 for antibodies to clostridium difficile spores and uses thereof.
This patent application is currently assigned to 3M Innovative Properties Company. The applicant listed for this patent is 3M Innovative Properties Company. Invention is credited to RAJ RAJAGOPAL, AI-PING WEI.
Application Number | 20140335543 14/205995 |
Document ID | / |
Family ID | 41016450 |
Filed Date | 2014-11-13 |
United States Patent
Application |
20140335543 |
Kind Code |
A1 |
RAJAGOPAL; RAJ ; et
al. |
November 13, 2014 |
ANTIBODIES TO CLOSTRIDIUM DIFFICILE SPORES AND USES THEREOF
Abstract
The present invention provides antibodies that bind to the
endospore of the bacterium Clostridium difficile, methods of making
such antibodies, and methods of using such antibodies, including
methods of detecting C. difficile endospores.
Inventors: |
RAJAGOPAL; RAJ; (WOODBURY,
MN) ; WEI; AI-PING; (WOODBURY, MN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
3M Innovative Properties Company |
St. Paul |
MN |
US |
|
|
Assignee: |
3M Innovative Properties
Company
St. Paul
MN
|
Family ID: |
41016450 |
Appl. No.: |
14/205995 |
Filed: |
March 12, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12919254 |
Mar 28, 2011 |
8697374 |
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PCT/US2009/035050 |
Feb 25, 2009 |
|
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14205995 |
|
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61032270 |
Feb 28, 2008 |
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Current U.S.
Class: |
435/7.32 |
Current CPC
Class: |
G01N 2800/067 20130101;
A61P 31/04 20180101; G01N 33/56911 20130101; C07K 16/40 20130101;
G01N 2333/33 20130101; G01N 2469/10 20130101; C07K 16/1282
20130101 |
Class at
Publication: |
435/7.32 |
International
Class: |
G01N 33/569 20060101
G01N033/569 |
Claims
1. A kit comprising at least two isolated antibodies or
antigen-binding fragments thereof, wherein each isolated antibody
binds to a distinct antigenic epitope of the Clostridium difficile
spore.
2. The kit of claim 1, wherein at least one isolated antibody or
antigen-binding fragment thereof binds to hypothetical protein
CD1021 of C. difficile strain 630 having SEQ ID NO: 1, or a
polypeptide fragment of hypothetical protein CD 1021.
3. The kit of claim 1, wherein at least one isolated antibody or
antigen-binding fragment thereof binds to putative
N-acetylmuramoyl-L-alanine amidase protein of C. difficile strain
630 having SEQ ID NO: 5, or a fragment of the putative
N-acetylmuramoyl-L-alanine amidase protein.
4. The kit of claim 1, wherein a first isolated antibody or
antigen-binding fragment thereof binds to hypothetical protein
CD1021 of C. difficile strain 630 having SEQ ID NO: 1, or a
polypeptide fragment of hypothetical protein CD 1021, and wherein a
second isolated antibody or antigen-binding fragment thereof binds
to putative N-acetylmuramoyl-L-alanine amidase protein of C.
difficile strain 630 having SEQ ID NO: 5, or a fragment of the
putative N-acetylmuramoyl-L-alanine amidase protein.
5. The kit of claim 2, wherein the polypeptide fragment of
hypothetical protein CD1021 is selected from the group consisting
of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:9, SEQ ID
NO:10, and fragments thereof.
6. The kit of claim 3, wherein the polypeptide fragment of the
putative N-acetylmuramoyl-L-alanine amidase protein is selected
from the group consisting of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8,
and fragments thereof.
7. A method of detecting the presence of a Clostridium difficile
spore in a sample, the method comprising contacting the sample with
at least two isolated antibodies or antigen-binding fragments
thereof, wherein each isolated antibody binds to a distinct
antigenic epitope of the C. difficile spore.
8. The method of claim 7, wherein at least one isolated antibody or
antigen-binding fragment thereof binds to hypothetical protein
CD1021 of C. difficile strain 630 having SEQ ID NO: 1, or a
polypeptide fragment of hypothetical protein CD 1021.
9. The method of claim 7, wherein at least one isolated antibody or
antigen-binding fragment thereof binds to putative
N-acetylmuramoyl-L-alanine amidase protein of C. difficile strain
630 having SEQ ID NO: 5, or a fragment of the putative
N-acetylmuramoyl-L-alanine amidase protein.
10. The method of claim 7, wherein a first isolated antibody or
antigen-binding fragment thereof binds to hypothetical protein
CD1021 of C. difficile strain 630 having SEQ ID NO: 1, or a
polypeptide fragment of hypothetical protein CD 1021, and wherein a
second isolated antibody or antigen-binding fragment thereof binds
to putative N-acetylmuramoyl-L-alanine amidase protein of C.
difficile strain 630 having SEQ ID NO: 5, or a fragment of the
putative N-acetylmuramoyl-L-alanine amidase protein.
11. The method of claim 8, wherein the polypeptide fragment of
hypothetical protein CD1021 is selected from the group consisting
of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:9, SEQ ID
NO:10, and fragments thereof.
12. The method of claim 9, wherein the polypeptide fragment of the
putative N-acetylmuramoyl-L-alanine amidase protein is selected
from the group consisting of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8,
and fragments thereof.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a division of U.S. patent application
Ser. No. 12/919,254, filed Mar. 28, 2011, now allowed, which is a
national stage filing under 35 U.S.C. 371 of PCT/US2009/035050,
filed Feb. 25, 2009, which claims priority to U.S. Provisional
Application Ser. No. 61/032,270, filed Feb. 28, 2008, the
disclosure of which is incorporated by reference in their entirety
herein.
[0002] This application has associated with it a sequence listing
with the file name Sequence_Listing.sub.--63842US013.TXT, created
Feb. 18, 2014. The sequence listing file contains 98,928 bytes and
it is incorporated herein by reference in its entirety.
BACKGROUND
[0003] Clostridium difficile, an anaerobic spore forming
Gram-positive bacteria, is the major cause of pseudomembraneous
colitis and antibiotic associated diarrhea in humans and is one of
the most widespread bacterium implicated in hospital acquired,
nosocomial infections (see, for example, Wren, 2006, Future
Microbiol; 1(3):243-245). According to the Center for Disease
Control (CDC), C. difficile is responsible for tens of thousands of
cases of diarrhea and at least 5,000 deaths each year in the United
States. The number of C. difficile infections doubled between 1993
and 2003, with the largest increase coming after 2000.
[0004] Individuals with a C. difficile-associated disease shed
spores in the stool. C. difficile infections are frequently
transmitted between hospitalized patients and the organism is often
present on the hands of hospital personnel (see, for example,
McFarland et al., 1989, N Engl J Med; 320:204-210). Patients
infected with a C. difficile infection are isolated and precautions
are taken to avoid outbreaks. Asymptomatic carriers can shed spores
and need to be screened for isolation purposes (see, for example,
Kyne et al., 2000, N Engl J Med; 342:390-397).
[0005] C. difficile spores are resistant to heat, drying, and
cleaning agents and can survive up to seventy days on environmental
surfaces, such as cart handles, bedrails, bedpans, toilets, bathing
tubs, floors, furniture, linens, telephones, stethoscopes,
thermometers, and remote controls. Thus, environmental surfaces are
a ready source of infection. The thorough cleaning of patient's
rooms during hospitalization is needed.
[0006] There is a clear need to monitor cleaning effectiveness and
to verify that patient rooms and environmental surfaces are free of
C. difficile spores. Currently, there are no easy to use, rapid
methods for detecting C. difficile spores in environmental and
patient samples. While kits (both immunoassay and molecular assays)
are currently commercially available for the detection of C.
difficile toxin, these kits do not detect C. difficile spores.
Thus, there is a need for rapid and easy to use systems for the
detection of C. difficile spores.
SUMMARY OF THE INVENTION
[0007] The present invention includes an isolated antibody that
binds to a Clostridium difficile spore. In some embodiments, the
spore is an ungerminated spore. In some embodiments, the spore is a
germinated spore. In some embodiments, the antibody does not bind
to Clostridium difficile vegetative cells. In some embodiments, the
antibody does not bind to C. difficile toxin.
[0008] The present invention includes an isolated antibody that
binds to hypothetical protein CD1021 of Clostridium difficile
strain 630 having SEQ ID NO: 1, or a fragment of hypothetical
protein CD 1021. In some embodiments, the isolated antibody binds a
fragment of hypothetical protein CD1021 including amino acid
residues 505 to 604. In some embodiments, the isolated antibody
binds a fragment of hypothetical protein CD1021 including amino
acid residues 30 to 120. In some embodiments, the isolated antibody
binds a fragment of hypothetical protein CD1021 including amino
acid residues 194 to 293. In some embodiments, the isolated
antibody binds to a fragment of hypothetical protein CD 1021
including amino acid residues 203 to 217. In some embodiments, the
isolated antibody binds to a fragment of hypothetical protein
CD1021 including amino acid residues 333 to 347.
[0009] The present invention includes an isolated antibody that
binds to the amino acid sequence SEQ ID NO:2.
[0010] The present invention includes an isolated antibody that
binds to the amino acid sequence SEQ ID NO:9.
[0011] The present invention includes an isolated antibody that
binds to the amino acid sequence SEQ ID NO:10.
[0012] The present invention includes an isolated antibody that
binds to the amino acid sequence EGSSLQYKGDDPESY (SEQ ID NO:3).
[0013] The present invention includes an isolated antibody that
binds to the amino acid sequence LKNETYKTKYHKYLE (SEQ ID NO:4).
[0014] The present invention includes an isolated antibody that
binds to putative N-acetylmuramoyl-L-alanine amidase protein of
Clostridium difficile strain 630 having SEQ ID NO: 5 or a fragment
of the putative N-acetylmuramoyl-L-alanine amidase protein. In some
embodiments, the isolated antibody binds to a fragment of the
putative N-acetylmuramoyl-L-alanine amidase protein including amino
acid residues 294 to 393. In some embodiments, the isolated
antibody binds to a fragment of the putative
N-acetylmuramoyl-L-alanine amidase protein including amino acid
residues 582 to 596. In some embodiments, the isolated antibody
binds to a fragment of the putative N-acetylmuramoyl-L-alanine
amidase protein including amino acid residues 64 to 78.
[0015] The present invention includes an isolated antibody that
binds to the amino acid sequence SEQ ID NO:6.
[0016] The present invention includes an isolated antibody that
binds to the amino acid sequence YKLKDKNGGTTKTVA (SEQ ID NO:7).
[0017] The present invention includes an isolated antibody that
binds to the amino acid sequence KFKEKPDADSIKLKY (SEQ ID NO:8).
[0018] The present invention includes a monoclonal antibody, or
antigen binding fragment thereof, wherein the monoclonal antibody
or antigen binding fragment inhibits the binding of an antibody of
the present invention to its antigen target.
[0019] The present invention includes an antigen binding fragment
of an isolated antibody of the present invention.
[0020] In some embodiments, an isolated antibody of the present
invention is a polyclonal antibody. In some embodiments, an
isolated antibody of the present invention is a monoclonal
antibody. In some embodiments, the isolated antibody of the present
invention does not bind to Bacillus subtilis spores or Clostridium
sporogenes spores. In some embodiments, the antibodies and antigen
binding fragments of the present invention are labeled.
[0021] The present invention includes a composition including one
or more of the isolated antibodies of the present invention, or
antigen binding fragments thereof.
[0022] The present invention includes a kit including one or more
of the isolated antibodies of the present invention, or antigen
binding fragments thereof.
[0023] The present invention includes a hybridoma cell line or
transformed B cell line that produces a monoclonal antibody of the
present invention.
[0024] The present invention includes an isolated polynucleotide
sequence including the nucleic acid sequence coding for the heavy
chain, the light chain, the heavy chain variable region, the light
chain variable region, or one or more complementarity determining
regions of a monoclonal antibody of the present invention. The
present invention includes an expression vector including such an
isolated polynucleotide sequence. The present invention includes a
host cell including such an expression vector.
[0025] The present invention includes a method of preparing an
anti-Clostridium difficile antibody, the method including
immunizing a host organism with a polypeptide including at least a
portion of a protein encoded by the C. difficile genome in an
amount effective to generate an antibody response to the
polypeptide. In some embodiments, the polypeptide including at
least a portion of a protein encoded by the C. difficile genome is
selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4,
SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,
SEQ ID NO:10, and fragments thereof. In some embodiments, the
method further includes purifying the antibody preparation.
[0026] The present invention includes a method of preparing an
anti-Clostridium difficile antibody, the method including
expressing a nucleic acid sequence encoding at least a portion of a
protein encoded by the C. difficile genome in an immunocompetent
host organism. In some embodiments, the nucleic acid sequence
encoding at least a portion of a protein encoded by the C.
difficile genome encodes an amino acid sequence selected from SEQ
ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID
NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, and
fragments thereof. In some embodiments, the method further includes
purifying the antibody preparation.
[0027] The present invention includes a composition including at
least two isolated antibodies or antigen-binding fragments thereof,
wherein each isolated antibody binds to a distinct antigenic
epitope of the Clostridium difficile spore.
[0028] In some embodiments of the composition, at least one
isolated antibody or antigen-binding fragment thereof binds to
hypothetical protein CD 1021 of C. difficile strain 630 having SEQ
ID NO: 1, or a polypeptide fragment of hypothetical protein CD1021.
In some embodiments, the polypeptide fragment of hypothetical
protein CD 1021 is selected from SEQ ID NO:2, SEQ ID NO:3, SEQ ID
NO:4, SEQ ID NO:9, SEQ ID NO:10, and fragments thereof.
[0029] In some embodiments of the composition, at least one
isolated antibody or antigen-binding fragment thereof binds to
putative N-acetylmuramoyl-L-alanine amidase protein of C. difficile
strain 630 having SEQ ID NO: 5, or a fragment of the putative
N-acetylmuramoyl-L-alanine amidase protein. In some embodiments,
the polypeptide fragment of the putative N-acetylmuramoyl-L-alanine
amidase protein is selected from the group consisting of SEQ ID
NO:6, SEQ ID NO:7, SEQ ID NO:8, and fragments thereof.
[0030] In some embodiments of the composition, a first isolated
antibody or antigen-binding fragment thereof binds to hypothetical
protein CD1021 of C. difficile strain 630 having SEQ ID NO: 1, or a
polypeptide fragment of hypothetical protein CD1021, and a second
isolated antibody or antigen-binding fragment thereof binds to
putative N-acetylmuramoyl-L-alanine amidase protein of C. difficile
strain 630 having SEQ ID NO: 5, or a fragment of the putative
N-acetylmuramoyl-L-alanine amidase protein. In some embodiments,
the polypeptide fragment of hypothetical protein CD1021 is selected
from SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:9, SEQ ID
NO:10, and fragments thereof. In some embodiments, the polypeptide
fragment of the putative N-acetylmuramoyl-L-alanine amidase protein
is selected from the group consisting of SEQ ID NO:6, SEQ ID NO:7,
SEQ ID NO:8, and fragments thereof.
[0031] The present invention includes a kit including at least two
isolated antibodies or antigen-binding fragments thereof, wherein
each isolated antibody binds to a distinct antigenic epitope of the
Clostridium difficile spore.
[0032] In some embodiments of the kit, at least one isolated
antibody or antigen-binding fragment thereof binds to hypothetical
protein CD 1021 of C. difficile strain 630 having SEQ ID NO: 1, or
a polypeptide fragment of hypothetical protein CD1021. In some
embodiments, the polypeptide fragment of hypothetical protein
CD1021 is selected from SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ
ID NO:9, SEQ ID NO:10, and fragments thereof.
[0033] In some embodiments of the kit, at least one isolated
antibody or antigen-binding fragment thereof binds to putative
N-acetylmuramoyl-L-alanine amidase protein of C. difficile strain
630 having SEQ ID NO: 5, or a fragment of the putative
N-acetylmuramoyl-L-alanine amidase protein. In some embodiments,
the polypeptide fragment of the putative N-acetylmuramoyl-L-alanine
amidase protein is selected from SEQ ID NO:6, SEQ ID NO:7, SEQ ID
NO:8, and fragments thereof.
[0034] In some embodiments of the kit, a first isolated antibody or
antigen-binding fragment thereof binds to hypothetical protein
CD1021 of C. difficile strain 630 having SEQ ID NO: 1, or a
polypeptide fragment of hypothetical protein CD1021, and a second
isolated antibody or antigen-binding fragment thereof binds to
putative N-acetylmuramoyl-L-alanine amidase protein of C. difficile
strain 630 having SEQ ID NO: 5, or a fragment of the putative
N-acetylmuramoyl-L-alanine amidase protein. In some embodiments,
the polypeptide fragment of hypothetical protein CD1021 is selected
from SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:9, SEQ ID
NO:10, and fragments thereof. In some embodiments, the polypeptide
fragment of the putative N-acetylmuramoyl-L-alanine amidase protein
is selected from SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, and
fragments thereof.
[0035] The present invention includes a method of detecting the
presence of a Clostridium difficile spore in a sample, the method
including contacting the sample with one or more isolated
antibodies of the present invention.
[0036] The present invention includes a method of detecting the
presence of a Clostridium difficile spore in a sample, the method
including contacting the sample with at least two isolated
antibodies or antigen-binding fragments thereof, wherein each
isolated antibody binds to a distinct antigenic epitope of the C.
difficile spore.
[0037] The present invention includes a method of detecting the
presence of a Clostridium difficile spore in a sample, the method
including: contacting the sample with a first isolated antibody or
antigen-binding fragment thereof, wherein the first isolated
antibody binds to a first antigenic epitope of the C. difficile
spore; and contacting the sample with a second isolated antibody or
antigen-binding fragment thereof, wherein the second isolated
antibody binds to a second antigenic epitope of the C. difficile
spore.
[0038] In some embodiments of the methods of the present invention,
at least one isolated antibody or antigen-binding fragment thereof
binds to hypothetical protein CD 1021 of C. difficile strain 630
having SEQ ID NO: 1, or a polypeptide fragment of hypothetical
protein CD1021. In some embodiments, the polypeptide fragment of
hypothetical protein CD1021 is selected from SEQ ID NO:2, SEQ ID
NO:3, SEQ ID NO:4, SEQ ID NO:9, SEQ ID NO:10, and fragments
thereof.
[0039] In some embodiments of the methods of the present invention,
at least one isolated antibody or antigen-binding fragment thereof
binds to putative N-acetylmuramoyl-L-alanine amidase protein of C.
difficile strain 630 having SEQ ID NO: 5, or a fragment of the
putative N-acetylmuramoyl-L-alanine amidase protein. In some
embodiments, the polypeptide fragment of the putative
N-acetylmuramoyl-L-alanine amidase protein is selected from SEQ ID
NO:6, SEQ ID NO:7, SEQ ID NO:8, and fragments thereof.
[0040] In some embodiments of the methods of the present invention,
a first isolated antibody or antigen-binding fragment thereof binds
to hypothetical protein CD 1021 of C. difficile strain 630 having
SEQ ID NO: 1, or a polypeptide fragment of hypothetical protein
CD1021, and a second isolated antibody or antigen-binding fragment
thereof binds to putative N-acetylmuramoyl-L-alanine amidase
protein of C. difficile strain 630 having SEQ ID NO: 5, or a
fragment of the putative N-acetylmuramoyl-L-alanine amidase
protein. In some embodiments, the polypeptide fragment of
hypothetical protein CD 1021 is selected from SEQ ID NO:2, SEQ ID
NO:3, SEQ ID NO:4, SEQ ID NO:9, SEQ ID NO:10, and fragments
thereof. In some embodiments, the polypeptide fragment of the
putative N-acetylmuramoyl-L-alanine amidase protein is selected
from SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, and fragments
thereof.
[0041] Unless otherwise specified, "a," "an," "the," and "at least
one" are used interchangeably and mean one or more than one.
BRIEF DESCRIPTION OF THE FIGURES
[0042] FIGS. 1a and 1b represent the homology between the amino
acid sequences for hypothetical CD1021 proteins from C. difficile
strain 630 (SEQ ID NO:1, corresponding to GenBank Accession No.
YP.sub.--001087502), C. difficile QCD-32g58 (SEQ ID NO:11,
corresponding to GenBank Accession No. ZP.sub.--01804840), C.
difficile QCD-32g58 (SEQ ID NO:12, corresponding to GenBank
Accession No. ZP.sub.--01804841), C. difficile QCD-32g58 (SEQ ID
NO:21, translated from the region corresponding to nucleotides
461827 to 462825 of GenBank Accession No. NZ_AAML04000007), C.
difficile QCD-32g58 (SEQ ID NO:22, translated from region
corresponding to nucleotides 462824 to 463732 of GenBank Accession
No. NZ_AAML04000007), and from C. difficile QCD-66c26 (SEQ ID
NO:23, translated from the complement of the region corresponding
to nucleotides 15690 to 17597 of GenBank Accession No.
NZ_ABFD01000037). The sequences were aligned using the multiple
sequence alignment program CustalW, which is publicly available.
The consensus sequence as shown is SEQ ID NO:38. Amino acid
residues that are identical in at least four of the six
hypothetical proteins are shown in the consensus sequence. An "X"
residue in the consensus sequence indicates that two or more of the
aligned sequences showed nonidentity at the respective residue or
it indicates that sequence information was lacking for the
respective residue in three or more of the aligned sequences. A "."
symbol located at any given position in one of the aligned
sequences indicates that amino acid position was unreported in the
corresponding GenBank entry.
DETAILED DESCRIPTION
[0043] The present invention relates to antibodies that bind to the
endospore of the bacterium Clostridium difficile (also referred to
herein as "C. difficile," "C. diff," "c. diff," "C-diff," or
"C.D"). Such spore-specific antibodies are useful, for example, in
the detection of C. difficile endospores in environmental,
biological, and food samples. Only a few genera of bacteria, such
as, for example, Bacillus and Clostridium, are capable of forming
endospores. Bacterial endospores are highly resistant to hostile
physical and chemical conditions, proving to be one of the most
durable types of cells found in nature. They can survive high heat,
drying, radiation, and many damaging chemicals and are a dormant
form of the bacterium that allows it to survive sub-optimal
environmental conditions. Endospores can survive for a very long
time and then return to a growing state, a process termed
germination. Because endospores are resistant to heat, radiation,
disinfectants, and desiccation, they are difficult to eliminate
from medical and pharmaceutical materials and are a frequent cause
of contamination.
[0044] Antibodies of the present invention bind to the endospore
(also referred to herein as "spore") of the bacterium C. difficile.
As used herein, the terms "antibody" or "antibodies" are used
interchangeably. An antibody of the present invention may bind to
both viable spores and inactivated C. difficile spores. Spores may
be inactivated by any of a variety of methods, including, but not
limited to, for example, treatment with formalin, formaldehyde,
glutaraldehydes, chemical disinfectants, autoclaving, and
ultraviolet radiation. Antibodies of the present invention may bind
to both germinated and ungerminated C. difficile spores. Antibodies
of the present invention may bind to ungerminated C. difficile
spores and not bind to germinated C. difficile spores. Antibodies
of the present invention may bind to germinated C. difficile spores
and not bind to ungerminated C. difficile spores. Methods for the
preparation of ungerminated spores and germinated spores are well
known to the skilled artisan. Briefly, bacterial spores are
generally prepared by growing the bacteria on media such as tryptic
soy agar or in tryptic soy broth until most cells turn into spores.
Spores are collected by centrifugation and washed several times
with a buffer such as PBS. The suspension can be treated with
alcohol to kill vegetative cells and washed to collect spores (see,
for example, Long and Williams, 1958, J Bacteriol: 76:332 and
Powers, 1968, Appl Microbiol; 16:180-181). Spore germination can be
triggered by a variety of methods. See, for example, Gould, 1970, J
Appl Bacteriol; 33:34-49; Foerster and Foster, 1966, J Bacteriol;
91:1168-1177; Moir and Smith, 1990, Ann Rev Microbiol; 44:531-553;
and U.S. Patent Application Publication No. 2003/0175318A1.
[0045] Antibodies of the present invention may bind C. difficile
spores and not bind to the spores of other endospore-forming
bacteria. Antibodies of the present invention may bind C. difficile
spores and not bind to the spores of other endospore-forming
bacteria of the Firmicute phylum, such as for example, endospores
produced by any of the various species of the Clostridium or
Bacillus genera. Species of the Clostridium and Bacillus genera of
bacteria include, but are not limited to, Clostridium aceticum,
Clostridium acetobutylicum, Clostridium botulinum, Clostridium
butyricum, Clostridium carnis, Clostridium chauvoei, Clostridium
denitrificans, Clostridium fervidus, Clostridium formicoaceticum,
Clostridium novyi, Clostridium pasteurianum, Clostridium
perfringens, Clostridium septicum, Clostridium sporogenes,
Clostridium tetani, Clostridium thermoaceticum, Clostridium
thermocellum, Clostridium thermosacchrolyticum, Clostridium
tyrobutyricum, Clostridium welchii, Bacillus agaradhaerens,
Bacillus alcalophilus, Bacillus amyloliquefaciens, Bacillus
anthracia, Bacillus atrophaeus, Bacillus azotoformans, Bacillus
badius, Bacillus benzoevorans, Bacillus carboniphilus, Bacillus
cereus, Bacillus chitinolyticus, Bacillus circulans, Bacillus
clarkii, Bacillus clausii, Bacillus coagulans, Bacillus cohnii,
Bacillus edaphicus, Bacillus ehimensis, Bacillus fastidiosus,
Bacillus firmus, Bacillus flexus, Bacillus fumarioli, Bacillus
fusiformis, Bacillus gibsonii, Bacillus globisporus, Bacillus
halmapalus, Bacillus haloalkaliphilus, Bacillus halodenitrificans,
Bacillus halodurans, Bacillus halophilus, Bacillus horikoshii,
Bacillus horti, Bacillus infernos, Bacillus insolitus, Bacillus
kaustophilus, Bacillus laevolacticus, Bacillus lentus, Bacillus
licheniformis, Bacillus marinus, Bacillus megaterium, Bacillus
methanolicus, Bacillus mojavensis, Bacillus mucilaginosus, Bacillus
mycoides, Bacillus naganoensis, Bacillus niacini, Bacillus
oleronius, Bacillus pallidus, Bacillus pasteurii, Bacillus
pseudalcaliphilus, Bacillus Pseudofirmus, Bacillus pseudomycoides,
Bacillus psychrophilus, Bacillus psychrosaccharolyticus, Bacillus
pumilus, Bacillus schlegelii, Bacillus silvestris, Bacillus
simplex, Bacillus siralis, Bacillus smithii, Bacillus sphaericus,
Bacillus sporothermodurans, Bacillus stearothermophilus, Bacillus
subtilis, Bacillus thermoamylovorans, Bacillus thermocatenulatus,
Bacillus thermocloaceae, Bacillus thermodenitrificans, Bacillus
thermoglucosidasius, Bacillus thermoleovorans, Bacillus
thermosphaericus, Bacillus thuringiensis, Bacillus tusciae,
Bacillus vallismortis, Bacillus vedderi, Bacillus vulcani, and
Bacillus weihenstephanensis.
[0046] Antibodies of the present invention may bind C. difficile
spores and not bind to the spores of other bacteria, such as, for
example, Desulfotomaculum, Sporolactobacillus, Brevibacillus,
Sporosarcina, and Thermoactinomyces.
[0047] In some embodiments, antibodies of the present invention
bind to C. difficile spores and do not bind to the spores of
Bacillus subtilis (also referred to herein as B. subtilis) and
Clostridium sporogenes (also referred to herein as C.
sporogenes).
[0048] Antibodies of the present invention may bind to C. difficile
spores and not bind to C. difficile vegetative cells. Antibodies of
the present invention may bind to C. difficile spores and not bind
to vegetative cells of other endospore-forming bacteria, including
any of those described herein. In some embodiments, antibodies of
the present invention do not bind to vegetative cells of C.
difficile, B. subtilis, and C. sporogenes. Methods for culturing
vegetative cells of a wide variety of Clostridium and Bacillus
species, including, but not limited C. difficile, C. sporogenes,
and B. subtilis, are well known to the skilled artisan. See, for
example, Madigan et al., 2003, Brock Biology of Microorganisms,
Prentice Hall; and Cappucino, 2005, Microbiology Laboratory Manual,
Benjamin Cummings.
[0049] Pathogenic C. difficile strains produce various toxins. The
best characterized are enterotoxin (toxin A) and cytotoxin (toxin
B) and these two toxins are responsible for the diarrhea and
inflammation seen in infected patients (see, for example,
Gianfrilli et al., 1984, Microbiologica; 7:375-9). Antibodies of
the present invention may bind to C. difficile spores and not bind
to a toxin produced by C. difficile, for example, the antibody may
not bind to toxin A and/or toxin B. Methods for preparing C.
difficile toxin A and toxin B and determining if an antibody binds
to C. difficile toxin A and/or toxin B are well known to the
skilled artisan. See, for example, U.S. Pat. Nos. 4,530,833;
4,533,630; 4,863,852; 4,879,218; 5,231,003; 5,610,023; 5,965,375;
6,503,722; 6,939,548; and 7,179,611.
[0050] Bacterial endospores, including C. difficile endospores, are
encased in a multilayered protein structure formed by the ordered
assembly of many polypeptides. The endospore contains four
protective layers, the core, the cortex, the coat, and the
exosporium. The outermost layer of the spore is the exosporium, a
thin covering made of protein. Interior to this is the spore coat
which is made up of highly cross-linked keratin and layers of
spore-specific proteins. The spore coat is impermeable to many
toxic molecules and may also contain enzymes that are involved in
germination. The cortex lies beneath the spore coat and consists of
peptidoglycan. The core wall lies beneath the cortex and surrounds
the protoplast or core of the endospore. The core has normal cell
structures, such as DNA and ribosomes, but is metabolically
inactive.
[0051] Some embodiments of the present invention include antibodies
that bind to a spore-specific protein, such as, for example, an
exosporium protein, a spore coat protein, a spore cortex protein, a
spore inner membrane protein, or a spore core protein of C.
difficile. Such an antibody may bind to a spore-specific protein
found on one or more endospore-forming bacteria of the Firmicute
phylum described herein. In some embodiments, the antibody binds to
a spore-specific protein found in C. difficile, but does not bind
to the spore-specific protein other endospore-forming bacteria of
the Firmicute phylum, such as, for example, B. subtilis and C.
sporogenes.
[0052] Some embodiments of the present invention include antibodies
that bind to a spore coat assembly protein of C. difficile. One
such spore coat assembly protein is the CotH protein (also referred
to herein as "cotH"). The CotH protein is a structural component of
the spore coat and has been well characterized in B. subtilis. It
is involved in directing the assembly of coat proteins and in
stabilizing coat proteins. See, for example, Naclerio et al., 1996,
J Bacteriol; 178(15):4375-4380 and Zilha et al., 1999, J Bacteriol;
181:2631-2633). The present invention includes antibodies that bind
to a putative CotH protein in C. difficile.
[0053] The complete genome sequence of C. difficile strain 630 has
been determined and is available in the GenBank.RTM. sequence
database maintained by the National Center for Biotechnology
Information (NCBI), National Library of Medicine (NLM), National
Institutes of Health (NIH). See also, Sebaihia et al., 2006, Nat.
Genet; 38 (7):779-786). Strain 630 is multi-drug resistant and was
isolated from a patient with severe pseudomembraneous colitis that
had spread to dozens of other patients on the same ward in Zurich,
Switzerland in 1982 (Wren, 2006, Future Microbiol; 1(3):243-245).
Thus, strain 630 has the genetic attributes of a fully virulent,
highly transmissible, drug resistant strain.
[0054] Efforts are currently under way to obtain the complete
genome sequences of other C. difficile strains. The Sanger
Institute (Wellcome Trust Genome Campus, Hinxton, Cambridge, UK) is
sequencing the genome of C. difficile strain 820291. C. difficile
strain R20291 was isolated in Stoke Mandeville, UK, and is closely
related to the North American hypervirulent BI strains. Washington
University in St. Louis (St. Louis, Mo.) is sequencing the genome
of C. difficile QCD-32g58.
[0055] A thorough search of all the GenBank.RTM. entries for C.
difficile strain 630 identified hypothetical protein CD 1021
(YP.sub.--001087502), which demonstrates a conserved domain (amino
acid residues 90 to 393) which is homologous to the spore coat
assembly protein H (cotH) of B. subtilis. The analysis was
performed using conserved domain search tools available from the
NCBI. Hypothetical Protein CD1021 of C. difficile 630 (GenBank
Accession No. YP.sub.--001087502) has the amino acid sequence SEQ
ID NO:1. See Sebaihia et al., 2006, Nat. Genet; 38 (7):779-786.
[0056] Some embodiments of the present invention include antibodies
that bind to the hypothetical protein CD1021 of C. difficile, and
fragments thereof. An antibody of the present invention may bind to
a hypothetical protein CD1021 in a variety of C. difficile strains,
including, but not limited to, any of the C. difficile strains
discussed herein. For example, an antibody of the present invention
may bind to the hypothetical protein CD1021 of C. difficile strain
630, C. difficile strain R20291, C. difficile strain QCD-32q58, C.
difficile strain QCD-66c26, C. difficile ATCC 43255, C. difficile
ATCC 43593, C. difficile ATCC 43594, C. difficile ATCC 43596, C.
difficile ATCC 43597, C. difficile ATCC 43598, C. difficile ATCC
43603, C. difficile ATCC 9689, and/or C. difficile ATCC 700792.
Antibodies of the present invention include antibodies that bind to
the hypothetical protein CD1021 of C. difficile strain 630 having
SEQ ID NO:1.
[0057] Some embodiments of the present invention include antibodies
that bind to polypeptide fragments of the hypothetical protein
CD1021 of C. difficile. A polypeptide fragment may be, for example,
about 50, about 100, about 200, about 300, about 400, about 500, or
about 600 amino acids in length. A polypeptide fragment may be, for
example, about 10, about 15, about 20, about 25, about 30, about
35, about 40, or about 45 amino acids in length. A polypeptide
fragment may be about 8-20, about 12-15, or about 10-20 amino acids
in length.
[0058] Some embodiments of the present invention include antibodies
that bind to polypeptide fragments of the hypothetical protein
CD1021 of C. difficile strain 630 (SEQ ID NO:1). For example, the
present invention includes antibodies that bind to a polypeptide
including residues 505-604 of hypothetical protein CD1021 of C.
difficile strain 630 (SEQ ID NO:2), antibodies that bind to a
polypeptide including residues 30-120 of hypothetical protein
CD1021 of C. difficile strain 630 (SEQ ID NO:9), antibodies that
bind to a polypeptide including residues 194-293 of hypothetical
protein CD1021 of C. difficile strain 630 (SEQ ID NO:10),
antibodies that bind to a polypeptide including residues 203 to 217
of hypothetical protein CD 1021 of C. difficile strain 630 (SEQ ID
NO:3), and antibodies that bind to a polypeptide including residues
333 to 347 of hypothetical protein CD1021 of C. difficile strain
630 (SEQ ID NO:4).
[0059] The spore cortex, a thick layer of peptidoglycan, is
responsible for maintaining the highly dehydrated state of the
spore and contributes to the extreme dormancy and heat resistance
of the spores. Bacterial spore germination includes a series of
degradation events that lead to the irreversible loss of spore
dormancy and the rehydration of the core. The spore contains
enzymes that are involved in germination. Thus, an antibody that
binds to a spore-specific protein involved in germination may be
used to identify germinating spores. The present invention includes
antibodies that bind to a spore-specific protein involved in
germination. Such an antibody may bind to germinated spores but not
bind to ungerminated spores. Such an antibody may bind to
ungerminated spores but not bind to germinated spores.
[0060] Cortex lytic enzymes, including the amidase N-acetylmuramoyl
L-alanine amidase, play a key role in germination, resulting in
hydrolysis of the cortex (see, for example, Moriyama et al., 1996,
J Bacteriol; 181:2373-2378). The present invention includes
antibodies that bind to a C. difficile amidase, including
antibodies that bind to the N-acetylmuramoyl-L-alanine amidase of
C. difficile.
[0061] A thorough search of all the GenBank.RTM. entries for C.
difficile strain 630 identified cell surface protein (putative
N-acetylmuramoyl-L-alanine amidase, YP.sub.--001087517, also
referred to herein as "CD1036") which has conserved domains
CW_binding.sub.--2 (putative cell wall binding repeat 2; 174 to
265, 275 to 368, 381 to 461) and Amidase.sub.--3
(N-acetylmuramoyl-L-alanine amidase, 493 to 673). The putative
N-acetylmuramoyl-L-alanine amidase cell surface protein of C.
difficile 630 (GenBank Accession No. YP.sub.--001087517) has the
amino acid sequence SEQ ID NO:5. See Sebaihia et al., 2006, Nat.
Genet; 38 (7):779-786.
[0062] The present invention includes antibodies that bind to the
putative N-acetylmuramoyl-L-alanine amidase of C. difficile, and
fragments thereof. An antibody of the present invention may bind to
the putative N-acetylmuramoyl-L-alanine amidase in a variety of C.
difficile strains, including, but not limited to, any of the C.
difficile strains discussed herein. For example, an antibody of the
present invention may bind to the putative
N-acetylmuramoyl-L-alanine amidase of C. difficile strain 630, C.
difficile strain R20291, C. difficile strain QCD-32q58, C.
difficile ATCC 43255, C. difficile ATCC 43593, C. difficile ATCC
43594, C. difficile ATCC 43596, C. difficile ATCC 43597, C.
difficile ATCC 43598, C. difficile ATCC 9689, and/or C. difficile
ATCC 700792. Antibodies of the present invention include antibodies
that bind to the putative N-acetylmuramoyl-L-alanine amidase of C.
difficile strain 630 having SEQ ID NO:5.
[0063] Some embodiments of the present invention include antibodies
that bind to polypeptide fragments of the putative
N-acetylmuramoyl-L-alanine amidase. A polypeptide fragment may be,
for example, about 50, about 100, about 200, about 300, about 400,
about 500, or about 600 amino acids in length. A polypeptide
fragment may be, for example, about 10, about 15, about 20, about
25, about 30, about 35, about 40, or about 45 amino acids in
length. A polypeptide fragment may be about 8-20, about 12-15, or
about 10-20 amino acids in length. The present invention includes
antibodies that bind to polypeptide fragments of the putative
N-acetylmuramoyl-L-alanine amidase of C. difficile strain 630
having SEQ ID NO:5. For example, the present invention includes
antibodies that bind to a polypeptide including residues 294-393 of
putative N-acetylmuramoyl-L-alanine amidase of C. difficile strain
630 (SEQ ID NO:6), antibodies that bind to a polypeptide including
residues 582-596 of putative N-acetylmuramoyl-L-alanine amidase of
C. difficile strain 630 (SEQ ID NO:7), and antibodies that bind to
a polypeptide including residues 64-78 of putative
N-acetylmuramoyl-L-alanine amidase of C. difficile strain 630 (SEQ
ID NO:8).
[0064] Antibodies of the present invention include, but are not
limited to, polyclonal antibodies, affinity-purified polyclonal
antibodies, monoclonal antibodies, human antibodies, humanized
antibodies, chimeric antibodies, anti-idiotypic antibodies,
multispecific antibodies, single chain antibodies, single-chain Fvs
(scFv), disulfide-linked Fvs (sdFv), Fab fragments, F(ab')
fragments, F(ab')2 fragments, Fv fragments, diabodies, linear
antibodies fragments produced by a Fab expression library,
fragments comprising either a VL or VH domain, intracellularly-made
antibodies (i.e., intrabodies), and antigen-binding antibody
fragments thereof. Any of a wide variety of target antigens may be
used to produce the antibodies of the present invention, including,
but not limited to, C. difficile cells, spores or toxins, proteins,
peptides, carbohydrates and combinations thereof. Proteins and
peptides may be, for example, naturally occurring, chemically
synthesized, or recombinantly produced. An antigen may be
conjugated to a carrier.
[0065] Also included in the present invention are various antibody
fragments, also referred to as antigen binding fragments, which
include only a portion of an intact antibody, generally including
an antigen binding site of the intact antibody and thus retaining
the ability to bind antigen. Fragments can be obtained via chemical
or enzymatic treatment of an intact or complete antibody or
antibody chain. Fragments can also be obtained by recombinant
means. Examples of antibody fragments include, for example, Fab,
Fab', Fd, Fd', Fv, dAB, and F(ab')2 fragments produced by
proteolytic digestion and/or reducing disulfide bridges and
fragments produced from an Fab expression library. Such antibody
fragments can be generated by techniques well known in the art.
Antibodies of the present invention can include the variable
region(s) alone or in combination with the entirety or a portion of
the hinge region, CH1 domain, CH2 domain, CH3 domain and/or Fc
domain(s). The term "antigen-binding fragment" refers to a
polypeptide fragment of an immunoglobulin or antibody that binds
antigen or competes with the intact antibody for antigen
binding.
[0066] The antibodies of the present invention can be of any type
(such as, for example, IgG, IgE, IgM, IgD, IgA and IgY), class
(such as, for example, IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or
subclass of immunoglobulin molecule. In some embodiments, the
immunoglobulin is an IgG. Immunoglobulins can have both heavy and
light chains. An array of IgG, IgE, IgM, IgD, IgA, and IgY heavy
chains can be paired with a light chain of the kappa or lambda
form.
[0067] The antibodies of the invention can be from any animal
origin, including birds and mammals. In some embodiments, the
antibodies are human, murine, rat, donkey, sheep, rabbit, goat,
guinea pig, camel, horse, llama, camel, or chicken antibodies. As
used herein, "human" antibodies include antibodies having the amino
acid sequence of a human immunoglobulin and include antibodies
isolated from human immunoglobulin libraries or from animals
transgenic for one or more human immunoglobulins.
[0068] Antibodies of the present invention may be a polyclonal
antibody. The term "polyclonal antibody" refers to an antibody
produced from more than a single clone of plasma cells. In contrast
"monoclonal antibody" refers to an antibody produced from a single
clone of plasma cells. The preparation of polyclonal antibodies is
well known.
[0069] A polyclonal antibody to a target antigen may be obtained by
immunizing any of a variety of host animals with an immunogen. Any
of a wide variety of immunization protocols may be used. The host
animal may be any mammal, for example, a mouse, hamster, rat,
rabbit, guinea pig, goat, sheep, horse, cow, buffalo, bison, camel,
or llama. A host animal may be a bird, for example, a chicken or a
turkey. In some embodiments, an antibody preparation, rather than
obtained from a blood sample, is obtained from another fluid
source, for example, from milk, colostrums, egg white, or egg yolk.
In some embodiments, an antibody preparation is obtained, not by
immunizing a host animal with the target antigen, but rather, from
an individual with a prior exposure to the antigen or from pooled
serum, for example, from pooled human serum.
[0070] It may be useful to conjugate the immunizing agent to a
protein known to be immunogenic in the mammal being immunized
Examples of such immunogenic proteins include, but are not limited
to, keyhole limpet hemocyanin (KLH), serum albumin, bovine
thyroglobulin, and soybean trypsin inhibitor. Examples of adjuvants
which may be employed include Freund's complete adjuvant and
MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose
dicorynomycolate). The immunization protocol may be selected by one
skilled in the art without undue experimentation.
[0071] Some embodiments of the present invention include antiserum
that binds to a C. difficile spore. As used herein, antiserum
refers to the blood from an immunized host animal from which the
clotting proteins and red blood cells (RBCs) have been removed. An
antiserum (also referred to herein as an "antiserum preparation,"
"crude antiserum," or "raw antiserum") still possesses
immunoglobulins of all classes as well as other various serum
proteins. Thus, in addition to antibodies that recognize the target
antigen, the antiserum also contains antibodies to various
non-target antigens that can sometimes react non-specifically in
immunological assays.
[0072] In some embodiments of the present invention, an antibody
may be enriched. Such enrichment may eliminate non-immunoglobulin
proteins from the preparation and/or enrich for one or more classes
of immunoglobulin (such as, for example, IgG) within the sample.
Any of a variety of methods may be used to obtain such an enriched
antibody, including, but not limited to, those described herein.
Methods of eliminating non-immunoglobulin serum proteins from an
antibody preparation and methods for enriching for the IgG fraction
are well known in the art. For example, ammonium sulfate
precipitation, Protein A binding, Protein G binding, or caprylic
acid precipitation may be used to enrich for the IgG class of
antibodies.
[0073] Antibodies of the present invention include antibodies with
enhanced avidity for the target antigen. Such antibodies may be
prepared by antigen affinity immunoadsorption. Antigen affinity
immunoadsorption may be carried out by any of a variety of means.
For example, antigen affinity immunoadsorption may be carried out
by antigen affinity column chromatography. Column chromatography
may be carried out by any mechanical means, for example, carried
out in a column run with or without pressure, carried out in a
column run from top to bottom or bottom to top, or the direction of
the flow of fluid in the column may be reversed during the
chromatography process. Alternatively, antigen affinity
immunoadsorption may be carried out by means other than column
chromatography. For example, affinity immunoadsorption may be
carried out using a batch process in which the solid support is
separated from the liquid used to load, wash, and elute the sample
by any suitable means, including gravity, centrifugation, or
filtration. Affinity immunoadsorption also may be carried out by
contacting the sample with a filter that adsorbs or retains some
molecules in the sample more strongly than others. The antigen
affinity column may be prepared by any of a variety of methods,
including, but not limited to, those described herein. The binding
of an antibody preparation to an antigen affinity column may be
carried out by any of a wide variety of immunoadsorption methods,
including, but not limited to, those described herein. The binding
of the antibody preparation to the antigen affinity column may
occur in a variety of buffers or salts including, but not limited
to, sodium, potassium, ammonium, chloride, acetate, phosphate,
citrate, Tris buffers and/or organic buffers with a buffering
capacity near neutrality. Specific examples of such buffers and
salts include, for example, Tris, sodium phosphate, potassium
phosphate, ammonium phosphate, sodium chloride, potassium chloride,
ammonium chloride, sodium citrate, potassium citrate, ammonium
citrate, sodium acetate, potassium acetate, or ammonium
acetate.
[0074] Antibodies of the present invention include monoclonal
antibodies. A population of monoclonal antibodies is homogeneous.
All of the monoclonal antibodies in the preparation recognize the
same epitope on the target molecule and all of the monoclonal
antibodies have the same affinity. As used herein, "affinity" is
the binding strength of the interaction of a monoclonal antibody
with its antigenic epitope. As used herein, an "epitope" is the
portion of an antigen bound by an antibody. The higher the
affinity, the tighter the association between antigen and antibody,
and the more likely the antigen is to remain in the binding
site.
[0075] Monoclonal antibodies of the present invention include, but
are not limited to, humanized antibodies, chimeric antibodies,
single chain antibodies, single-chain Fvs (scFv), disulfide-linked
Fvs (sdFv), Fab fragments, F(ab') fragments, F(ab')2 fragments, Fv
fragments, diabodies, linear antibodies fragments produced by a Fab
expression library, fragments including either a VL or VH domain,
intracellularly-made antibodies (i.e., intrabodies), and
antigen-binding antibody fragments thereof.
[0076] Monoclonal antibodies of the present invention can be
produced by an animal (including, but not limited to, human, mouse,
rat, rabbit, hamster, goat, horse, chicken, or turkey), chemically
synthesized, or recombinantly expressed. Monoclonal antibodies of
the present invention can be purified by any method known in the
art for purification of an immunoglobulin molecule, for example, by
chromatography (e.g., ion exchange, affinity, and sizing column
chromatography), centrifugation, differential solubility, or by any
other standard technique for the purification of proteins. In
addition, the antibodies of the present invention or fragments
thereof can be fused to heterologous polypeptide sequences
described herein or otherwise known in the art, to facilitate
purification.
[0077] Monoclonal antibodies of the present invention may be of any
isotype. The monoclonal antibodies of the present invention may be,
for example, murine IgM, IgG1, IgG2a, IgG2b, IgG3, IgA, IgD, or
IgE. The monoclonal antibodies of the present invention may be, for
example, human IgM, IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, or
IgE. In some embodiments, the monoclonal antibody may be murine
IgG2a, IgG1, or IgG3. With the present invention, a given heavy
chain may be paired with a light chain of either the kappa or the
lambda form.
[0078] Monoclonal antibodies can be obtained by various techniques
familiar to those skilled in the art. For example, spleen cells
from an animal immunized with a desired antigen are immortalized,
commonly by fusion with a myeloma cell (see, for example, Kohler
and Milstein, 1976, Eur J Immunol; 6:511-519; J. Goding In
"Monoclonal Antibodies: Principles and Practice," Academic Press,
pp 59-103 (1986); and Harlow et al., Antibodies: A Laboratory
Manual, page 726 (Cold Spring Harbor Pub. (1988)). Monoclonal
antibodies can be isolated and purified from hybridoma cultures by
techniques well known in the art. Other known methods of producing
transformed B cell lines that produce monoclonal antibodies may
also be used. Monoclonal antibodies of the present invention may be
produced by recombinant DNA techniques, for example, produced by
phage display or by combinatorial methods. See, for example, U.S.
Pat. No. 5,223,409; WO 92/18619; WO 91/17271; WO 92/20791; WO
92/15679; WO 93/01288; WO 92/01047; WO 92/09690; or WO 90/02809.
Such methods can be used to generate human monoclonal
antibodies.
[0079] Monoclonal antibodies of the present invention include
chimeric antibodies. A chimeric antibody is one in which different
portions are derived from different animal species. For example,
chimeric antibodies can be obtained by splicing the genes from a
mouse antibody molecule with appropriate antigen specificity
together with genes from a human antibody molecule of appropriate
biological specificity. See, for example, Takeda et al., 1985,
Nature; 314:544-546.
[0080] A therapeutically useful antibody may be derived from a
"humanized" monoclonal antibody. Humanized monoclonal antibodies
are produced by transferring one or more CDRs from the heavy and
light variable chains of a mouse (or other species) immunoglobulin
into a human variable domain, then substituting human residues into
the framework regions of the murine counterparts. The use of
antibody components derived from humanized monoclonal antibodies
obviates potential problems associated with immunogenicity of
murine constant regions. Techniques for producing humanized
monoclonal antibodies can be found, for example, in Jones et al.,
1986, Nature; 321:522 and Singer et al., 1993, J Immunol: 150:2844.
The constant region of a humanized monoclonal antibody of the
present invention can be that from human immunoglobulin belonging
to any isotype. It may be, for example, the constant region of
human IgG. The framework regions of the constant region derived
from human immunoglobulin are not particularly limited.
[0081] An intact antibody molecule has two heavy (H) chain variable
regions (abbreviated herein as VH) and two light (L) chain variable
regions (abbreviated herein as VL). The VH and VL regions can be
further subdivided into regions of hypervariability, termed
"complementarity determining regions" ("CDR"), interspersed with
regions that are more conserved, termed "framework regions" (FR).
The extent of the framework region and CDR's has been precisely
defined (see, Kabat, E. A., et al. (1991) Sequences of Proteins of
Immunological Interest, Fifth Edition, U.S. Department of Health
and Human Services, NIH Publication No. 91-3242, and Chothia, C. et
al., J. Mol. Biol. 1987; 196: 901-917). Each VH and VL is composed
of three CDR's and four FRs, arranged from amino-terminus to
carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,
CDR3, FR4. The present invention includes an antibody with the
heavy chain, the light chain, the heavy chain variable region, the
light chain variable region, and/or one or more complementarity
determining regions of a monoclonal antibody of the present
invention.
[0082] The present invention includes bispecific or bifunctional
antibodies. A bispecific or bifunctional antibody is an artificial
hybrid antibody having two different heavy/light chain pairs and
two different binding sites. Bispecific antibodies can be produced
by a variety of methods including fusion of hybridomas or linking
of F(ab') fragments. See, for example, Songsivilai and Lachmann,
1990, Clin Exp Immunol; 79:315-321 and Kostelny et al., 1992, J
Immunol; 148:1547-1553. In addition, bispecific antibodies can be
formed as "diabodies" (Holliger et al., 1993, PNAS USA:
90:6444-6448) or "Janusins" (Traunecker et al., 1991, EMBO J;
10:3655-3659 and Traunecker et al., 1992, Int J Cancer Suppl;
7:51-52).
[0083] Also included in the present invention are hybridoma cell
lines, transformed B cell lines, and host cells that produce the
monoclonal antibodies of the present invention; the progeny or
derivatives of these hybridomas, transformed B cell lines, and host
cells; and equivalent or similar hybridomas, transformed B cell
lines, and host cells. Progeny or derivatives thereof may produce
an antibody with one or more of the identifying characteristics,
such as, for example, isotype and antigen specificity, of the
antibody produced by the parental line.
[0084] The present invention further provides an isolated
polynucleotide molecule having a nucleotide sequence encoding a
monoclonal antibody of the invention. The present invention is
further directed to an isolated polynucleotide molecule having a
nucleotide sequence that has at least 75%, at least 80%, at least
85%, at least 90%, at least 95%, at least 96%, at least 97%, at
least 98%, or at least 99% sequence identity to nucleotide sequence
encoding a monoclonal antibody of the invention. The invention also
encompasses polynucleotides that hybridize under high stringency to
a nucleotide sequence encoding an antibody of the invention, or a
complement thereof. As used herein "stringent conditions" refer to
the ability of a first polynucleotide molecule to hybridize, and
remain bound to, a second, filter-bound polynucleotide molecule in
0.5 M NaHPO4, 7% sodium dodecyl sulfate (SDS), and 1 mM EDTA at
65.degree. C., followed by washing in 0.2.times.SSC/0.1% SDS at
42.degree. C. (see Ausubel et al. (eds.), Current Protocols in
Molecular Biology, Vol. 1, Green Publishing Associates, Inc., and
John Wiley & Sons, Inc., NY, at p. 2.10.3 (1989)). Also
included in the present invention are polynucleotides that encode
one or more of the CDR regions or the heavy and/or light chains of
a monoclonal antibody of the present invention. General techniques
for cloning and sequencing immunoglobulin variable domains and
constant regions are well known. See, for example, Orlandi et al.,
1989, PNAS USA; 86:3833.
[0085] The present invention also includes recombinant vectors
including an isolated polynucleotide of the present invention. The
vector can be, for example, in the form of a plasmid, a viral
particle, or a phage. The appropriate DNA sequence can be inserted
into a vector by a variety of procedures. In general, the DNA
sequence is inserted into an appropriate restriction endonuclease
site(s) in a vector by procedures known in the art. Such procedures
are deemed to be within the scope of those skilled in the art.
Large numbers of suitable vectors and promoters are known to those
of skill in the art, and are commercially available. The following
vectors are provided by way of example. Bacterial vectors include,
for example, pQE70, pQE60, pQE-9, pBS, pD10, phagescript, psiX174,
pbluescript SK, pbsks, pNH8A, pNH16a, pNH18A, pNH46A, ptrc99a,
pKK223-3, pKK233-3, pDR540, and pRIT5. Eukaryotic vectors include,
for example, pWLNEO, pSV2CAT, pOG44, pXT1, pSG, pSVK3, pBPV, pMSG,
and pSVL. However, any other plasmid or vector can be used.
[0086] Some embodiments of the present invention also include host
cells containing the above-described vectors. The host cell can be
a higher eukaryotic cell, such as a mammalian or insect cell, or a
lower eukaryotic cell, such as a yeast cell. Or, the host cell can
be a prokaryotic cell, such as a bacterial cell, or a plant cell.
Introduction of a vector construct into the host cell can be
effected by any suitable techniques, such as, for example, calcium
phosphate transfection, DEAE-Dextran mediated transfection, or
electroporation. (Davis, L., et al., Basic Methods in Molecular
Biology (1986)).
[0087] Monoclonal antibodies of the present invention can be
expressed in mammalian cells, yeast, bacteria, or other cells under
the control of appropriate promoters. Cell-free translation systems
can also be employed to produce such proteins using RNAs derived
from the DNA constructs of the present invention. Appropriate
cloning and expression vectors for use with prokaryotic and
eukaryotic hosts are described by Sambrook, et al., Molecular
Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor,
N.Y. (1989).
[0088] Also included in the present invention are phage display
libraries expressing one or more hypervariable regions from a
monoclonal antibody of the present invention, and clones obtained
from such a phage display library. A phage display library is used
to produce antibody derived molecules. Gene segments encoding the
antigen-binding variable domains of antibodies are fused to genes
encoding the coat protein of a bacteriophage. Bacteriophage
containing such gene fusions are used to infect bacteria, and the
resulting phage particles have coats that express the
antibody-fusion protein, with the antigen-binding domain displayed
on the outside of the bacteriophage. Phage display libraries can be
prepared, for example, using the Ph.D.TM.-7 Phage Display Peptide
Library Kit (Catalog No. E8100S) or the Ph.D.TM.-12 Phage Display
Peptide Library Kit (Catalog No. E8110S) available from New England
Biolabs Inc., Ipswich, Mass. See also, Smith and Petrenko, 1997,
Chem Rev; 97:391-410.
[0089] The antibodies of the present invention may be coupled
directly or indirectly to a substrate or detectable marker by
techniques well known in the art. A detectable marker is an agent
detectable, for example, by spectroscopic (e.g., u.v., i.r.,
visible, Raman, surface enhanced Raman scattering (SERS), mass
spectroscopy), photochemical, biochemical, immunochemical, or
chemical means. Useful detectable markers include, but are not
limited to, fluorescent dyes, chemiluminescent compounds,
radioisotopes, electron-dense reagents, enzymes, colored particles,
biotin, or dioxigenin. A detectable marker often generates a
measurable signal, such as radioactivity, fluorescent light, color,
or enzyme activity. Antibodies conjugated to detectable agents may
be used for diagnostic or therapeutic purposes. Examples of
detectable agents include various enzymes, naturally-occurring
spore-associated biomolecules (e.g., dipicolinic acid, calcium
dipicolinate), prosthetic groups, fluorescent materials,
luminescent materials, bioluminescent materials, radioactive
materials, positron emitting metals using various positron emission
tomographies, nonradioactive paramagnetic metal ions, Raman labels
and SERS labels. The detectable substance can be coupled or
conjugated either directly to the antibody or indirectly, through
an intermediate such as, for example, a linker known in the art,
using techniques known in the art. See, for example, U.S. Pat. No.
4,741,900, describing the conjugation of metal ions to antibodies
for diagnostic use. Examples of suitable enzymes include
horseradish peroxidase, alkaline phosphatase, beta-galactosidase,
and acetylcholinesterase; examples of suitable prosthetic group
complexes include streptavidin/biotin and avidin/biotin; examples
of suitable fluorescent materials include umbelliferone,
fluorescein, fluorescein isothiocyanate, rhodamine,
dichlorotriazinylamine fluorescein, dansyl chloride and
phycoerythrin; an example of a luminescent material includes
luminol; examples of bioluminescent materials include luciferin,
and aequorin; and examples of suitable radioactive material include
iodine (.sup.121I, .sup.123I, .sup.125I, .sup.131I), carbon
(.sup.14C), sulfur (.sup.35S), tritium (.sup.3H), indium
(.sup.111In, .sup.112In, .sup.113mIn, .sup.115mIn), technetium
(.sup.99Tc, .sup.99 mTc), thallium (.sup.201Ti), gallium
(.sup.68Ga, .sup.67Ga), palladium (.sup.103Pd) molybdenum
(.sup.99Mo), xenon (.sup.133Xe), fluorine (.sup.18F), .sup.153Sm,
.sup.177Lu, .sup.159Gd, .sup.149Pm, .sup.140La, .sup.175Yb,
.sup.166Ho, .sup.90Y, .sup.47Sc, .sup.186Re, .sup.188Re,
.sup.142Pr, .sup.105Rh, and .sup.97Ru. Techniques for conjugating
such moieties to antibodies are well-known.
[0090] Antibodies of the present invention include derivatives of
antibodies that are modified or conjugated by the covalent
attachment of any type of molecule to the antibody. Such antibody
derivatives include, for example, antibodies that have been
modified by glycosylation, acetylation, pegylation,
phosphorylation, amidation, derivatization by known
protecting/blocking groups, proteolytic cleavage, or linkage to a
cellular ligand or other protein. Any of numerous chemical
modifications can be carried out by known techniques, including,
but not limited to, specific chemical cleavage, acetylation,
formylation, and metabolic synthesis of tunicamycin. Additionally,
the derivatives can contain one or more non-classical amino
acids.
[0091] Antibodies of the present invention can be assayed for
immunospecific binding by the methods described herein and by any
suitable method known in the art. The immunoassays that can be used
include, but are not limited to, competitive and non-competitive
assay systems using techniques such as BIAcore analysis,
fluorescence activated cell sorter (FACS) analysis,
immunofluorescence, immunocytochemistry, Western blots,
radio-immunoassays, enzyme linked immunosorbent assay (ELISA),
"sandwich" immunoassays, immunoprecipitation assays, precipitin
reactions, gel diffusion precipitin reactions, immunodiffusion
assays, agglutination assays, complement-fixation assays,
immunoradiometric assays, fluorescent immunoassays, and protein A
immunoassays. Such assays are routine and well known in the art
(see, for example, Ausubel et al., eds, Current Protocols in
Molecular Biology, Vol. 1, John Wiley & Sons, Inc., NY
(1994)).
[0092] Also included in the present invention are compositions
including one or more of the antibodies described herein. A
composition may also include, for example, buffering agents to help
to maintain the pH in an acceptable range or preservatives to
retard microbial growth. A composition may include, for example,
carriers, excipients, stabilizers, chelators, salts, or
antimicrobial agents. Acceptable carriers, excipients, stabilizers,
chelators, salts, preservatives, buffering agents, or antimicrobial
agents, include, but are not limited to, buffers such as phosphate,
citrate, and other organic acids; antioxidants including ascorbic
acid and methionine; preservatives, such as sodium azide,
octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;
benzalkonium chloride, benzethonium chloride; phenol, butyl or
benzyl alcohol; alkyl parabens such as methyl or propyl paraben;
catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol;
polypeptides; proteins, such as serum albumin, gelatin, or
non-specific immunoglobulins; hydrophilic polymers such as
olyvinylpyrrolidone; amino acids such as glycine, glutamine,
asparagine, histidine, arginine, or lysine; monosaccharides,
disaccharides, and other carbohydrates including glucose, mannose,
or dextrins; chelating agents such as EDTA; sugars such as sucrose,
mannitol, trehalose or sorbitol; salt-forming counter-ions such as
sodium; metal complexes (for example, Zn-protein complexes); and/or
non-ionic surfactants such as TWEEN, PLURONICS, or polyethylene
glycol (PEG). As used herein, a composition is not a polyclonal
antiserum.
[0093] The invention also provides a kits or detection systems
including one or more antibodies of the present invention. The kit
may include one or more containers filled with one or more of the
antibodies of the invention. Additionally, the kit may include
other reagents such as buffers and solutions needed to practice the
invention are also included. Optionally associated with such
container(s) can be a notice or printed instructions. A kit can
include packaging material. As used herein, the phrase "packaging
material" refers to one or more physical structures used to house
the contents of the kit. The packaging material is constructed by
well known methods, which can provide a sterile, contaminant-free
environment.
[0094] The present invention includes isolated antibodies.
"Isolated," when used to describe the various antibodies disclosed
herein, means the antibody that has been identified and separated
and/or recovered from a component of its natural environment.
Contaminant components of its natural environment are materials
that would typically interfere with diagnostic or therapeutic uses
for the polypeptide, and may include enzymes, hormones, and other
proteinaceous or non-proteinaceous solutes.
[0095] The antibodies of the present invention may "specifically
bind to" or be "specific for" a particular polypeptide or an
epitope on a particular polypeptide. Such an antibody is one that
binds to that particular polypeptide or epitope on a particular
polypeptide without substantially binding to any other polypeptide
or polypeptide epitope.
[0096] Antibodies of the present invention can be produced by an
animal, chemically synthesized, or recombinantly expressed.
Antibodies of the present invention can be purified by any method
known in the art for purification of an immunoglobulin molecule,
for example, by chromatography (including, but not limited to, ion
exchange, affinity, and sizing column chromatography),
centrifugation, differential solubility, or by any other standard
technique for the purification of proteins. In addition, the
antibodies of the present invention or fragments thereof can be
fused to heterologous polypeptide sequences described herein or
otherwise known in the art, to facilitate purification or
detection.
[0097] The antibodies of the present invention may be used in a
wide variety of diagnostic and therapeutic methods, including, but
not limited to, methods for detecting C. difficile spores, and
polypeptide fragments thereof, and methods for isolating or
purifying C. difficile spores, or polypeptide fragments
thereof.
[0098] The antibodies of the present invention may be used in any
of the wide variety of immunoassay techniques known in the art to
determine the presence or absence of C. difficile spores in a
sample. As used herein, an immunoassay is a test that identifies
the presence of an analyte, such as C. difficile spores, in a
sample, using the reaction of an antibody to its antigen target.
The assay takes advantage of the specific binding of an antibody to
its antigen. Also included in the present invention are such
methods of detection.
[0099] In an immunoassay, a sample is contacted with one or more
antibodies and the antibodies are allowed to bind to their
antigenic target, if present in the sample. Then, the binding of
the one or more antibodies to their antigenic targets is
determined, by detecting antibody bound to its antigen target. Such
detection may be accomplished, for example, colorimetrically,
fluorimetrically, enzymatically, or with radioactive isotopes.
Depending on the format of the assay, detectable labels can be
bound to an antigen or an antibody. The detectable moiety should be
capable of producing, either directly or indirectly, a detectable
signal. For example, the detectable moiety may be a radioisotope,
such as, for example, .sup.3H, .sup.14C, .sup.32P, .sup.35S, or
.sup.125I, a fluorescent or chemiluminescent compound, such as, for
example, fluorescein isothiocyanate, rhodamine, or luciferin, or an
enzyme, such as, for example, alkaline phosphatase,
beta-galactosidase or horseradish peroxidase, a Raman label, or a
SERS label. Any method known in the art for conjugating an antibody
or antigen to a detectable moiety may be employed. A detectable
moiety (also referred to herein as a detectable label) may be
conjugated directly or indirectly to the antibody so as to generate
a "labeled" antibody. The label may be detectable by itself (e.g.
radioisotope labels or fluorescent labels) or, in the case of an
enzymatic label, may catalyze chemical alteration of a substrate
compound or composition which is detectable.
[0100] An immunoassay of the present invention includes, but is not
limited to, competitive and non-competitive assay systems, using
techniques such as BIAcore analysis, fluorescence activated cell
sorter (FACS) analysis, immunofluorescence, immunocytochemistry,
Western blots, radio-immunoassays, enzyme linked immunosorbent
assay (ELISA), "sandwich" immunoassays, immunoprecipitation assays,
precipitin reactions, gel diffusion precipitin reactions,
immunodiffusion assays, agglutination assays, complement-fixation
assays, immunoradiometric assays, fluorescent immunoassays, and
protein A immunoassays. Such assays are routine and well known in
the art (see, for example, Ausubel et al., eds, Current Protocols
in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., NY
(1994)).
[0101] An immunoassay of the present invention may be homogeneous
or heterogeneous. A heterogeneous immunoassay requires a step to
remove unbound antibody or antigen from the sample, usually using a
solid phase reagent. Because homogeneous assays do not require this
step, they are typically faster and easier to perform. Separation
methods include, for example, precipitation (for example, with a
second antibody) and removal on a coated tube, coated bead, coated
well, magnetic particles or glass particles.
[0102] An immunoassay of the present invention may be, for example,
a competitive binding assay. In a competitive immunoassay, the
antigen in the sample competes with labeled antigen to bind with
antibodies. The amount of labeled antigen bound to the antibody
site is then measured. In this method, the response will be
inversely proportional to the concentration of antigen in the
unknown. This is because the greater the response, the less antigen
in the sample was available to compete with the labeled antigen. An
example of a competitive immunoassay is a radioimmunoassay
(RIA).
[0103] An immunoassay of the present invention maybe be, for
example, an Enzyme-Linked ImmunoSorbent Assay (ELISA). In an ELISA,
an unknown amount of antigen is affixed to a surface and then a
specific antibody is washed over the surface so that it can bind to
the antigen. This antibody is linked to an enzyme, and in the final
step a substance is added that the enzyme can convert to some
detectable signal and the amount of antigen in the sample can be
measured. Performing an ELISA involves at least one antibody with
specificity for a particular antigen. The sample with an unknown
amount of antigen is immobilized on a solid support (for example, a
polystyrene microtiter plate) either non-specifically (via
adsorption to the surface) or specifically (via capture by another
antibody specific to the same antigen, in a "sandwich" ELISA).
After the antigen is immobilized, the detection antibody is added,
forming a complex with the antigen. The detection antibody can be
covalently linked to an enzyme, or can itself be detected by a
secondary antibody which is linked to an enzyme through
bioconjugation. Between each step the plate is typically washed
with a mild detergent solution to remove any proteins or antibodies
that are not specifically bound. After the final wash step the
plate is developed by adding an enzymatic substrate to produce a
visible signal, which indicates the quantity of antigen in the
sample. ELISAs may utilize chromogenic, luminescent, or fluorigenic
substrates.
[0104] An immunoassay of the present invention may be a sandwich
assay. In sandwich assays, the analyte is a "sandwich" between two
antibodies that bind to different antigenic epitopes on the target
analyte. One antibody serves as a capture antibody and a second
antibody serves as a detection antibody. The capture antibody may
be coated to a solid phase, such as a tube or well, and the
detection antibody may be detectably labeled.
[0105] An immunoassay of the present invention may be, for example,
an immunochromatographic lateral flow assay (also referred to
herein as a lateral flow assay, a lateral flow test, or
immunochromatographic strip test). Lateral flow assays use a simple
device to quickly detect the presence (or absence) of a target
analyte in sample. These tests are commonly used for medical
diagnostics either for home testing, point of care testing, or
laboratory use. Often produced in a dipstick format, lateral flow
tests are a form of immunoassay in which the test sample, which may
be suspended in an aqueous solution, flows through a porous
substrate (for example, a nitrocellulose membrane) via capillary or
wicking action towards an absorbent pad. After the sample is
applied to the substrate, it encounters a colored reagent (for
example, gold or latex particles) which mixes with the sample and
binds to the analyate, if present in the sample. The mixture
transits the substrate encountering lines or zones which have been
pretreated to immobilize an antibody capable of binding the
analyte. If the analyte is present in the sample, the colored
reagent can become bound at the test line or zone. In alternative
embodiments, the lateral flow assay can be used to detect specific
antibodies present in a sample. In those embodiments, the colored
reagent can be antigen-coated particles and the detection lines or
zones can be pretreated with the antigen. See, for example U.S.
Pat. Nos. 5,753,517, 6,485,982, 6,509,196, 7,189,522, and RE39664,
U.S. Patent Application Publication No. 2006/0275920, and Jeong et
al., 2003, Korean J Biol Sci; 7:89-92).
[0106] Also included in the present invention are detection methods
in which one or more antibodies described herein are used to bind
C. difficile spores, if present in a sample, to a substrate and the
presence of spores is then detected and/or quantified by any of a
variety of means. The presence of spores may be determined by, for
example, microscopy, culturing, enzymatic activity antibody binding
(as, for example, in an ELISA assay), calcium molecular
fluorescence or luminescence, or lanthanide metal mediated
luminescence. Dipicolinic acid in a 1:1 complex with calcium ions
is present in high concentrations in bacterial spores and has not
been observed in any other life forms. A lanthanide metal, such as
for example, terbium or europium, will combine with dipicolinic
acid (DPA) present in any bacterial spores in a sample to produce a
lanthanate chelate, such as, for example, terbium or europium
dipicolinate. Such lanthanate chelates have distinctive absorbance
and emission spectra that can be detected using photoluminescence
testing. Lanthanide metal mediated luminescence may also be
utilized as a detection signal in any of the various immunoassay
methods described herein, for example, in a lateral flow assay.
Upon spore germination, Ca-DPA is released and calcium can be
detected by a number of means. Fluorimetric detection of calcium by
the use of molecular fluorescence or luminescence for sensing
offers high sensitivity. Calcium indicator dyes can be categorized
into two groups; the first are the dyes that increase their
fluorescence in the presence of calcium, while the second group are
dyes that have different excitation and/or emission wavelengths in
the presence of calcium than they have in its absence. The calcium
indicator dyes, calcium green-1, calcium green-2, and Fluo-4 are
representative of the dyes that increase their fluorescence in the
presence of calcium ion (Ca2.sup.+) without changing wavelengths.
Fura-2 and Indo-1 are ratiometric Ca2.sup.+ indicators that are
generally considered interchangeable in most experiments. Fura-2,
upon binding Ca2.sup.+, exhibits a shift in its absorption or
excitation peak from 338 nm to 366 nm. Indo-1 on the other hand has
a shift in the emission from 485 nm to 405 nm in the presence of
calcium. Calcium can also be detected using calcium-activated
photoproteins, such as aequorin and obelin. Since there is no need
for excitation from external irradiation for the emission of
bioluminescence, the signal produced has virtually no background.
This allows for detection limits at extremely low levels, making
these photoproteins attractive labels for analytical applications.
Calcium mediated signaling may also be utilized as a detection
signal in any of the various immunoassay methods described herein,
for example, in a lateral flow assay. Such methods, and the other
methods described herein, also allow for the quantification of
spores present in a sample. See, for example, U.S. Pat. Nos.
5,876,960; 6,498,041; 6,815,178; and 7,306,942, U.S. Patent
Application Publication Nos. 2003/0138876; 2004/0014154; and
2005/0136508; and Ponce, 2003, NASA Tech Brief; 27(3):pp. i-ii,
1-3.
[0107] With the detection methods of the present invention, one or
more antibodies may be used, including one or more of the
antibodies described herein that bind to a C. difficile spore.
Further, one or more additional antibodies of known specificity may
be used, for example, one or more antibodies that bind to C.
difficile vegetative cells, that bind to vegetative cells of a
different bacterial species, such as, for example, C. clostridium
or B. subtilis, or that bind to spores of a different bacterial
species, such as, for example, spores of C. clostridium or B.
subtilis, may be used.
[0108] Samples may be obtained from a wide variety of source and
include, but are not limited to, environmental or food samples and
medical or veterinary samples. Examples of environmental samples,
include, but are not limited to, water samples, soil samples, plant
samples, and air samples. Examples of foods include, but are not
limited to: meats, poultry, eggs, fish, seafood, vegetables,
fruits, prepared foods (e.g., soups, sauces, pastes), grain
products (e.g., flour, cereals, breads), canned foods, milk, other
dairy products (e.g., cheese, yogurt, sour cream), fats, oils,
desserts, condiments, spices, pastas, beverages, water, and animal
feed. Medical or veterinary samples include, but are not limited
to: clinical samples, cell lysates, whole blood or a portion
thereof (e.g., serum), other bodily fluids or secretions (e.g.,
saliva, sputum, sweat, sebum, urine, cerebrospinal fluid), feces,
cells, tissues, organs, biopsies, and different types of swabs.
[0109] A sample may be obtained from a fomite. The term "fomite" is
generally used to refer to an inanimate object or substrate capable
of carrying infectious organisms and/or transferring them. A fomite
serves to transmit an infectious agent, such as C. difficile, from
person to person. Fomites can include, but are not limited to,
cloths, mop heads, towels, sponges, wipes, eating utensils, coins,
paper money, cell phones, clothing (including shoes), doorknobs,
feminine products, diapers, etc., portions thereof, and
combinations thereof. There are many examples of fomites with
respect to medicine; tools such as laryngoscopes that are not
properly disinfected between uses, dirty towels, eating utensils,
and surfaces such as floors, walls, and tables may all serve to
spread disease. The surface of interest can include at least a
portion of a variety of surfaces, including, but not limited to,
walls (including doors), floors, ceilings, drains, refrigeration
systems, ducts (e.g., airducts), vents, toilet seats, handles,
doorknobs, handrails, bedrails (e.g., in a hospital), countertops,
tabletops, eating surfaces (e.g., trays, dishes, etc.), working
surfaces, equipment surfaces, clothing, etc., and combinations
thereof.
[0110] Samples may be liquid, solid, or semi-solid. Samples may be
swabs of solid surfaces. Samples may be used directly in the
detection methods of the present invention, without preparation or
dilution. For example, liquid samples, may be assayed directly.
Samples may be diluted or suspended in solution, which may include,
but is not limited to a buffered solution or a bacterial culture
medium. A sample that is a solid or semi-solid may be suspending in
a liquid by mincing, mixing or macerating the solid in the liquid.
A sample may by further concentrated or enriched.
[0111] The immunoassays of the present invention may include
various appropriate control samples. For example, negative control
samples containing no bacterial spores, cells or toxin, or positive
control samples containing bacterial cells, spores or toxin may be
assayed. An immunoassay of the present invention may take as little
as a few minutes to develop and may require little or no sample or
reagent preparation. An immunoassay of the present invention may be
performed in a qualitative or quantitative format. Qualitative
results provide a simple positive or negative result for a sample.
The cutoff between positive and negative is determined by the
analyst and may be statistical. Two or three times the standard
deviation is often used to distinguish positive and negative
samples. In quantitative formats, results may be interpolated into
a standard curve, which is typically a serial dilution of the
target.
[0112] The present invention is illustrated by the following
examples. It is to be understood that the particular examples,
materials, amounts, and procedures are to be interpreted broadly in
accordance with the scope and spirit of the invention as set forth
herein.
EXAMPLES
Example 1
Generation of C. difficile Spores
[0113] One tube of Brain Heart Infusion Broth (BHI) was inoculated
with C. difficile (ATCC.RTM. No. 700792, American Type Culture
Collection, Manassas, Va.) and incubated for twenty-four hours at
35-37.degree. C. under anaerobic conditions. Following incubation,
one milliliter (ml) aliquots of the broth culture were transferred
to a minimum of four tubes containing BHI broth and then incubated
for twelve days at 35-37.degree. C. under anaerobic conditions. The
broth cultures were centrifuged at 10,000 rotations per minute
(rpm) for 10 minutes (min) and the cell pellet was resuspended in
10 ml of absolute ethanol for one hour at room temperature to kill
vegetative cells. After one hour the suspension was centrifuged at
10,000 RPM for 10 minutes and the cell pellet was washed at least
twice with sterile Butterfield's buffer. The pellet was resuspended
in sterile Butterfield's Buffer and spore number per milliliter was
determined by plating serial dilution on anaerobic blood agar.
Resuspended spores were used in the following examples as both an
immunogen in the generation of polyclonal antibodies to inactivated
spores and as an antigenic target in ELISA assays to determine the
binding specificity of anti-spore antibodies.
Example 2
Generation of Polyclonal Antibody to Inactivated C. difficile
Spores
[0114] C. difficile spores, at a concentration of about 10.sup.6
spores per ml, were treated with 5% formalin for 10 minutes to
inactivate the spores. The inactivated spores were washed twice
with sterile Butterfield's buffer and then resuspended in sterile
Butterfield's buffer. A polyclonal antibody was raised against the
inactivated spores in rabbits using standard protocols (Antagene,
Inc, Mountain View, Calif.). Briefly, New Zealand White rabbits
(two individual rabbits) were immunized with 1.times.10.sup.6/ml
equivalent of inactivated spores per immunization. The immunogen
was diluted to 1 ml with sterile saline and combined with 1 ml of
the appropriate adjuvant. The antigen and adjuvant were mixed to
form a stable emulsion which was injected subcutaneously. Rabbits
were immunized on day one with antigen in Complete Freund's
Adjuvant (CFA), followed by immunizations on days 20, 40, and 60
with antigen in Incomplete Freund's Adjuvant (IFA) for all
subsequent injections.
[0115] Ten days after the last immunization, the blood was
collected from the rabbit and allowed to clot and retract at
37.degree. C. overnight. The clotted blood was then refrigerated
for 24 hours and the serum was decanted and clarified by
centrifugation at 2500 rpm for 20 minutes. The preimmune and immune
serums were tested against inactivated spores by ELISA. For the
ELISA protocol, inactivated spores of C. difficile were diluted to
10.sup.5 spores per milliliter (ml) in coating buffer (0.1 M
bicarbonate buffer 1.59 grams (g) Na.sub.2CO.sub.3 and 2.93 g
NaHCO.sub.3 per liter of sterile distilled water, pH 9.6). 100
microliter (.mu.l) of the spore solution was added to wells of an
ELISA plate (ELISA Enhanced Surface plate, BD Falcon, Franklin
Lakes, N.J.). To the control wells, 100 .mu.l of coating buffer was
added. The plates were wrapped with PARAFILM and incubated at
4.degree. C. for 15 to 16 hours. The plates were emptied and washed
three times with wash buffer (Phosphate buffered saline with 0.05%
Tween 20). The plates were blocked with 100 .mu.l of blocking
buffer (1% BSA and 0.05% Tween-20 in PBS) for two hours with
shaking at room temperature (RT). The plates were emptied, washed
three times with wash buffer and incubated with 100 .mu.l primary
antibody in blocking buffer for one to two hours with shaking at
RT. The plates were emptied, washed three times with wash buffer
and incubated with 100 .mu.l of secondary antibody with HRP label
(goat anti-rabbit HRP conjugate; 1:10,000 dilution, Pierce,
Rockford, Ill.) for one hour with shaking at RT. The plates were
emptied and washed four times with wash buffer. 50 .mu.l of the
substrate solution (1-step Ultra TMB, Pierce, Rockford, Ill.) was
added to each of the wells and incubated with shaking at RT for 15
to 30 minutes. The color was stopped by adding 50 .mu.l of stop
solution (1.5 M phosphoric acid) and absorbance of each plate was
read in a SpectraMax plus 384 (Molecular Devices, Sunnyvale,
Calif.) at 450 nm. The absorbances were compared to determine
fold-enhancement of signal for target. The immunized serum showed a
good response to inactivated spores, approximately 13 to 15 fold
that obtained with the preimmune serum (Table 1).
TABLE-US-00001 TABLE 1 Characterization of antiserum generated
against inactivated C. difficile spores Absorbance at 450 nm*
Antibody Rabbit #1 Rabbit #2 dilution Preimmune serum Antisera
Preimmune serum Antisera 1:1000 0.07 1.73 0.04 1.92 1:10,000 0.06
1.22 0.07 1.27 1:100,000 0.06 0.78 0.06 0.82 *Average no antigen
control readings with preimmune and antiserum were 0.12 at 450
nm.
[0116] The antibody was purified by ammonium sulfate precipitation
followed by protein A column chromatography. Briefly, antisera were
precipitated by drop-wise addition of ice-cold saturated ammonium
sulfate to a final ratio of 1:1 (50% saturated ammonium sulfate).
This procedure was done in an ice-cold beaker, with constant
stirring. The supernatant was transferred to a 50-mL centrifuge
tube and placed on a reciprocating mixer overnight at 4.degree. C.
The suspension was centrifuged at 10,000.times.g for 30 minutes at
2.degree. C. The supernatant was removed and the pellet was
resuspended in an equal volume of deionized water. The resuspended
protein was dialyzed (12,000-14,000 MW cut-off dialysis tubing)
against two liters (L) of PBS. The dialysis buffer was removed and
replaced after about two hours and again after about 24 hours.
After about 48 hours of dialysis, the protein dialysate was removed
and filtered with a 0.22 .mu.m filter.
[0117] The IgG fraction was recovered from the filtrate by
chromatography, using a Protein A affinity column from BioRad
(Hercules, Calif.) according to the manufacturer's instructions.
Briefly, a Shimadzu HPLC system (Model SCL-10AVP, Shimadzu
Corporation, Shimadzu Scientific Instruments, Inc., Columbia, Md.)
was used for all preparative chromatography runs. The solvents used
for the binding and elution of the antibodies are listed below were
Binding Buffer A (Phosphate Buffered Saline, pH 7.3) and Elution
Buffer B (20 mM Sodium Acetate, 0.5 M NaCl, pH 3.5). All buffers
were prepared with deionized water using a MILLI Q filtration
system (Millipore Corp., Billerica, Mass.) and were filtered
through a 0.22-.mu.m (pore size) membrane filter. The column was
pre-equilibrated with binding buffer A prior to sample injection.
The antisera samples were manually injected into the column via a
sample injection loop at T=0 minutes. After sample injection, the
solvents were run through the column as follows: Binding Buffer B
starting at T=0 at a flow rate of 0.6 ml/min; Elution Buffer B
starting at T=20 minutes at a flow rate of 1.0 ml/min; and Binding
Buffer B starting at T=60 minutes at a flow rate of 1.0 ml/min.
Fractions of the column eluate from each of the mobile phase
solvents were collected. Purified anti-C. difficile antibody
protein eluted at around 51 minutes. Each run was 70 minutes long
after which the second peak was put into dialysis for three changes
of buffer to change the pH. The fractions were then filtered and
pooled and the OD measured. At 20 minutes the buffer was changed to
elution buffer. The purified antibody was dialysed against PBS with
three changes of buffer at 4.degree. C. The dialysed antibody was
concentrated using Centricon filters (10,000 molecular weight cut
off; Millipore Corporation, Billerica, Mass.)
[0118] The purified antibody was tested by ELISA against C.
difficile, C. sporogenes (ATCC 3584) and B. subtilis (ATCC 19659)
spores. The C. sporogenes and B. subtilis spores were obtained from
Presque Isle Cultures (Erie, Pa.). Spores were diluted in coating
buffer. Various levels of spores (10.sup.3 to 10.sup.5 per ml) of
B. subtilis, C. sporogenes and C. difficile were coated and tested
with the antibody in ELISA assays performed as described above. The
antigen antibody interaction was detected using anti-rabbit HRP
antibody (Pierce, Rockford, Ill.) and the spore antibody showed
good reaction with C. difficile spores, about 20-fold response over
background at 10.sup.5 spores per ml, and showed a weak,
approximately 2-fold with C. sporogenes, and no reaction over
background with B. subtilis spores (see Table 2). The data
presented in Table 2 are the average of readings from three wells
from one of the experiment and are representative of at least three
separate experiments.
TABLE-US-00002 TABLE 2 Characterization of purified rabbit C.
difficile spore antibody Absorbance at 450 nm Spores/ml B. subtilis
C. sporogenes C. difficile 1.00E+03 0.21 0.24 0.17 1.00E+04 0.16
0.25 0.8 1.00E+05 0.16 0.41 4.13 No antigen 0.17 0.17 0.17
control
Example 3
Selection of C. difficile Spore-Specific Proteins for Generation of
Antibodies
[0119] The complete genome sequence of C. difficile strain 630 has
been determined and is available on the GenBank.RTM. sequence
database maintained by the National Center for Biotechnology
Information (NCBI), National Library of Medicine (NLM), National
Institutes of Health (NIH). See also, Sebaihia et al., 2006, Nat.
Genet; 38 (7):779-786). A thorough search of all the GenBank.RTM.
entries for C. difficile was made and two spore specific proteins
were picked for antibody production. One of the proteins selected
was hypothetical protein CD1021 (YP.sub.--001087502), which
demonstrates a conserved domain (amino acid residues 90 to 393) to
the spore coat assembly protein H (cotH). The other protein
selected was cell surface protein (putative
N-acetylmuramoyl-L-alanine amidase, YP.sub.--001087517) which has
conserved domains CW_binding.sub.--2 (putative cell wall binding
repeat 2; 174 to 265, 275 to 368, 381 to 461) and Amidase.sub.--3
(N-acetylmuramoyl-L-alanine amidase, 493 to 673). The analysis was
performed using conserved domain search tools available from the
NCBI.
[0120] Hypothetical Protein CD1021 of C. difficile 630 (GenBank
Accession No. YP.sub.--001087502) has the following amino acid
sequence:
TABLE-US-00003 (SEQ ID NO: 1) MKDKKFTLLI SIMIVFLCAV VGVYSTSSNK
SVDLYSDVYI EKYFNRDKVM EVNIEIDESD LKDMNENAIK EEFKVAKVTV DGDTYGNVGI
RTKGNSSLIS VANSDSDRYS YKINFDKYNT SQSMEGLTQL NLNNCYSDPS YMREFLTYSI
CEEMGLATPE FAYAKVSING EYHGLYLAVE GLKESYLENN FGNVTGDLYK SDEGSSLQYK
GDDPESYSNL IVESDKKTAD WSKITKLLKS LDTGEDIEKY LDVDSVLKNI AINTALLNLD
SYQGSFAHNY YLYEQDGVFS MLPWDFNMSF GGFSGFGGGS QSIAIDEPTT GNLEDRPLIS
SLLKNETYKT KYHKYLEEIV TKYLDSDYLE NMTTKLHDMI ASYVKEDPTA FYTYEEFEKN
ITSSIEDSSD NKGFGNKGFD NNNSNNSDSN NNSNSENKRS GNQSDEKEVN AELTSSVVKA
NTDNETKNKT TNDSESKNNT DKDKSGNDNN QKLEGPMGKG GKSIPGVLEV AEDMSKTIKS
QLSGETSSTK QNSGDESSSG IKGSEKFDED MSGMPEPPEG MDGKMPPGMG NMDKGDMNGK
NGNMNMDRNQ DNPREAGGFG NRGGGSVSKT TTYFKLILGG ASMIIMSIML VGVSRVKRRR
FIKSK. See also, Sebaihia et al., 2006, Nat. Genet; 38 (7):
779-786.
[0121] The putative N-acetylmuramoyl-L-alanine amidase cell surface
protein of C. difficile 630 (GenBank Accession No.
YP.sub.--001087517) has the following amino acid sequence:
TABLE-US-00004 (SEQ ID NO: 5) MLSKEINMRR NTKLLTTGIL SMAIVAPTMA
FATESNAMEN NADLNINLEK KSIVLGSKSK VSVKFKEKPD ADSIKLKYKC YDMPLNTTLN
YNQSTGAYEG IINYNKDPEY LNVWELQGIT INSKTNPKTL NRQDLEKMGL NLKDYNVTQE
CIIEDITSRK DVNKYLRKTS SPITELTGSD RYETAVKISK EGWKNGSDKV VIINGDVSID
GIISTPLATT YNAPILLVEK NNVPNSVKSE LKRLNPKDII IIGDENAISK TTANQIKSTV
NASQTRLNGS NRYETSLLIA KEIDKNHDVE KVYITNANGG EVDALTIAAK AGQDKQPIIL
TDKDSITDNT YKWLKSEDLQ NAYFIGGPQM ISTNVINKVN GITKDSVTNN RVYGADRHET
NANVIKKFYT DDELEAVLVA KSDVLVDALA AGPLAANLKS PILITPKTYV SAYHKDNLEA
KSANKVYKIG GGLTSKVMSS IASSLSKHNT TPTEPGNSGG KTVMIDPGHG GSAPGNSSGG
MIEKDYNLNT SLATTEYLRS KGFNVIMTRD TDKTLSLGNR TALSNSLKPD LFTSIHYNGS
TNKQGHGVEV FYKLKDKNGG TTKTVATNIL NRILEKFKLT NRGIKTRVLP SDSTKDYLYV
LRSNDMPAVL VECAFLDNEN DMSLINSSAK VKEMGTQIGK GIEDSLK. See also,
Sebaihia et al., 2006, Nat. Genet; 38 (7): 779-786.
Example 4
GAT Polyclonal Antibody Against Hypothetical Protein CD1021
[0122] A polyclonal antibody against hypothetical protein CD1021
was generated using Strategic Diagnostics Inc.'s (SDI) (Newark,
Del.) proprietary Genomic Antibody Technology.TM. (GAT). A unique
amino acid sequence from the protein was identified and this
sequence information was placed in SDI's proprietary plasmid
vector. The vector was introduced into a mouse. With this
technology, cells of the host animal take up the plasmid. In these
cells, the immunogen is synthesized and secreted by the host cells
and immediately recognized by the immune system leading to
production of polyclonal antibodies against the expressed protein
sequence. Both polyclonal and monoclonal antibodies can be
developed using GAT. The protein immunogen is produced in the host
animal using natural protein synthesis machinery. Since the
immunogen is not synthesized and purified in a laboratory, it does
not have the opportunity to denature or degrade. A native immunogen
is presented immediately to the immune system, resulting in a
mature antibody response.
[0123] The protein sequence of hypothetical protein CD 1021 used
for polyclonal antibody generation was residues 505 to 604 and had
the amino acid sequence: SKTIKSQLSG ETSSTKQNSG DESSSGIKGS
EKFDEDMSGM PEPPEGMDGK MPPGMGNMDK GDMNGKNGNM NMDRNQDNPR EAGGFGNRGG
GSVSKTTTYF (SEQ ID NO:2).
[0124] The sera from two immunized mice was pooled and tested
against the immunogen by Western blot to determine specificity of
the antibody. Later, the antibody was purified by ammonium sulfate
precipitation followed by protein A column chromatography, as
described in Example 2.
[0125] Initially, various levels of spores (10.sup.3 to 10.sup.5
per ml) of B. subtilis, C. sporogenes and C. difficile were coated
and tested with the antibody. The antigen-antibody interaction was
detected using anti-mouse HRP antibody (Pierce, Rockford, Ill.).
The mouse antibody against the hypothetical protein CD1021 (SEQ ID
NO:2) showed about a 10-fold increased response over background at
10.sup.5 spores per ml, and showed a weak response (about 2-fold)
with C. sporogenes spores, and no reaction over background with B.
subtilis spores (see Table 3). The data presented in Table 3 are
the average of readings of three wells and are representative of
two separate experiments.
TABLE-US-00005 TABLE 3 Characterization of C. difficile
hypothetical protein CD1021 mouse antibody Absorbance at 450 nm
Spores/ml B. subtilis C. sporogenes C. difficile 1.00E+03 0.13 0.13
0.2 1.00E+04 0.15 0.17 0.2 1.00E+05 0.15 0.32 1.61 No antigen 0.14
0.14 0.14 control
[0126] The antibody was further tested using sandwich ELISA. Plates
were coated with the polyclonal CD1021 antibody followed by binding
of spores (B. subtilis, C. sporogenes and C. difficile). The
antibody antigen interaction was detected using the anti-C.
difficile inactivated spore antibody from example 2, above, as the
second antibody, followed by anti-rabbit HRP antibody (BD
Pharmingen). The spores of B. subtilis, C. sporogenes, and C.
difficile were used at various levels and antibody is specific for
detection of C. difficile spores. The results are shown in Table 4,
below.
[0127] In the sandwich ELISA protocol, the purified mouse CD 1021
antibody was diluted in antigen coating buffer at 1 .mu.g/ml
concentration. 100 .mu.l of the of the antibody solution was added
to wells of an ELISA plate (ELISA Enhanced Surface plate, BD
Falcon, Franklin Lakes, N.J.). The plates were wrapped with
Parafilm and incubated at 4.degree. C. for 15 to 16 hours. The
plates were emptied and washed four times with wash buffer. The
plates were blocked with 100 .mu.l of blocking buffer for 2 hours
with shaking at room temperature (RT). The plates were emptied,
washed four times with wash buffer and spore solutions of C.
difficile, C. sporogenes, and B. subtilis (100 .mu.l of 10.sup.3,
10.sup.4, and 10.sup.5 spores per ml) in blocking buffer were
added. For control wells, 100 .mu.l of blocking buffer was added.
After incubation at RT for two hours, the plates were emptied,
washed four times with wash buffer and incubated with 100 .mu.l of
secondary antibody (anti-rabbit C. difficile spore antibody,
descried in Example 2) in blocking buffer for one hour with shaking
at RT. The plates were emptied, washed four times with wash buffer
and incubated with 100 .mu.l of secondary antibody with HRP label
(goat anti-rabbit HRP conjugate; 1;10,000 dilution, BD Pharmingen)
for one hour with shaking at RT. The plates were emptied and washed
three times with wash buffer. 50 .mu.l of the substrate solution
(1-step Ultra TMB, Pierce) was added to each of the wells and
incubated with shaking at RT for 15 to 30 minutes. The color was
stopped by adding 50 .mu.l of stop solution (1.5 M phosphoric acid)
and absorbance of each plate was read at 450 nm. The absorbances
were compared against control to determine fold-enhancement of
signal for target (see Table 4). The data presented in Table 4 are
the average of readings of three wells and are representative of
two separate experiments.
TABLE-US-00006 TABLE 4 Characterization of C. difficile
hypothetical protein CD1021 mouse antibody by Sandwich ELISA with
spore antibody Fold-change over control* Spores/ml B. subtilis C.
sporogenes C. difficile 1.00E+03 1.1 1.03 1.22 1.00E+04 1.12 1.08
1.4 1.00E+05 1.14 1.04 2.35 *No antigen control readings were 1.05
at 450 nm
Example 5
Polyclonal Antibodies Against Hypothetical Protein CD1021
Peptides
[0128] Two unique amino acid sequences were identified from the
hypothetical protein CD1021. A search in both protein and DNA
databases showed that both sequences are specific to C. difficile
and have no homology with other bacteria such as Bacillus. These
two sequences are:
TABLE-US-00007 (SEQ ID NO: 3) Peptide 1 EGSSLQYKGDDPESY (residues
203 to 217 of CD1021) (SEQ ID NO: 4) Peptide 2 LKNETYKTKYHKYLE
(residues 333 to 347 of CD1021).
[0129] The peptides were synthesized with a free cysteine at the
N-terminal end and conjugated to keyhole limpet hemocyanin (KLH) to
elicit high titer antibodies. Each of the two KLH conjugated
peptides was used separately to immunize two individual rabbits
according to standard protocols with antigen by Epitomics
(Burlingame, Calif.). Briefly, a primary injection of
KLH-conjugated peptide (0.5 mg/ml) with 1 ml of CFA was followed by
four boosts of KLH-conjugated peptide (0.25 mg/ml) with 1 ml of
IFA.
[0130] Blood was collected after each immunization and sera was
tested by ELISA using the respective peptides. Bleed three and four
showed a good response to the peptides, approximately 5 to 7-fold
response over the preimmune serum (Table 5).
TABLE-US-00008 TABLE 5 Characterization of C. difficile CD1021
peptide serum (bleed 3) Absorbance at 450 nm* Peptide 1 Peptide 2
Rabbit #1 Rabbit #2 Rabbit #1 Rabbit #2 Antibody Preimmune
Preimmune Preimmune Preimmune dilution serum Antisera serum
Antisera serum Antisera serum Antisera 1:1000 0.18 1.72 0.20 1.62
0.200 1.82 0.20 1.81 1:10,000 0.15 1.21 0.15 1.12 0.120 1.18 0.14
1.05 1:100,000 0.10 0.68 0.08 0.72 0.070 0.64 0.08 0.74 *Average no
antigen control readings with preimmune and antiserum were 0.15 at
450 nm.
[0131] The serum from bleed three and four were purified by
ammonium sulfate precipitation followed by protein A column
chromatography, as described in Example 2. The purified antibody
was tested by ELISA for binding to plate bound peptides.
[0132] The peptides were diluted in antigen coating buffer at 1
.mu.g/ml concentration. 100 .mu.l of the of the peptide solution
was added to wells of an ELISA plate (ELISA Enhanced Surface plate,
BD Falcon, Franklin Lakes, N.J.). To the control wells 100 .mu.l of
coating buffer was added. The plates were wrapped with Parafilm and
incubated at 4.degree. C. for 15 to 16 hours. The plates were
emptied and washed three times with wash buffer. The plates were
blocked 100 .mu.l of blocking buffer for two hours with shaking at
room temperature (RT). The plates were emptied, washed three times
with wash buffer and incubated with 100 .mu.l primary antibody in
blocking buffer for one to two hours with shaking at RT. The plates
were emptied, washed three times with wash buffer and incubated
with 100 .mu.l of secondary antibody with HRP label (1;10,000
dilution) for 1 hour with shaking at RT. The plates were emptied
and washed three times with wash buffer. 50 .mu.l of the substrate
solution (1-step Ultra TMB, Pierce) was added to each of the wells
and incubated with shaking at RT for 15 to 30 minutes. The color
was stopped by adding 50 .mu.l of stop solution (1.5 M phosphoric
acid) and absorbance of each plate was read at 450 nm. The
absorbances were compared against control to determine
fold-enhancement of signal for target.
[0133] Various levels of spores (10.sup.3 to 10.sup.5 per ml) of B.
subtilis, C. sporogenes and C. difficile were coated and tested
with the antibody. The antigen antibody interaction was detected
using anti-rabbit HRP antibody (Pierce, Rockford, Ill.) and the
antibodies were specific for detection of C. difficile spores,
demonstrating a 6 to 10-fold response over background with C.
difficile spores (see Table 6). The data presented in Table 6 are
the average of readings of three wells and are representative of at
least three separate experiments.
TABLE-US-00009 TABLE 6 Characterization of rabbit antibodies to
peptide sequences of C. difficile hypothetical protein CD1021
Absorbance at 450 nm Peptide 1 Ab Peptide 2 Ab B. C. C. B. C. C.
Spores/ml subtilis sporogenes difficile subtilis sporogenes
difficile 1.00E+03 0.15 0.14 0.2 0.14 0.14 0.21 1.00E+04 0.16 0.17
0.21 0.17 0.17 0.48 1.00E+05 0.16 0.29 1.53 0.16 0.27 0.95 No 0.22
0.22 0.22 0.2 0.2 0.2 Antigen control
Example 6
GAT Polyclonal Antibody Against a Putative
N-Acetylmuramoyl-L-Alanine Amidase Cell Surface Protein
[0134] A polyclonal antibody against a cell surface protein that is
a putative N-acetylmuramoyl-L-alanine amidase protein was generated
using Strategic Diagnostics Inc.'s (Newark, Del.) proprietary
Genomic Antibody Technology.TM. (GAT). A unique sequence from the
protein was identified and the immunogen was expressed in vivo in
mouse, as described in Example 4. The expressed immunogen is
recognized by the host immune system leading to production of a
polyclonal antibody against the expressed protein. The protein
sequence of cell surface protein (putative
N-acetylmuramoyl-L-alanine amidase) used for antibody generation
was residues 294 to 393, having the amino acid sequence of
DKNHDVEKV YITNANGGEV DALTIAAKAG QDKQPIILTD KDSITDNYKW LKSEDLQNAY
FIGGPQMIST NVINKVNGIT KDSVTNNRVY GADRHETNAN (SEQ ID:6).
[0135] The sera was pooled from immunized animals and tested
against the immunogen by Western blot to determine specificity of
the antibody. Later, the antibody was purified by ammonium sulfate
precipitation followed by protein A column chromatography.
[0136] Initially, various levels of ungerminated spores (10.sup.3
to 10.sup.5 per ml) of B. subtilis, C. sporogenes and C. difficile
were coated and tested with the antibody. The antigen antibody
interaction was detected using anti-mouse HRP antibody (Pierce,
Rockford, Ill.). The antibody against cell surface protein
(putative N-acetylmuramoyl-L-alanine amidase) did not show any
reaction with C. difficile or other spores.
[0137] The spores of B. subtilis, C. sporogenes and C. difficile
were germinated using various germinant solutions. The germinated
spores (10.sup.3 to 10.sup.5 per ml) were coated and tested with
the antibody. The antigen antibody interaction was detected using
anti-mouse HRP antibody (Pierce, Rockford, Ill.). The antibody was
able to detect germinated C. difficile spores, demonstrating about
a 6-fold response over background, but not germinated B. subtilis
or C. sporogenes spores (see Table 7). The data presented in Table
7 are the average of readings of three wells and are representative
of two separate experiments.
[0138] Germination of spores has been well studied with B. subtilis
spores which can be induced to germinate by specific germinants,
including L-alanine and a combination of asparagine glucose,
fructose and potassium ions ("AGFK") (Moir and Smith, 1990, Ann Rev
Microbiol; 44:531-553). Initial attempts to germinate C. difficile
spores were using various germinants (combination of AGFK with
alanine and Inosine), but spore germination was not efficient. The
C. difficile spores germinate in a nutrient medium with addition of
1% sodium taurocholate (Sorg and Sonenshein, 2008, J Bacteriol
(published online ahead of print on 1 Feb. 2008)
doi:10.1128/JB.01765-07), the spores were germinated in Brain
Brain-Heart Infusion broth supplemented with yeast extract (5
mg/ml), L-cysteine (0.1%) and 1% sodium taurocholate. The
germination of spores was followed by measuring the OD600 of spore
cultures (OD600 decreases upon germination) and by phase contrast
microscopy. Spores of C. sporogenes and B. subtilis were germinated
following previously as described before (Broussolle et. al., 2002,
Anaerobe; 8:89-100; Moir and Smith, 1990, Ann Rev Microbiol; 44,
531-553).
TABLE-US-00010 TABLE 7 Characterization of C. difficile putative
amidase mouse antibody Absorbance at 450 nm B. subtilis C.
sporogenes C. diffcile Spores/ml Ungerminated Germinated
Ungerminated Germinated Ungerminated Germinated 1.00E+03 0.13 0.17
0.14 0.16 0.2 0.18 1.00E+04 0.12 0.2 0.13 0.22 0.2 0.39 1.00E+05
0.15 0.22 0.15 0.27 0.2 0.82 No antigen control 0.13 0.13 0.13 0.13
0.13 0.13
Example 7
Polyclonal Antibody Against Cell Surface Protein (Putative
N-Acetylmuramoyl-L-Alanine Amidase) Peptides
[0139] Two unique sequences were identified from the protein
sequence of C. difficile N-acetylmuramoyl-L-alanine amidase and
upon BLAST search in both protein and DNA database the sequences
are specific to C. difficile and have no homology with other
bacteria such as Bacillus.
TABLE-US-00011 Peptide 1 (SEQ ID NO: 7) YKLKDKNGGTTKTVA (amino acid
residues 582 to 596) Peptide 2 (SEQ ID NO: 8) KFKEKPDADSIKLKY
(amino acid residues 64 to 78)
[0140] The peptides were synthesized with a free cysteine at the
N-terminal end and conjugated to KLH to elicit high titer
antibodies. Both KLH conjugated peptides were combined and used
together to immunize two individual rabbits according to standard
protocols by Antagene (Mountain View, Calif.). Ten days after the
last immunization, the blood was collected from the rabbit and
allowed to clot and retract at 37.degree. C. overnight. The clotted
blood was then refrigerated for 24 hours and the serum was decanted
and clarified by centrifugation at 2500 rpm for 20 minutes. Initial
ELISA was done with preimmune and immunized serum against the
peptides Immunized serum against the peptides showed good response
to peptides, demonstrating about a 7 to 10-fold response over the
preimmune serum (Table 8).
TABLE-US-00012 TABLE 8 Characterization of C. difficile putative
amidase peptide serum Absorbance at 450 nm* Rabbit #1 Rabbit #2
Antibody Preimmune Preimmune dilution serum Antisera serum Antisera
1:1000 0.25 1.83 0.22 1.70 1:10,000 0.20 1.55 0.15 1.81 1:100,000
0.12 0.91 0.10 0.98 *Average no antigen control readings with
preimmune and antiserum were 0.20 at 450 nm.
[0141] The antibody was purified by ammonium sulfate precipitation
followed by protein A column chromatography, as described in
Example 2. The purified antibody was tested by ELISA. Initially,
various levels of ungerminated spores (10.sup.3 to 10.sup.5 per ml)
of B. subtilis, C. sporogenes and C. difficile were coated and
tested with the antibody. The antigen antibody interaction was
detected using anti-rabbit HRP antibody (Pierce, Rockford, Ill.).
The antibody against cell surface protein (putative
N-acetylmuramoyl-L-alanine amidase) did not show any reaction with
C. difficile or other spores. The spores of B. subtilis, C.
sporogenes and C. difficile were germinated using various germinant
solutions. The germinated spores (10.sup.3 to 10.sup.5 per ml) were
coated and tested with the antibody. The antigen antibody
interaction was detected using anti-rabbit HRP antibody (Pierce,
Rockford, Ill.). The antibody was able to detect germinated C.
difficile spores, demonstrating about a 6-fold response over
background, but not B. subtilis or C. sporogenes germinated spores
(see Table 9). The data presented in Table 9 are the average of
readings of three wells and are representative of two separate
experiments.
TABLE-US-00013 TABLE 9 Characterizationof C. difficile putative
amidase rabbit antibody Absorbance at 450 nm B. subtilis C.
sporogenes C. difficile Spores/ml Ungerminated Germinated
Ungerminated Germinated Ungerminated Germinated 1.00E+03 0.13 0.18
0.14 0.17 0.2 0.18 1.00E+04 0.13 0.19 0.12 0.21 0.18 0.44 1.00E+05
0.14 0.22 0.15 0.26 0.19 0.93 No antigen control 0.13 0.13 0.13
0.13 0.13 0.13
Example 8
Polyclonal Antibody Against C. difficile Common Antigen for
Detection of C. difficile Spores
[0142] The commercially available rabbit C. difficile common
antigen (glutamate dehydrogenase) antibody (Meridian Life Science,
Saco, Me.) reacts with both toxigenic and nontoxigenic strains and
is used to detect vegetative cells of C. difficile. In this
example, the commercially available rabbit C. difficile common
antigen antibody was tested for detection of C. difficile spores by
ELISA. The antibody was purified by ammonium sulfate precipitation
followed by protein A column chromatography. Various levels of
spores (10.sup.3 to 10.sup.5 per ml) of B. subtilis, C. sporogenes
and C. difficile were coated to a plate and tested with the
antibody. The antigen antibody interaction was detected using
anti-rabbit HRP antibody (Pierce, Rockford, Ill.). The common
antigen antibody showed good reaction with C. difficile spores
(about a 10-fold response with 10.sup.4 spores per ml and an
18-fold response over background at 10.sup.5 spores per ml) and
showed a weak (about 2-fold response over background) with C.
sporogenes spores and no reaction over background with B. subtilis
spores (see Table 10). The data presented in Table 10 are the
average of readings of three wells and are representative of at
least three separate experiments. This antibody may be used as a
capture antibody in sandwich ELIZA assays.
TABLE-US-00014 TABLE 10 Characterization of rabbit C. difficile
common antigen antibody Absorbance at 450 nm Spores/ml B. subtilis
C. sporogenes C. difficile 1.00E+03 0.14 0.15 0.44 1.00E+04 0.15
0.15 2.13 1.00E+05 0.2 0.31 3.69 No antigen 0.19 0.19 0.19
control
Example 9
Binding of C. difficile Antibodies to Vegetative Cells
[0143] The rabbit polyclonal antibody to inactivated spores
(described in more detail in Example 2), rabbit polyclonal antibody
to CD1021 sequence SEQ ID NO:3 (described in more detail in Example
5), rabbit polyclonal antibody to the two amidase peptides SEQ ID
NO:7 and SEQ ID NO:8 (described in more detail in Example 7), and
the commercially available anti-GDH antibody (described in more
detail in Example 8) were screened by ELISA for binding to
vegetative cells of C. difficile. C. difficile ATCC strains 43594,
43596, and 43603 were grown in thioglycollate medium under
anaerobic conditions for 16 to 18 hours. The cells were serially
diluted in antigen coating buffer and 100 .mu.l of various
dilutions was added to the ELISA plate and the plate was incubated
under anaerobic condition for one hour at 37.degree. C., to prevent
sporulation. The plates were emptied and washed three times with
wash buffer and blocked with blocking buffer for one hour under
anaerobic conditions at 37.degree. C. The plate was emptied and
washed three times with wash buffer and incubated with primary
antibody at RT for one hour. The plates were emptied, washed three
times with wash buffer, incubated with secondary antibody with HRP
label (1:10,000) for one hour at RT. The plates were washed and
substrate was added to develop color and plates were read at 450 nm
after adding the stop solution. The rabbit polyclonal antibodies to
inactivated spores, CD 1021 (SEQ ID NO:3) and amidase (SEQ ID NO:7
and SEQ ID NO:8) did not show any binding with vegetative cells
(about 10.sup.3 to 10.sup.7 cells per ml). However, the
commercially available anti-GDH antibody showed good binding at
about 10.sup.7 cells per ml, with a four-fold enhancement of signal
over control (see Table 11). The data presented in Table 11 are the
average of readings of three wells and are representative of two
separate experiments.
TABLE-US-00015 TABLE 11 Binding of antibodies with vegetative
cells. ATCC 43594 Absorbance at 450 nm No antigen Antibody control
10.sup.3 cells/ml 10.sup.4 cells/ml 10.sup.5 cells/ml 10.sup.6
cells/ml 10.sup.7 cells/ml Spore Ab 0.12 0.11 0.13 0.12 0.12 0.16
Amidase Ab 0.15 0.14 0.11 0.11 0.12 0.27 CD1021 0.11 0.11 0.11 0.12
0.12 0.24 peptide 1 Ab GDH Ab 0.12 0.11 0.10 0.14 0.35 0.85 ATCC
43596 Absorbance at 450 nm No antigen Antibody control 10.sup.3
cells/ml 10.sup.4 cells/ml 10.sup.5 cells/ml 10.sup.6 cells/ml
10.sup.7 cells/ml Spore Ab 0.12 0.12 0.12 0.11 0.11 0.14 Amidase Ab
0.15 0.11 0.11 0.11 0.12 0.22 CD1021 peptide 1 Ab 0.11 0.11 0.12
0.11 0.13 0.21 GDH Ab 0.12 0.11 0.12 0.16 0.40 0.76 ATCC 43603
Absorbance at 450 nm No antigen Antibody control 10.sup.3 cells/ml
10.sup.4 cells/ml 10.sup.5 cells/ml 10.sup.6 cells/ml 10.sup.7
cells/ml Spore Ab 0.12 0.12 0.13 0.12 0.12 0.15 Amidase Ab 0.15
0.11 0.12 0.11 0.13 0.25 CD1021 peptide 1 Ab 0.11 0.11 0.12 0.11
0.12 0.22 GDH Ab 0.12 0.09 0.10 0.14 0.43 0.80
Example 10
Use of Antibodies in Lateral Flow Devices for C. difficile Spore
Detection
[0144] Various antibodies described in the above examples, were
labeled with Cy3 (Cy3 Ab labeling kit, Amersham Biosciences,
Piscataway, N.J.) and tested for ability to detect C. difficile
spores. Specifically, the rabbit polyclonal antibody to inactivated
spores (described in more detail in Example 2 and referred to as
"spore" antibody in Table 12), the rabbit polyclonal antibody to
CD1021 sequence SEQ ID NO:3 (described in more detail in Example 5
and referred to as CD1021 peptide 1 Ab in Table 12), the rabbit
polyclonal antibody to a mix of the two amidase peptides SEQ ID
NO:7 and SEQ ID NO:8 (described in more detail in Example 7 and
referred to as amidase peptide Ab in Table 12), and the
commercially available rabbit antibody to C. difficile glutamate
dehydrogenase common antigen (described in more detail in Example 8
and referred to as "GDH" antibody in Table 12) were labeled and
tested.
[0145] A typical lateral flow strip with conjugate pad,
nitrocellulose membrane, and absorbent pad was prepared. Antibodies
were spotted on the nitrocellulose and allowed to dry. Germinated
and ungerminated spores (10.sup.5 per ml) of C. difficile, C.
sporogenes and B. subtilis were mixed with of each of the labeled
antibodies (50 .mu.l of 10 .mu.g/ml Cy3 labeled antibody)
separately and applied to the conjugate pad. The antibody-spore
mixture was allowed to wick for ten minutes. Phosphate buffered
saline was used as a control. After ten minutes, the strips were
scanned using a microarray scanner (Tecan, Durham, N.C.).
[0146] The lateral flow strips using the C. difficile spore
antibody, amidase antibody, or the CD1021 antibody, showed that the
antibodies are specific in detecting C. difficile spores. The
amidase antibody did not detect ungerminated spores, but was able
to detect germinated spores. Based on these observations one can
design lateral flow strips for detecting C. difficile spores. For
example, labeled GDH antibody could be used as a detection reagent
and spore antibody or CD 1021 antibody as capture reagent to detect
ungerminated spores and amidase antibody as capture reagent to
detect germinated spores.
[0147] As outlined in Table 12, various pairings of antibodies can
be used to detect the C. difficile spores, detecting, spores or
germinated spores.
TABLE-US-00016 TABLE 12 Antibody pairings for detection of C.
difficile spores Antibody 1 Antibody2 Specificity Amidase GDH C.
difficile germinated spores Peptide Ab Amidase CD1021 peptide 1 Ab
C. difficile germinated spores Peptide Ab Amidase Spore C.
difficile germinated spores Peptide Ab CD1021 peptide 1 GDH C.
difficile spores Ab CD1021 Amidase Peptide Ab C. difficile
germinated spores Peptide 1 Ab CD1021 Spore C. difficile spores
Peptide 1 Ab Spore GDH C. difficile spores Spore Amidase Peptide Ab
C. difficile germinated spores Spore CD1021 C. difficile spores
Peptide 1 Ab Spore Spore C. difficile spores CD1021 CD1021 C.
difficile spores Peptide 1 Ab Peptide 1 Ab Amidase Amidase Peptide
Ab C. difficile germinated spores Peptide Ab
Example 11
Detection of C. difficile Spores on a Surface
[0148] 100 .mu.l 10.sup.6 per ml of C. difficile spores were spread
on sterile aluminum coupons (1''.times.3'') and allowed to dry at
RT for about an hour. The spores were recovered by rubbing
vigorously for 5 to 10 seconds with a sterile swab moistened with
about 50 .mu.l of sterile PBS. The swabs containing the spores were
immersed in 1 ml of coating buffer and vortexed vigorously for one
to two minutes. The cotton swab was removed from the solution and
recovery of spores was determined by ELISA. For control, the
coupons were spread with sterile PBS and processed similarly. As
seen in Table 13, the C. difficile spore antibody and the CD1021
peptide 1 Ab were able to detect the presence of spores on a
surface.
TABLE-US-00017 TABLE 13 Detection of C. difficile spores from
aluminum coupons Absorbance at 450 nm C. difficile No antigen
CD1021 control Spore peptide 1 Spore CD1021 Ab Ab Ab Ab1 Coupon 1
1.35 1.12 0.15 0.18 Coupon 2 1.64 1.24 0.18 0.21 Coupon 3 1.22 0.93
0.14 0.2
Example 12
Antigen Affinity Purification of Antibodies
[0149] The purification of an antibody specific for a particular
antigen and free of cross reactants from other immunoglobulins is
often beneficial. Any of the polyclonal antibodies described in the
above examples may be purified by affinity chromatography using the
peptide or protein antigen covalently bound to an affinity matrix
through NH.sub.2 linkages. Purification can be achieved using
peptide/protein bound affinity matrix such as Affygel (Biorad,
Hercules, Calif.), AminoLink resin (Pierce, Rockford, Ill.) or CNBr
activated Sepharose 4B (Amersham Biosciences, Piscataway, N.J.) in
a column. The antibodies that are specific to the antigen bind to
the column. The unbound antibodies and other serum proteins pass
through the column. The antigen bound antibodies are then eluted
from the column. The resulting purified antibody is highly
specific.
Example 13
Antibodies to Additional Regions of C. difficile Hypothetical
Protein CD1021
[0150] Following the procedures described in more detail in Example
4, murine polyclonal and monoclonal antibodies can be developed to
additional polypeptide sequences of the hypothetical protein CD1021
C. difficile strain 630. For example, a polypeptide having residues
30 to 120 of the hypothetical protein CD1021 C. difficile strain
630 having the amino acid sequence of KSVDLYSDVY IEKYFNRDKV
MEVNIEIDES DLKDMNENAI KEEFKVAKVT VDGDTYGNVG IRTKGNSSLI SVANSDSDRY
SYKINFDKYN T (SEQ ID NO: 9) or a polypeptide having residues 194 to
293 of the hypothetical protein CD1021 C. difficile strain 630
having the amino acid sequence of VTGDLYKSDE GSSLQYKGDD PESYSNLIVE
SDKKTADWSK ITKLLKSLDT GEDIEKYLDV DSVLKNIAIN TALLNLDSYQ GSFAHNYYLY
EQDGVFSMLP (SEQ ID NO: 10) can be used as immunogens. Polyclonal
and monoclonal antibodies can also be developed to fragments
thereof, including, for example, fragments of about 10-20 amino
acids and about 14 amino acids of SEQ ID NO:9 and SEQ ID NO:10.
Example 14
Monoclonal Antibodies
[0151] Monoclonal antibodies that bind to C. difficile endospores
may be produced by a variety of known methods. Monoclonal
antibodies may be produced using any of SEQ ID NO:1-12, 16-23, 38,
and 45-50, and fragments thereof, as an antigen. For example, mice
that have been immunized with the hypothetical CD 1021 protein
sequence SEQ ID NO:2, as described in Example 4, or the putative
amidase protein sequence SEQ ID NO:6, as described in Example 6,
may be used for the generation of murine monoclonal antibodies.
Further, rabbits that have been immunized with the hypothetical
CD1021 peptides SEQ ID NO:3 or SEQ ID NO:4, as described in Example
5, or the putative amidase peptide sequences SEQ ID NO:7 or SEQ ID
NO:8, as described in Example 7, may be used for the generation of
rabbit monoclonal antibodies. Epitomic's proprietary method for
making monoclonal antibodies from rabbits rather than the
conventional method of starting with mice may be used. The basic
principal for making the antibody is the same as for mouse
monoclonals. A proprietary rabbit fusion partner is used that can
fuse to rabbit B-cells to create the rabbit hybridoma cells.
Hybridomas are then screened to select for clones with specific and
sensitive antigen recognition and the antibodies are characterized
using a variety of methods.
Example 15
Blast Analysis
[0152] The C. difficile strain 630 protein sequences for the
hypothetical protein CD1021 (YP.sub.--001087502) (SEQ ID NO:1) and
the putative N-acetylmuramoyl-L-alanine amidase cell surface
protein (YP.sub.--001087517) (SEQ ID NO:5) were searched for
homologies against the approximately 988 microbial genomic
sequences available at GenBank, RefSeq Nucleotides, EMBL, DDBJ, and
PDB sequences (excluding HTGS0,1,2, EST, GSS, STS, PAT, WGS).
Sequences producing significant alignments (E value of 0.0) were
found only in the genomic sequence databases for C. difficile
strain 630, C. difficile QCD-66c26, and C. difficile QCD-32g58.
Hypothetical Protein CD1021 in C. difficile QCD-32g58.
[0153] For hypothetical protein CD1021 of C. difficile strain 630
(SEQ ID NO:1) the following sequences with significant homology (E
value of 0.0) were found in C. difficile QCD-32g58.
[0154] GenBank Accession No. ZP.sub.--01804840, hypothetical
protein CdifQ.sub.--04001048 in C. difficile QCD-32g58 having the
amino acid sequence:
TABLE-US-00018 (SEQ ID NO: 11) MIIFLCAVVG VYSTSSNKSV DLYSDVYIEK
YFNRDKVMEV NIEIDESDLK DMNENAIKEE FKVAKVTVDG DTYGNVGIRT KGNSSLTSVA
NSDSDRYSYK INFDKYNTSQ SMEGLTQLNL NNCYSDPSYM REFLTYSICE EMGLATPEFA
YAKVSINGEY HGLYLAVEGL KESYLENNFG NVTGDLYKSD EGSSLQYKGD DPESYSNLIV
ESDKKTADWS KITKLLKSLD TGEDIEKYLD VDSVLKNIAI NTALLNLDSY QGSFAHNYYL
YEQDGVFSML PWDFNMSFGG FSGFGGGSQS IAIDEPTTGN LEDRPLISSL LKK.
[0155] GenBank Accession No. ZP.sub.--01804841, hypothetical
protein CdifQ.sub.--04001049 in C. difficile QCD-32g58 having the
amino acid sequence:
TABLE-US-00019 (SEQ ID NO: 12) MTTKLHDMIA SYVKEDPTAF YTYEEFEKNI
TSSIEDSSDN KGFGNKGFDN NNSNNSDSNN NSNSENKRSG NQSDKKEVNA ELTSSVVKTN
TDNETENKTT NDSESKNNTD KDKSGNDNNQ KLEGPRGKGG KSIPGVLEVA EDMSKTIKSQ
LSGETSSTKQ NSGDESSSGI KGSEKFDEDM SGMPEPPEGM DGKMPPGMGN MDKGDMNGKN
GNMNMDRNQD NPREAGGFGN RGGGSVSKTT TYFKLILGGA SMIIMSIMLV GVSRVKRRRF
IKSK.
[0156] REGION 461827 to 462825 of GenBank Accession No.
NZ_AAML04000007; C. difficile QCD-32g58 C_difficile_bld4_cont00007
having the nucleotide sequence:
TABLE-US-00020 (SEQ ID NO: 13) AAGATAAAAA AATTTACCCT TCTTATCTCT
ATTATGATTA TATTTTTATG TGCTGTAGTT GGAGTTTATA GTACATCTAG CAACAAAAGT
GTTGATTTAT ATAGTGATGT ATATATTGAA AAATATTTTA ACAGAGACAA GGTTATGGAA
GTTAATATAG AGATAGATGA AAGTGACTTG AAGGATATGA ATGAAAATGC TATAAAAGAA
GAATTTAAGG TTGCAAAAGT AACTGTAGAT GGAGATACAT ATGGAAACGT AGGTATAAGA
ACTAAAGGAA ATTCAAGTCT TACATCTGTA GCAAATAGTG ATAGTGATAG ATACAGCTAT
AAGATTAATT TTGATAAGTA TAATACTAGT CAAAGTATGG AAGGGCTTAC TCAATTAAAT
CTTAATAACT GTTACTCTGA CCCATCTTAT ATGAGAGAGT TTTTAACATA TAGTATTTGC
GAGGAAATGG GATTAGCGAC TCCAGAATTT GCATATGCTA AAGTCTCTAT AAATGGCGAA
TATCATGGTT TGTATTTGGC AGTAGAAGGA TTAAAAGAGT CTTATCTTGA AAATAATTTT
GGTAATGTAA CTGGAGACTT ATATAAGTCA GATGAAGGAA GCTCGTTGCA ATATAAAGGA
GATGACCCAG AAAGTTACTC AAACTTAATC GTTGAAAGTG ATAAAAAGAC AGCTGATTGG
TCTAAAATTA CAAAACTATT AAAATCTTTG GATACAGGTG AAGATATTGA AAAATATCTT
GATGTAGATT CTGTCCTTAA AAATATAGCA ATAAATACAG CTTTATTAAA CCTTGATAGC
TATCAAGGCA GTTTTGCCCA TAACTATTAT TTATATGAGC AAGATGGAGT ATTTTCTATG
TTACCATGGG ATTTTAATAT GTCATTTGGT GGATTTAGTG GTTTTGGTGG AGGTAGTCAA
TCTATAGCAA TTGATGAACC TACGACAGGT AATTTAGAAG ACAGACCTCT CATATCCTCG
TTATTAAAA.
[0157] An amino acid sequence encoded by nucleotide sequence SEQ ID
NO:13 is:
TABLE-US-00021 (SEQ ID NO: 21) KIKKFTLLIS IMIIFLCAVV GVYSTSSNKS
VDLYSDVYIE KYFNRDKVME VNIEIDESDL KDMNENAIKE EFKVAKVTVD GDTYGNVGIR
TKGNSSLTSV ANSDSDRYSY KINFDKYNTS QSMEGLTQLN LNNCYSDPSY MREFLTYSIC
EEMGLATPEF AYAKVSINGE YHGLYLAVEG LKESYLENNF GNVTGDLYKS DEGSSLQYKG
DDPESYSNLI VESDKKTADW SKITKLLKSL DTGEDIEKYL DVDSVLKNIA INTALLNLDS
YQGSFAHNYY LYEQDGVFSM LPWDFNMSFG GFSGFGGGSQ SIAIDEPTTG NLEDRPLISS
LLK.
[0158] And, REGION 462824 to 463732 of GenBank Accession No.
NZ_AAML04000007; C. difficile QCD-32g58_difficile_bld4_cont00007
having the nucleotide sequence:
TABLE-US-00022 (SEQ ID NO: 14) AAAAATGAGA CACACAAAAC AAAATACCAT
AAATATCTGG AAGAGATAGT AACAAAATAC CTAGATTCAG ACTATTTAGA GAATATGACA
ACAAAATTGC ATGACATGAT AGCATCATAT GTAAAAGAAG ACCCAACAGC ATTTTATACT
TATGAAGAAT TTGAAAAAAA TATAACATCT TCAATTGAAG ATTCTAGTGA TAATAAGGGA
TTTGGTAATA AAGGGTTTGA CAACAATAAC TCTAATAACA GTGATTCTAA TAATAATTCT
AATAGTGAAA ATAAGCGCTC TGGAAATCAA AGTGATAAAA AAGAAGTTAA TGCTGAATTA
ACATCAAGCG TAGTCAAAAC TAATACAGAT AATGAAACTG AAAATAAAAC TACAAATGAT
AGCGAAAGTA AGAATAATAC AGATAAAGAT AAAAGTGGAA ATGATAATAA TCAAAAGCTA
GAAGGTCCTA GGGGTAAAGG AGGTAAGTCA ATACCAGGGG TTTTGGAAGT TGCAGAAGAT
ATGAGTAAAA CTATAAAATC TCAATTAAGT GGAGAAACTT CTTCGACAAA GCAAAACTCT
GGTGATGAAA GTTCAAGTGG AATTAAAGGT AGTGAAAAGT TTGATGAGGA TATGAGTGGT
ATGCCAGAAC CACCTGAGGG AATGGATGGT AAAATGCCAC CAGGAATGGG TAATATGGAT
AAGGGAGATA TGAATGGTAA AAATGGCAAT ATGAATATGG ATAGAAATCA AGATAATCCA
AGAGAAGCTG GAGGTTTTGG CAATAGAGGA GGAGGCTCTG TGAGTAAAAC AACAACATAC
TTCAAATTAA TTTTAGGTGG AGCTTCAATG ATAATAATGT CGATTATGTT AGTAGGTGTA
TCAAGGGTAA AGAGAAGAAG ATTTATAAAG TCAAAATAA.
[0159] An amino acid sequence encoded by nucleotide sequence SEQ ID
NO:14 is:
TABLE-US-00023 (SEQ ID NO: 22) KNETHKTKYH KYLEEIVTKY LDSDYLENMT
TKLHDMIASY VKEDPTAFYT YEEFEKNITS SIEDSSDNKG FGNKGFDNNN SNNSDSNNNS
NSENKRSGNQ SDKKEVNAEL TSSVVKTNTD NETENKTTND SESKNNTDKD KSGNDNNQKL
EGPRGKGGKS IPGVLEVAED MSKTIKSQLS GETSSTKQNS GDESSSGIKG SEKFDEDMSG
MPEPPEGMDG KMPPGMGNMD KGDMNGKNGN MNMDRNQDNP REAGGFGNRG GGSVSKTTTY
FKLILGGASM IIMSIMLVGV SRVKRRRFIK SK.
Hypothetical Protein CD1021 in C. difficile QCD-66c26
[0160] For hypothetical protein CD1021 of C. difficile strain 630
(SEQ ID NO:1) one sequence with significant homology (E value of
0.0) was found in C. difficile QCD-66c26.
[0161] The complement of REGION 15690 to 17597 of GenBank Accession
No. NZ_ABFD01000037; C. difficile QCD-66c26 contig00122 having the
nucleotide sequence:
TABLE-US-00024 (SEQ ID NO: 23) ATGAAAGATA AAAAATTTAC CCTTCTTATC
TCTATTATGA TTATATTTTT ATGTGCTGTA TTGGAGTTT ATAGTACATC TAGCAACAAA
AGTGTTGATT TATATAGTGA TGTATATATT GAAAAATATT TTAACAGAGA CAAGGTTATG
GAAGTTAATA TAGAGATAGA TGAAAGTGAC TTGAAGGATA TGAATGAAAA TGCTATAAAA
GAAGAATTTA AGGTTGCAAA AGTAACTGTA GATGGAGATA CATATGGAAA CGTAGGTATA
AGAACTAAAG GAAATTCAAG TCTTACATCT GTAGCAAATA GTGATAGTGA TAGATACAGC
TATAAGATTA ATTTTGATAA GTATAATACT AGTCAAAGTA TGGAAGGGCT TACTCAATTA
AATCTTAATA ACTGTTACTC TGACCCATCT TATATGAGAG AGTTTTTAAC ATATAGTATT
TGCGAGGAAA TGGGATTAGC GACTCCAGAA TTTGCATATG CTAAAGTCTC TATAAATGGC
GAATATCATG GTTTGTATTT GGCAGTAGAA GGATTAAAAG AGTCTTATCT TGAAAATAAT
TTTGGTAATG TAACTGGAGA CTTATATAAG TCAGATGAAG GAAGCTCGTT GCAATATAAA
GGAGATGACC CAGAAAGTTA CTCAAACTTA ATCGTTGAAA GTGATAAAAA GACAGCTGAT
TGGTCTAAAA TTACAAAACT ATTAAAATCT TTGGATACAG GTGAAGATAT TGAAAAATAT
CTTGATGTAG ATTCTGTCCT TAAAAATATA GCAATAAATA CAGCTTTATT AAACCTTGAT
AGCTATCAAG GCAGTTTTGC CCATAACTAT TATTTATATG AGCAAGATGG AGTATTTTCT
ATGTTACCAT GGGATTTTAA TATGTCATTT GGTGGATTTA GTGGTTTTGG TGGAGGTAGT
CAATCTATAG CAATTGATGA ACCTACGACA GGTAATTTAG AAGACAGACC TCTCATATCC
TCGTTATTAA AAAATGAGAC ACACAAAACA AAATACCATA AATATCTGGA AGAGATAGTA
ACAAAATACC TAGATTCAGA CTATTTAGAG AATATGACAA CAAAATTGCA TGACATGATA
GCATCATATG TAAAAGAAGA CCCAACAGCA TTTTATACTT ATGAAGAATT TGAAAAAAAT
ATAACATCTT CAATTGAAGA TTCTAGTGAT AATAAGGGAT TTGGTAATAA AGGGTTTGAC
AACAATAACT CTAATAACAG TGATTCTAAT AATAATTCTA ATAGTGAAAA TAAGCGCTCT
GGAAATCAAA GTGATAAAAA AGAAGTTAAT GCTGAATTAA CATCAAGCGT AGTCAAAACT
AATACAGATA ATGAAACTGA AAATAAAACT ACAAATGATA GCGAAAGTAA GAATAATACA
GATAAAGATA AAAGTGGAAA TGATAATAAT CAAAAGCTAG AAGGTCCTAG GGGTAAAGGA
GGTAAGTCAA TACCAGGGGT TTTGGAAGTT GCAGAAGATA TGAGTAAAAC TATAAAATCT
CAATTAAGTG GAGAAACTTC TTCGACAAAG CAAAACTCTG GTGATGAAAG TTCAAGTGGA
ATTAAAGGTA GTGAAAAGTT TGATGAGGAT ATGAGTGGTA TGCCAGAACC ACCTGAGGGA
ATGGATGGTA AAATGCCACC AGGAATGGGT AATATGGATA AGGGAGATAT GAATGGTAAA
AATGGCAATA TGAATATGGA TAGAAATCAA GATAATCCAA GAGAAGCTGG AGGTTTTGGC
AATAGAGGAG GAGGCTCTGT GAGTAAAACA ACAACATACT TCAAATTAAT TTTAGGTGGA
GCTTCAATGA TAATAATGTC GATTATGTTA GTAGGTGTAT CAAGGGTAAA GAGAAGAAGA
TTTATAAAGT CAAAATAA.
[0162] An amino acid sequence encoded by nucleotide sequence SEQ ID
NO:15 is:
TABLE-US-00025 (SEQ ID NO: 23) MKDKKFTLLI SIMIIFLCAV VGVYSTSSNK
SVDLYSDVYI EKYFNRDKVM EVNIEIDESD LKDMNENAIK EEFKVAKVTV DGDTYGNVGI
RTKGNSSLTS VANSDSDRYS YKINFDKYNT SQSMEGLTQL NLNNCYSDPS YMREFLTYSI
CEEMGLATPE FAYAKVSING EYHGLYLAVE GLKESYLENN FGNVTGDLYK SDEGSSLQYK
GDDPESYSNL IVESDKKTAD WSKITKLLKS LDTGEDIEKY LDVDSVLKNI AINTALLNLD
SYQGSFAHNY YLYEQDGVFS MLPWDFNMSF GGFSGFGGGS QSIAIDEPTT GNLEDRPLIS
SLLKNETHKT KYHKYLEEIV TKYLDSDYLE NMTTKLHDMI ASYVKEDPTA FYTYEEFEKN
ITSSIEDSSD NKGFGNKGFD NNNSNNSDSN NNSNSENKRS GNQSDKKEVN AELTSSVVKT
NTDNETENKT TNDSESKNNT DKDKSGNDNN QKLEGPRGKG GKSIPGVLEV AEDMSKTIKS
QLSGETSSTK QNSGDESSSG IKGSEKFDED MSGMPEPPEG MDGKMPPGMG NMDKGDMNGK
NGNMNMDRNQ DNPREAGGFG NRGGGSVSKT TTYFKLILGG ASMIIMSIML VGVSRVKRRR
FIKSK.
[0163] FIGS. 1a and 1b demonstrate the high degree of homology
between the amino acid sequences for hypothetical CD1021 proteins
YP.sub.--001087502 from C. difficile strain 630 (SEQ ID NO:1),
ZP.sub.--01804840, from C. difficile QCD-32g58 (SEQ ID NO:11),
ZP.sub.--01804841 from C. difficile QCD-32g58 (SEQ ID NO:12),
Region 461827 to 462825 NZ_AAML04000007 from C. difficile QCD-32g58
(SEQ ID NO:21), Region 462824 to 463732 of NZ_AAML04000007 C.
difficile QCD-32g58 (SEQ ID NO:22), and the complement of Region
15690 to 17597 NZ-ABFD01000037 from C. difficile QCD-66c26 (SEQ ID
NO:23). A consensus sequence is shown as SEQ ID NO:38.
Putative N-Acetylmuramoyl-L-Alanine Amidase Cell Surface
Proteins
[0164] For putative N-acetylmuramoyl-L-alanine amidase cell surface
protein of C. difficile strain 630 (SEQ ID NO:5) the following
additional sequences with significant homology (E value of 0.0) in
C. difficile strain 630 were found.
[0165] GenBank Accession No. YP.sub.--001087516, cell surface
protein putative N-acetylmuramoyl-L-alanine amidase, C. difficile
strain 630, having the amino acid sequence:
TABLE-US-00026 (SEQ ID NO: 16) MLSKEINMRR NTKLLTTGIL SMAIVTPTMA
FATESNAMEN NADLNINLEK KSIVLGSTSK VSVKFKEKPD ADSITLKYKC YDMPLDTTLN
YNQSTESYEG TINYNKDPEY LNVWELQGIT INSKNNPKTL NKQELEKMGL NLKDYNVTQE
CIIEDITSRK DVNKYLRKTS APITELTGSD RYETAVKISK EGWKNGSDKV VIINGDVSID
GIISTPLATT YNAPILLVEK NNVPNSVKSE LKRLNPRDVI IIGDENAISK TTANQIKSTV
NASQTRLKGS NRYETSLLIA KEIDKNHDVE KVYITNANGG EVDALTIAAK AGQDKQPIIL
TDKNSITDNT YKWLKSEDLQ NAYFIGGPQM ISTNVINKVN DITKDNVTNN RVYGADRHET
NANVIKKFYT DDELEAVLVA KSDVLVDALA AGPLAANLKS PILITPKTYV SAYHKENLEA
KSANKVYKIG GGLTSKVMSS IASSLSKHNT TPTEPGNSGG KTVMIDPGHG GSDTGTTGKP
LGGIREKDYT LNTSLATTEY LRSKGFNVIM TRDTDKTLSL GNRTALSNSL RPDLFTSIHY
NASDTTGNGV EVFYKLKDKD GGTTKTVATN ILNRILEKFN LKNRGAKTRT LSTDPTKDYL
YVLRNNDMPA VLVECAFLDN EKDMSLLNTS NKVKEMGTQI GKGIEDSLK.
[0166] And, GenBank Accession No. YP.sub.--001089297 (cell surface
protein C. difficile 630) having the amino acid sequence:
TABLE-US-00027 (SEQ ID NO: 17) MMKKTTKLLA TGMLSVAMVA PNVALAAENT
TANTESNSDI NINLQRKSVV LGSKSNASVK FKEKLNADSI TLNFMCYDMP LEATLNYNEK
TDSYEGVINY NKDPEYLNVW ELQSIKINGK DEQKVLNKED LESMGLNLKD YDVTQEFIIS
DANSTKAVNE YMRKTSAPVK KLAGATRFET AVEISKQGWK DGSSKVVIVN GELAADGITA
TPLASTYDAP ILLANKDDIP ESTKAELKRLNPSDVIIIGD DGSVSQKAVS QIKSAVNVNV
TRIGGVDRHE TSLLIAKEID KYHDVNKIYI ANGYAGEYDA LNISSKAGED QQPIILANKD
SVPQGTYNWL SSQGLEEAYY IGGSQSLSSK IIDQISKIAK NGTSKNRVSG ADRHETNANV
IKTFYPDKEL SAMLVAKSDI IVDSITAGPL AAKLKAPILI TPKTYVSAYH STNLSEKTAE
TVYQIGDGMK DSVINSIASS LSKHNAPTEP DNSGSAAGKT VVIDPGHGGS DSGATSGLNG
GAQEKKYTLN TALATTEYLR SKGINVVMTR DTDKTMALGE RTALSNTIKP DLFTSIHYNA
SNGSGNGVEI YYKVKDKNGG TTKTAASNIL KRILEKFNMK NRGIKTRTLD NGKDYLYVLR
NNNYPAILVE CAFIDNKSDM DKLNTAEKVK TMGTQIGIGI EDTVK.
[0167] For putative N-acetylmuramoyl-L-alanine amidase cell surface
protein of C. difficile strain 630 (SEQ ID NO:5) the following
sequences with significant homology (E value of 0.0) were found in
C. difficile QCD-32g58.
[0168] GenBank Accession No. ZP.sub.--01804350 (hypothetical
protein CdifQ.sub.--04001133; C. difficile QCD-32g58) having the
amino acid sequence:
TABLE-US-00028 (SEQ ID NO: 18) MFRFKEKPDA DSITLKYKCY DMPLDTTLNY
NQSTESYEGT INYNKDPEYL NVWELQGITI NSKNNPKTLN KQELEKMGLN LKDYNVTQEC
IIEDITSRKD VNKYLRKTSA PITELTGSDR YETAVKISKE GWKNGSDKVV IINGDVSIDG
IISTPLATTY NAPILLVEKN NVPNSVKSEL KRLNPRDVII IGDENAISKT TANQIKSTVN
ASQTRLKGSN RYETSLLIAK EIDKNHDVEK VYITNANGGE VDALTIAAKA GQDKQPIILT
DKNSITDNTY KWLKSEDLQN AYFIGGPQMI STNVINKVND ITKDNVTNNR VYGADRHETN
ANVIKKFYTD DELEAVLVAK SDVLVDALAA GPLAANLKSP ILITPKTYVS AYHKDNLEAK
SANKVYKIGG GLTSKVMNSI ASSLSKHNTT PTEPGNSGGK TVMIDPGHGG SDTGTTGKPL
GGIKEKDYTL NTSLATTEYL RSKGFNVIMT RDTDKTLSLG NRTALSNSLR PDLFTSIHYN
ASDTTGNGVE VFYKLKDKDG GTTKTVATNI LNRILEKFNL KNRGAKTRTL STDPTKDYLY
VLRNNDMPAV LVECAFLDNE KDMSLLNTSN KVKEMGTQIG KGIEDSLK.
[0169] GENEBANK Accession No. ZP.sub.--01804351 (hypothetical
protein CdifQ.sub.--04001134; C. difficile QCD-32g58) having the
amino acid sequence:
TABLE-US-00029 (SEQ ID NO: 19) MLSKEINMRR NTKLLTTGIL SMAIVAPTMA
FATESNAMEN NADLNINLEK KSIVLGSKSK VSVKFKEKPD ADSITLKYKC YDMPLDTTLN
YNQSTGAYEG TINYNQDPEY LNVWELQGIT INSKNNPKTL NGQDLEKMGL NLKDYNVTQE
CIIEDITSRK DVNKYLRKTS APITELTGSD RYETAVKISK EGWKNGSDKV VIINGDVSID
GIISTPLATT YNAPILLVEK NNVPNSVKSE LKRLNPKDII IIGDENAISK TTANQIKSTV
NASQTRLNGS NRYETSLLIA KEIDKNHDVE KVYITNANGG EVDALTIAAK AGQDKQPIIL
TDKDSITDNT YKWLKSEDLQ NAYFIGGPQM ISTNVINKVN GITKDSVTNN RVYGADRHET
NANVIKKFYT EDEIEAVLVA KSDVLVDALA AGPLAANLKS PILITPKTYV SAYHKDNLEA
KSANKVYKIG GGLTSKVMSS IASSLSKHNT TPTEPGNSGG KTVMIDPGHG GSAPGNSSGG
MIEKDYNLNT SLATTEYLRS KGFNVIMTRD TDKTLSLGNR TA.
[0170] And, GenBank Accession No. ZP.sub.--01802273 (hypothetical
protein CdifQ.sub.--04003247; C. difficile QCD-32g58) having the
amino acid sequence:
TABLE-US-00030 (SEQ ID NO: 20) MMKKTTKLLA TGMLSVAMVA PNVALAAENT
TANTESNSDI NINLQRKSVV LGSKSNASVK FKEKLNADSI TLNFMCYDMP LEATLNYNEK
TDSYEGVINY NKDPEYLNVW ELQSIKINGK DEQKVLNKED LESMGLNLKD YDVTQEFIIS
DANSTKAVNE YMRKTSAPVK KLAGATRFET AVEISKQGWK DGSSKVVIVN GELAADGITA
TPLASTYDAP ILLANKDDIP ESTKAELKRL NPSDVIIIGD DGSVSQKAVS QIKSAVNVNV
TRIGGVDRHE TSLLIAKEID KYHDVNKIYI ANGYAGEYDA LNISSKAGED QQPIILANKD
SVPQGTYNWL SSQGLEEAYY IGGSQSLSSK IIDQISKIAK NGTSKNRVSG ADRHETNANV
IKTFYPDKEL SAMLVAKSDI IVDSITAGPL AAKLKAPILI TPKTYVSAYH STNLSEKTAG
TVYQIGDGMK DSVINSIASS LSKHNAPTEP DNSGSAAGKT VVIDPGHGGS DSGATSGLNG
GAQEKKYTLN TALATTEYLR SKGINVVMTR DTDKTMALGE RTALSNTIKP DLFTSIHYNA
SNGAGNGVEI YYKVKDKNGG TTKTAASNIL KRILEKFNMK NRGIKTRTLD NGKDYLYVLR
NNNYPAILVE CAFIDNKSDM DKLNTAEKVK TMGTQIGIGI EDTVK.
[0171] The antibodies described herein may bind to one or more of
the amino acid sequences of SEQ ID NO:11, SEQ ID NO:12, SEQ ID
NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ
ID NO:21, SEQ ID NO:22, and SEQ ID NO:23, and fragments thereof.
Antibodies described herein may bind to one or more of the amino
acid sequences encoded by the genomic nucleotide sequences of SEQ
ID NO:13, SEQ ID NO:14, and SEQ ID NO:15 and polypeptide fragment
thereof.
[0172] Antibodies may be produced, by any of a variety methods,
including, but not limited to, any of those described herein, that
bind to one or more of the amino acid sequences of SEQ ID NO:11,
SEQ ID NO:12, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID
NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, and SEQ ID NO:23,
and fragment thereof. Antibodies may be produced, by any of the
various methods described herein, that bind to one or more of the
amino acid sequences encoded by the genomic nucleotide sequences of
SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15 and polypeptide
fragment thereof. Any such antibodies may be used in methods of
detecting C. difficile spores in a sample.
Example 16
Cloning and Sequencing CD1021 from C. difficile ATCC 9689
[0173] A CD1021 coding sequence was amplified by PCR using a
forward primer with NheI site
(5'-TAAGCTAGCATGAAAGATAAAAAATTTACC-3') (SEQ ID NO:24), a reverse
primer with XhoI site (5'-TTACTCGAGTTTTGACTTTATAAATCTTCT-3') (SEQ
ID NO:25) and genomic DNA from ATCC strain 9689 as the template. A
stop codon was removed from the reverse primer to facilitate
addition of 6-HIS tag to the end of the sequence. The resulting
fragment size was 1923 base pairs (bp).
[0174] Similarly, the region corresponding to amino acid residues
30 to 120 of CD1021 in C. difficile strain 630 (SEQ ID NO:9) (also
referred to herein as "fragment 1") was amplified using genomic DNA
from ATCC strain 9689 as the template with a forward primer with
NheI site (5'-ACAGCTAGCATGAAAAGTGTTGATTTATATAGT-3') (SEQ ID NO:26)
and a reverse primer with XhoI site (5'-ACTCTCGAGAGTATTATAC
TTATCAAAATTA-3') (SEQ ID NO:27). The resulting fragment size was
294 bp and included an ATG initiation codon.
[0175] The region corresponding to amino acid residues 194 to 293
of CD1021 in C. difficile strain 630 (SEQ ID NO:10) (also referred
to herein as "fragment 2") was amplified using genomic DNA from
ATCC strain 9689 as the template with a forward primer with NheI
site (5'-AATGCTAGCATGGTAACTGGAG ACTTATATAAGTCA-3') (SEQ ID NO:28)
and a reverse primer with XhoI site (5'-AAACTCGAGTGG TAACA
TAGAAAATACTCCAT-3') (SEQ ID NO:29). The resulting fragment size was
321 bp and included an ATG initiation codon.
[0176] And, the region corresponding to amino acid residues 505 to
604 of CD1021 in C. difficile strain 630 (SEQ ID NO:2) (also
referred to herein as "fragment 3") was amplified using genomic DNA
from ATCC strain 9689 as the template with a forward primer with
NheI site (5'-GCAGCTAGCATGAGTAAAACTATAAAATCTCAA-3') (SEQ ID NO:30)
and a reverse primer with XhoI site
(5'-AATCTCGAGGAAGTATGTTGTTGTTTTACT CAC-3') (SEQ ID NO:31). The
resulting fragment size was 321 bp and included an ATG initiation
codon.
[0177] The resultant PCR reactions were run on an agarose gel
(0.8%) and products of the expected size were observed. The PCR
fragments were cloned using Zero Blunt TOPO PCR Cloning Kit
(Invitrogen, Carlsbad, Calif.) according to manufacturer's
instructions. The transformed colonies were picked and grown in LB
with Kanamycin (100 .mu.g/ml) for 16 hours at 37.degree. C. The
plasmid was isolated from these cultures using Qiaprep spin
miniprep kit (Qiagen, Valencia, Calif.) and the plasmids were cut
with EcoRI (Invitrogen) and analyzed by agarose gel. The clones
having the insert were sequenced using BigDye.RTM. Terminator v1.1
Cycle Sequencing Kit (Applied Biosystems, Foster City, Calif.) with
M13 forward -20 primer (5'-GTAAAACGACGGCCAGT-3') (SEQ ID NO:32) and
M13 reverse -27 primer (5'-CAGGAAACAGCTATGAC-3') (SEQ ID NO:33).
Appropriate internal primers were used to obtain the complete
sequence of the 1923 bp CD1021 coding sequence. Three clones were
selected for further characterization for each of the four cloning
reactions. For the 1923 bp CD1021 coding sequence, the three clones
were pCD1021-1, pCD1021-2, and pCD1021-3. For fragment 1, the three
clones were pCD1021-Fr1-1, pCD1021-Fr1-2, and pCD1021-Fr1-3. For
fragment 2, the three clones were pCD1021-Fr2-1, pCD1021-Fr2-2, and
pCD1021-Fr2-3. For fragment 3, the three clones were pCD1021-F3-1,
pCD1021-Fr3-2, and pCD1021-Fr3-3.
[0178] The nucleotide sequence of C. difficile ATCC 9689 CD1021
fragment in plasmid pCD1021-2 is:
TABLE-US-00031 (SEQ ID NO: 39) ATGAAAGATA AAAAATTTAC CCTTCTTATC
TCGATTATGA TTATATTTTT ATGTGCTGTA GTTGGAGTTT ATAGTACATC TAGCAACAAA
AGTGTTGATT TATATAGTGA TGTATATATT GAAAAATATT TTAACAGAGA CAAGGTTATG
GAAGTTAATA TAGAGATAGA TGAAAGTGAC TTGAAGGATA TGAATGAAAA TGCTATAAAA
GAAGAATTTA AGGTTGCAAA AGTAACTGTA GATGGAGATA CATATGGAAA CGTAGGTATA
AGAACTAAAG GAAATTCAAG TCTTATATCT GTAGCAAATA GTGATAGTGA TAGATACAGC
TATAAGATTA ATTTTGATAA GTATAATACT AGTCAAAGTA TGGAAGGGCT TACTCAATTA
AATCTTAATA ACTGTTACTC TGACCCATCT TATATGAGAG AGTTTTTAAC ATATAGTATT
TGCGAGGAAA TGGGATTAGC GACTCCAGAA TTTGCATATG CTAAAGTCTC TATAAATGGC
GAATATCATG GTTTGTATTT GGCAGTAGAA GGATTAAAAG AGTCTTATCT TGAAAATAAT
TTTGGTAATG TAACTGGAGA CTTATATAAG TCAGATGAAG GAAGCTCGTT GCAATATAAA
GGAGATGACC CAGAAAGTTA CTCAAACTTA ATCGTTGAAA GTGATAAAAA GACAGCTGAT
TGGTCTAAAA TCACAAAACT ATTAAAATCT TTGGATACAG GTGAAGATAT TGAAAAATAT
CTTGATGTAG ATTCTGTCCT TAAAAATATA GCAATAAATA CAGCTTTATT AAACCTTGAT
AGCTATCAAG GGAGTTTTGC CCATAACTAT TATTTATATG AGCAAGATGG AGTATTTTCT
ATGTTACCAT GGGATTTTAA TATGTCATTT GGTGGATTTA GTGGTTTTGG TGGAGGTAGT
CAATCTATAG CAATTGATGA ACCTACGACA GGTAATTTAG AAGACAGACC TCTCATATCC
TCGTTATTAA AAAATGAGAC ATACAAAACA AAATACCATA AATATCTGGA AGAGATAGTA
ACAAAATACC TAGATTCAGA CTATTTAGAG AATATGACAA CAAAATTGCA TGACATGATA
GCATCATATG TAAAAGAAGA CCCAACAGCA TTTTATACTT ATGAAGAATT TGAAAAAAAT
ATAACATCTT CAATTGAAGA TTCTAGTGAT AATAAGGGAT TTGGTAATAA AGGGTTTGAC
AACAATAACT CTAATAACAG TGATTCTAAT AATAATTCTA ATAGTGAAAA TAAGCGCTCT
GGAAATCAAA GTGATGAAAA AGAAGTTAAT GCTGAATTAA CATCAAGCGT AGTCAAAGCT
AATACAGATA ATGAAACTAA AAATAAAACT ACAAATGATA GTGAAAGTAA GAATAATACA
GATAAAGATA AAAGTGGAAA TGATAATAAT CAAAAGCTAG AAGGTCCTAT GGGTAAAGGA
GGTAAGTCAA TACCAGGGGT TTTGGAAGTT GCAGAAGATA TGAGTAAAAC TATAAAATCT
CAATTAAGTG GAGAAACTTC TTCGACAAAG CAAAACTCTG GTGATGAAAG TTCAAGTGGA
ATTAAAGGTA GTGAAAAGTT TGATGAGGAT ATGAGTGGTA TGCCAGAACC ACCTGAGGGA
ATGGATGGTA AAATGCCACC AGGAATGGGT AATATGGATA AGGGAGATAT GAATGGTAAA
AATGGCAATA TGAATATGGA TAGAAATCAA GATAATCCAA GAGAAGCTGG AGGTTTTGGC
AATAGAGGAG GAGGCTCTGT GAGTAAAACA ACAACATACT TCAAATTAAT TTTAGGTGGA
GCTTCAATGA TAATAATGTC GATTATGTTA GTTGGTGTAT CAAGGGTAAA GAGAAGAAGA
TTTATAAAGT CAAAA.
The translated amino acid sequence of CD 1021 fragment in plasmid
pCD1021-2 is:
TABLE-US-00032 (SEQ ID NO: 45)
MKDKKFTLLISIMIIFLCAVVGVYSTSSNKSVDLYSDVYIEKYFNRDKV
MEVNIEIDESDLKDMNENAIKEEFKVAKVTVDGDTYGNVGIRTKGNSSL
ISVANSDSDRYSYKINFDKYNTSQSMEGLTQLNLNNCYSDPSYMREFLT
YSICEEMGLATPEFAYAKVSINGEYHGLYLAVEGLKESYLENNFGNVTG
DLYKSDEGSSLQYKGDDPESYSNLIVESDKKTADWSKITKLLKSLDTGE
DIEKYLDVDSVLKNIAINTALLNLDSYQGSFAHNYYLYEQDGVFSMLPW
DFNMSFGGFSGFGGGSQSIAIDEPTTGNLEDRPLISSLLKNETYKTKYH
KYLEEIVTKYLDSDYLENMTTKLHDMIASYVKEDPTAFYTYEEFEKNIT
SSIEDSSDNKGFGNKGFDNNNSNNSDSNNNSNSENKRSGNQSDEKEVNA
ELTSSVVKANTDNETKNKTTNDSESKNNTDKDKSGNDNNQKLEGPMGKG
GKSIPGVLEVAEDMSKTIKSQLSGETSSTKQNSGDESSSGIKGSEKFDE
DMSGMPEPPEGMDGKMPPGMGNMDKGDMNGKNGNMNMDRNQDNPREAGG
FGNRGGGSVSKTTTYFKLILGGASMIIMSIMLVGVSRVKRRRFIKSK.
[0179] The nucleotide sequence of C. difficile ATCC 9689 CD1021
fragment (30 to 120 amino acid residues) in plasmid pCD1021-Fr1-1
is:
TABLE-US-00033 (SEQ ID NO: 40) ATGAAAAGTG TTGATTTATA TAGTGATGTA
TATATTGAAA AATATTTTAA CAGAGACAAG GTTATGGAAG TTAATATAGA GATAGATGAA
AGTGACTTGA AGGATATGAA TGAAAATGCT ATAAAAGAAG AATTTAAGGT TGCAAAAGTA
ACTGTAGATG GAGATACATA TGGAAACGTA GGTATAAGAA CTAAAGGAAA TTCAAGTCTT
ATATCTGTAG CAAATAGTGA TAGTGATAGA TACAGCTATA AGATTAATTT TGATAAGTAT
AATACT.
[0180] The translated amino acid sequence of CD1021 fragment (30 to
120 amino acid residues) in plasmid pCD1021-Fr1-1 is:
TABLE-US-00034 (SEQ ID NO: 46)
MKSVDLYSDVYIEKYFNRDKVMEVNIEIDESDLKDMNENAIKEEFKVAK
VTVDGDTYGNVGIRTKGNSSLISVANSDSDRYSYKINFDKYNT.
[0181] The nucleotide sequence of C. difficile ATCC 9689 CD1021
fragment (194 to 293 amino acid residues) in plasmid pCD1021-Fr2-1
is:
TABLE-US-00035 (SEQ ID NO: 41) ATGGTAACTG GAGACTTATA TAAGTCAGAT
GAAGGAAGCT CGTTGCAATA TAAAGGAGAT GACCCAGAAA GTTACTCAAA CTTAATCGTT
GAAAGTGATA AAAAGACAGC TGATTGGTCT AAAATCACAA AACTATTAAA ATCTTTGGAT
ACAGGTGAAG ATATTGAAAA ATATCTTGAT GTAGATTCTG TCCTTAAAAA TATAGCAATA
AATACAGCTT TATTAAACCT TGATAGCTAT CAAGGGAGTT TTGCCCATAA CTATTATTTA
TATGAGCAaG ATGGAGTATT TTCTATGTTA CCA.
[0182] The translated amino acid sequence of CD1021 fragment (194
to 293 amino acid residues) in plasmid pCD1021-Fr2-1 is:
TABLE-US-00036 (SEQ ID NO: 47)
MVTGDLYKSDEGSSLQYKGDDPESYSNLIVESDKKTADWSKITKLLKSL
DTGEDIEKYLDVDSVLKNIAINTALLNLDSYQGSFAHNYYLYEQDGVFS MLP.
[0183] The nucleotide sequence of C. difficile ATCC 9689 CD1021
fragment (505 to 604 amino acid residues) in plasmid pCD1021-Fr3-1
is:
TABLE-US-00037 (SEQ ID NO: 42) ATGAGTAAAA CTATAAAATC TCAATTAAGT
GGAGAAACTT CTTCGACAAA GCAAAACTCT GGTGATGAAA GTTCAAGTGG AATTAAAGGT
AGTGAAAAGT TTGATGAGGA TATGAGTGGT ATGCCAGAAC CACCTGAGGG AATGGATGGT
AAAATGCCAC CAGGAATGGG TAATATGGAT AAGGGAGATA TGAATGGTAA AAATGGCAAT
ATGAATATGG ATAGAAATCA AGATAATCCA AGAGAAGCTG GAGGTTTTGG CAATAGAGGA
GGAGGCTCTG TGAGTAAAAC AACAACATAC TTC.
[0184] The translated amino acid sequence of CD1021 fragment (505
to 604 amino acid residues) in plasmid pCD1021-Fr3-1 is:
TABLE-US-00038 (SEQ ID NO: 48)
MSKTIKSQLSGETSSTKQNSGDESSSGIKGSEKFDEDMSGMPEPPEGMD
GKMPPGMGNMDKGDMNGKNGNMNMDRNQDNPREAGGFGNRGGGSVSKTT TYF.
Example 17
Expression of C. difficile ATCC 9689 CD1021 Clones
[0185] The recombinant plasmid pCD1021-2, obtained in Example 16,
and the expression vector pET21-a(+) (Novagen, Madison, Wis.) were
separately cut with the restriction enzymes NheI and XhoI (New
England Biolabs, Ipswich, Mass.) according to manufacturer's
instructions. The restriction enzyme-digested pCD1021-2 DNA was run
on an agarose gel (0.8%) and the full-length CD 1021 fragment was
purified from the gel using Qiaquick gel extraction kit (Qiagen).
The resultant fragment (1917 bp) was ligated into the restriction
enzyme-digested expression vector pET21-a(+) using T4 DNA ligase
(Invitrogen) according to manufacturer's instructions.
[0186] The ligated mixture was used to transform TOP10 chemically
competent E. coli cells and plated on LB Agar with ampicillin (50
.mu.g/ml). The plates were incubated at 37.degree. C. for 12 to 16
hours and the recombinant clones were picked and grown in LB with
ampicillin (100 .mu.g/ml) for 16 hours at 37.degree. C. The plasmid
was isolated from these cultures by miniprep using alkaline lysis
protocol (Miniprep kit, Qiagen) and the plasmids were cut with NheI
and XhoI and analyzed by agarose gel. The clones having the
full-length CD 1021 insert were selected and tested for expression
of proteins according to manufacturer's instruction.
[0187] Similarly, the fragment 1, fragment 2, and fragment 3 of
clones CD1021-Fr1-1, CD1021-Fr2-2, and CD1021-Fr3-1 obtained in
Example 16, were restriction digested with NheI and XhoI and cloned
into NheI/XhoI restricted pET21a+. The recombinant clones were
picked and analyzed as described above. The clones having the
inserts were selected and tested for expression of proteins
according to manufacturer's instruction.
[0188] The recombinant clones were transformed into competent cells
of BLR(DE3) and plated on LB Agar with ampicillin (50 .mu.g/ml).
The plates were incubated at 37.degree. C. for 12 to 16 hours.
Several colonies were picked and grown in 5 ml of LB with
ampicillin (100 .mu.g/ml) for 16 hours at 37.degree. C. The
overnight grown clones were diluted 1:100 into 5 ml LB with
ampicillin (100 .mu.g/ml) and grown to an OD600 of 0.6 to 0.7. The
clones were induced with 0.4 mM isopropyl
.beta.-D-1-thiogalactopyranoside (IPTG; Sigma, St. Louis, Mo.) for
3 hrs either at 37.degree. C. or 15.degree. C. using Lab-Line MaxQ
4000 Incubated and Refrigerated Shakers (Barnstead International,
Dubuque, Iowa).
[0189] The induced cells were spun at 5000 rpm at 4.degree. C. for
10 min and resuspended in 1 ml of 0.1 mM TRIS-HCl buffer, pH 8.0.
The cells were sonicated using Branson Digital Sonifier model
S-250D (Branson, Danbury, Conn.) using 1/8 inch tapered microtip
for 10 sec to lyse the cells. The cell extracts were analyzed by
SDS-PAGE. The gels were stained with Coomassie blue and the induced
cells showed better expression of recombinant protein of expected
molecular weight at 15.degree. C. than at 37.degree. C.
Example 18
Cloning and Sequencing Amidase (CD1036) from C. difficile ATCC
9689
[0190] A putative N-acetylmuramoyl-L-alanine amidase cell surface
protein coding sequence was amplified by PCR using a forward primer
with NcoI site (5'-AATCCATG G TAAGTAAGGAGATTAATATG-3') (SEQ ID
NO:34), a reverse primer with XhoI site
(5'-TTCCTCGAGTTTTAATGAATCTTCTATTCC-3') (SEQ ID NO:35) and genomic
DNA from C. difficile ATCC strain 9689 as the template. A stop
codon was removed from the reverse primer to facilitate addition of
6-HIS tag to the end of the sequence. The resulting fragment size
was 2045 bp.
[0191] Similarly, the region corresponding to amino acid residues
294 to 393 of the putative N-acetylmuramoyl-L-alanine amidase cell
surface protein of C. difficile 630 (SEQ ID NO:6) was amplified
using genomic DNA from ATCC strain 9689 as the template with a
forward primer with NcoI site (5'-AGG CCA
TGGATAAAAATCATGATGTGGAA-3') (SEQ ID NO:36) and a reverse primer
with XhoI site (5'-TTTCTCGAGGTTTGCATTTG TTTCGTGTCT-3') (SEQ ID
NO:37). The resulting fragment size was 317 bp and included an ATG
initiation codon.
[0192] The resultant PCR fragments were cloned using Zero Blunt
TOPO PCR Cloning Kit (Invitrogen, San Diego, Calif.) and
recombinant clones sequenced, using the procedures described in
Example 16. Three clones were characterized for each of the two
cloning reactions. For the 2045 bp CD1036 coding sequence, the
three clones were pCD1036-1, pCD1036-2, and pCD1036-3. For fragment
1, the three clones were pCD1036-Fr1-1, pCD1036-Fr1-2, and
pCD1036-Fr1-3.
[0193] The nucleotide sequence of C. difficile ATCC 9689 CD1036
fragment in plasmid pCD1036-2 is:
TABLE-US-00039 (SEQ ID NO: 43) ATGGTAAGTA AGGAGATTAA TATGAGAAGA
AATACAAAAT TATTAACAAC AGGGATTCTT TCAATGGCAA TCGTCGCACC TACAATGGCA
TTTGCTACTG AATCTAATGC TATGGAAAAT AACGCTGATT TAAATATAAA CTTAGAGAAA
AAAAGTATCG TTTTAGGTAG CAAATCAAAA GTTAGTGTCA AATTTAAAGA AAAACCAGAT
GCAGATAGCA TTAcATTAAA GTATAAATGC TATGACATGC CATTGAATAC AACTCTAAAT
TACAATCAAT CAACTGGGGC ATATGAAGGA ACTATCAATT ATAACCAAGA CCCAGAATAT
CTAAATGTTT GGGAACTACA AGGGATAACA ATAAACAGCA AAAATAATCa TAAAACTTTA
AACAGACAAG ACCTAGAAAA GCTGGGATTA AATTTAAAAG ACTATAATGT AACACAGGAA
TGTATAATTG AAGATATAAC TTCTAGAAAA GATGTAAATA AATATTTGAG AAAAACTTCT
TCACCTATTA CAGAACTTAC AGGAAGTGAT AGATATGAAA CAGCAGTTAA AATAAGTAAA
GAGGGCTGGA AAAATGGTTC AGATAAGGTA GTTATAATAA ATGGGGATGT AAGTATAGAT
GGCATTATAT CAACTCCACT GGCAACCACA TATAATGCAC CAATACTTTT GGTTGAAAAA
AACAATGTAC CTAATAGTGT AAAATCAGAA TTAAAGCGCC TAAACCCTAA AGATATAATT
ATAATTGGAG ATGAGAATGC TATTTCTAAA ACTACTGCTA ATCAAATTAA ATCAACTGTA
AATGCTAGTC AAACACGTTT AAATGGTTCT AATAGATATG AGACATCTTT ATTGATAGCA
AAGGAAATAG ATAAAAATCA TGATGTGGAA AAAGTATACA TAACAAATGC TAATGGCGGA
GAAGTGGATG CACTTACTAT AGCAGCAAAA GCAGGTCAAG ACAAGCAACC AATTATATTA
ACTGATAAAG ATAGTATTAC AGACAATACA TATAAATGGT TAAAGAGTGA GGATTTACAA
AATGCTTATT TTATAGGTGG TCCTCAAATG ATATCAACAA ATGTTATAAA TAAGGTAAAT
GGAATAACTA AAGATAGTGT TACTAATAAT AGAGTATACG GAGCAGATAG ACACGAAACA
AATGCAAACG TAATAAAAAA ATTCTATACA GATGATGAGT TAGAGGCTGT TTTAGTAGCT
AAATCAGATG TACTTGTTGA TGCTTTAGCA GCAGGTCCAT TGGCTGCGAA CTTAAAATCT
CCAATACTTA TAACACCAAA GACGTATGTA TCTGCATACC ATAAAGATAA TTTAGAAGCT
AAATCAGCTA ATAAGGTATA CAAAATAGGA GGAGGATTGA CTTCTAAGGT AATGAGCTCT
ATAGCATCAT CATTATCTAA ACACAATACG ACTCCAACAG AACCAGGAAA TAGTGGGGGC
AAGACAGTTA TGATTGACCC AGGGCATGGT GGTTCAGCAC CTGGAAATTC ATCTGGAGGA
ATGATTGAAA AAGATTACAA TTTAAATACT TCACTTGCAA CAACTGAATA TTTACGTTCA
AAGGGATTCA ATGTAATAAT GACAAGAGAC ACAGATAAGA CTTTATCTCT TGGAAATAGA
ACTGCTCTAT CTAATTCATT GAAACCAGAT TTATTTACAA GTATACATTA TAATGGCTCA
ACTAATAAAC AAGGTCATGG TGTAGAAGTA TTTTATAAGC TTAAAGATAA AAATGGAGGG
ACTACTAAAA CTGTAGCTAC CAATATATTA AATAGAATTT TAGAGAAATT TAAACTTACA
AATAGAGGTA TAAAAACAAG AGTACTTCCT AGTGATTCTA CAAAAGATTA TTTATACGTT
TTAAGAAGTA ATGATATGCC AGCTGTACTT GTAGAATGTG CATTTTTGGA TAATGAAAAT
GATATGAGTT TAATAAACTC ATCTGCAAAA GTAAAAGAAA TGGGTACACA AATAGGTAAA
GGAATAGAAG ATTCATTAAA A.
The translated amino acid sequence of CD1036 fragment in plasmid
pCD1036-2 is:
TABLE-US-00040 (SEQ ID NO: 49)
MVSKEINMRRNTKLLTTGILSMAIVAPTMAFATESNAMENNADLNINLE
KKSIVLGSKSKVSVKFKEKPDADSITLKYKCYDMPLNTTLNYNQSTGAY
EGTINYNQDPEYLNVWELQGITINSKNNHKTLNRQDLEKLGLNLKDYNV
TQECIIEDITSRKDVNKYLRKTSSPITELTGSDRYETAVKISKEGWKNG
SDKVVIINGDVSIDGIISTPLATTYNAPILLVEKNNVPNSVKSELKRLN
PKDIIIIGDENAISKTTANQIKSTVNASQTRLNGSNRYETSLLIAKEID
KNHDVEKVYITNANGGEVDALTIAAKAGQDKQPIILTDKDSITDNTYKW
LKSEDLQNAYFIGGPQMISTNVINKVNGITKDSVTNNRVYGADRHETNA
NVIKKFYTDDELEAVLVAKSDVLVDALAAGPLAANLKSPILITPKTYVS
AYHKDNLEAKSANKVYKIGGGLTSKVMSSIASSLSKHNTTPTEPGNSGG
KTVMIDPGHGGSAPGNSSGGMIEKDYNLNTSLATTEYLRSKGFNVIMTR
DTDKTLSLGNRTALSNSLKPDLFTSIHYNGSTNKQGHGVEVFYKLKDKN
GGTTKTVATNILNRILEKFKLTNRGIKTRVLPSDSTKDYLYVLRSNDMP
AVLVECAFLDNENDMSLINSSAKVKEMGTQIGKGIEDSLK.
[0194] The nucleotide sequence of C. difficile ATCC 9689 CD1036
fragment (294 to 393 amino acid residues) in plasmid pCD1036-Fr1-1
is:
TABLE-US-00041 (SEQ ID NO: 44) ATGGATAAAA ATCATGATGT GGAAAAAGTA
TACATAACAA ATGCTAATGG CGGAGAAGTG GATGCACTTA CTATAGCAGC AAAAGCAGGT
CAAGACAAGC AACCAATTAT ATTAACTGAT AAAGATAGTA TTACAGACAA TACATATAAA
TGGTTAAAGA GTGAGGATTT ACAAAATGCT TATTTTATAG GTGGTCCTCA AATGATATCA
ACAAATGTTA TAAATAAGGT AAATGGAATA ACTAAAGATA GTGTTACTAA TAATAGAGTA
TACGGAGCAG ATAGACACGA AACAAATGCA AAC.
[0195] The translated amino acid sequence 9689 CD1036 fragment (294
to 393 amino acid residues) in plasmid pCD1036-Fr1-1 is:
TABLE-US-00042 (SEQ ID NO: 50)
MDKNHDVEKVYITNANGGEVDALTIAAKAGQDKQPIILTDKDSITDNTY
KWLKSEDLQNAYFIGGPQMISTNVINKVNGITKDSVTNNRVYGADRHET NAN.
Example 19
Expression of C. difficile ATCC 9689 Amidase Clones
[0196] The entire coding sequence of a putative
N-acetylmuramoyl-L-alanine amidase cell surface protein from the
recombinant plasmid pCD 1036-2, as obtained in Example 18, and the
expression vector pET21-d(+) (Novagen) were cut with the
restriction enzymes NcoI and XhoI (New England Labs, Ipswich,
Mass.) and cloned into pET21-d(+), using the procedures described
in Example 17. Similarly, the fragment corresponding to amino acid
residues 294 to 393 of the putative N-acetylmuramoyl-L-alanine
amidase cell surface protein of C. difficile 630 (SEQ ID NO:6) from
the recombinant plasmid pCD1036-Fr-1 was cut with the restriction
enzymes NcoI and XhoI and cloned in to pET21-d(+), following the
procedures described in Example 17. The recombinant clones were
picked and analyzed as described in example 17. The clones having
the inserts were selected and tested for expression of proteins
according to manufacturer's instruction.
[0197] The recombinant clones were transformed into competent cells
of BLR(DE3) and plated on LB Agar with ampicillin (50 .mu.g/ml).
Several colonies were picked and analyzed for protein expression as
described in Example 17. The induced cells showed better expression
of recombinant protein of expected molecular weight at 15.degree.
C. than at 37.degree. C.
[0198] The complete disclosure of all patents, patent applications,
and publications, and electronically available material (including,
for instance, nucleotide sequence submissions in, e.g., GenBank and
RefSeq, and amino acid sequence submissions in, e.g., SwissProt,
PIR, PRF, PDB, and translations from annotated coding regions in
GenBank and RefSeq) cited herein are incorporated by reference. In
the event that any inconsistency exists between the disclosure of
the present application and the disclosure(s) of any document
incorporated herein by reference, the disclosure of the present
application shall govern. The foregoing detailed description and
examples have been given for clarity of understanding only. No
unnecessary limitations are to be understood therefrom. The
invention is not limited to the exact details shown and described,
for variations obvious to one skilled in the art will be included
within the invention defined by the claims.
[0199] Unless otherwise indicated, all numbers expressing
quantities of components, molecular weights, and so forth used in
the specification and claims are to be understood as being modified
in all instances by the term "about." Accordingly, unless otherwise
indicated to the contrary, the numerical parameters set forth in
the specification and claims are approximations that may vary
depending upon the desired properties sought to be obtained by the
present invention. At the very least, and not as an attempt to
limit the doctrine of equivalents to the scope of the claims, each
numerical parameter should at least be construed in light of the
number of reported significant digits and by applying ordinary
rounding techniques.
[0200] Notwithstanding that the numerical ranges and parameters
setting forth the broad scope of the invention are approximations,
the numerical values set forth in the specific examples are
reported as precisely as possible. All numerical values, however,
inherently contain a range necessarily resulting from the standard
deviation found in their respective testing measurements.
[0201] All headings are for the convenience of the reader and
should not be used to limit the meaning of the text that follows
the heading, unless so specified.
Sequence CWU 1
1
501635PRTClostridium difficile 1Met Lys Asp Lys Lys Phe Thr Leu Leu
Ile Ser Ile Met Ile Val Phe 1 5 10 15 Leu Cys Ala Val Val Gly Val
Tyr Ser Thr Ser Ser Asn Lys Ser Val 20 25 30 Asp Leu Tyr Ser Asp
Val Tyr Ile Glu Lys Tyr Phe Asn Arg Asp Lys 35 40 45 Val Met Glu
Val Asn Ile Glu Ile Asp Glu Ser Asp Leu Lys Asp Met 50 55 60 Asn
Glu Asn Ala Ile Lys Glu Glu Phe Lys Val Ala Lys Val Thr Val 65 70
75 80 Asp Gly Asp Thr Tyr Gly Asn Val Gly Ile Arg Thr Lys Gly Asn
Ser 85 90 95 Ser Leu Ile Ser Val Ala Asn Ser Asp Ser Asp Arg Tyr
Ser Tyr Lys 100 105 110 Ile Asn Phe Asp Lys Tyr Asn Thr Ser Gln Ser
Met Glu Gly Leu Thr 115 120 125 Gln Leu Asn Leu Asn Asn Cys Tyr Ser
Asp Pro Ser Tyr Met Arg Glu 130 135 140 Phe Leu Thr Tyr Ser Ile Cys
Glu Glu Met Gly Leu Ala Thr Pro Glu 145 150 155 160 Phe Ala Tyr Ala
Lys Val Ser Ile Asn Gly Glu Tyr His Gly Leu Tyr 165 170 175 Leu Ala
Val Glu Gly Leu Lys Glu Ser Tyr Leu Glu Asn Asn Phe Gly 180 185 190
Asn Val Thr Gly Asp Leu Tyr Lys Ser Asp Glu Gly Ser Ser Leu Gln 195
200 205 Tyr Lys Gly Asp Asp Pro Glu Ser Tyr Ser Asn Leu Ile Val Glu
Ser 210 215 220 Asp Lys Lys Thr Ala Asp Trp Ser Lys Ile Thr Lys Leu
Leu Lys Ser 225 230 235 240 Leu Asp Thr Gly Glu Asp Ile Glu Lys Tyr
Leu Asp Val Asp Ser Val 245 250 255 Leu Lys Asn Ile Ala Ile Asn Thr
Ala Leu Leu Asn Leu Asp Ser Tyr 260 265 270 Gln Gly Ser Phe Ala His
Asn Tyr Tyr Leu Tyr Glu Gln Asp Gly Val 275 280 285 Phe Ser Met Leu
Pro Trp Asp Phe Asn Met Ser Phe Gly Gly Phe Ser 290 295 300 Gly Phe
Gly Gly Gly Ser Gln Ser Ile Ala Ile Asp Glu Pro Thr Thr 305 310 315
320 Gly Asn Leu Glu Asp Arg Pro Leu Ile Ser Ser Leu Leu Lys Asn Glu
325 330 335 Thr Tyr Lys Thr Lys Tyr His Lys Tyr Leu Glu Glu Ile Val
Thr Lys 340 345 350 Tyr Leu Asp Ser Asp Tyr Leu Glu Asn Met Thr Thr
Lys Leu His Asp 355 360 365 Met Ile Ala Ser Tyr Val Lys Glu Asp Pro
Thr Ala Phe Tyr Thr Tyr 370 375 380 Glu Glu Phe Glu Lys Asn Ile Thr
Ser Ser Ile Glu Asp Ser Ser Asp 385 390 395 400 Asn Lys Gly Phe Gly
Asn Lys Gly Phe Asp Asn Asn Asn Ser Asn Asn 405 410 415 Ser Asp Ser
Asn Asn Asn Ser Asn Ser Glu Asn Lys Arg Ser Gly Asn 420 425 430 Gln
Ser Asp Glu Lys Glu Val Asn Ala Glu Leu Thr Ser Ser Val Val 435 440
445 Lys Ala Asn Thr Asp Asn Glu Thr Lys Asn Lys Thr Thr Asn Asp Ser
450 455 460 Glu Ser Lys Asn Asn Thr Asp Lys Asp Lys Ser Gly Asn Asp
Asn Asn 465 470 475 480 Gln Lys Leu Glu Gly Pro Met Gly Lys Gly Gly
Lys Ser Ile Pro Gly 485 490 495 Val Leu Glu Val Ala Glu Asp Met Ser
Lys Thr Ile Lys Ser Gln Leu 500 505 510 Ser Gly Glu Thr Ser Ser Thr
Lys Gln Asn Ser Gly Asp Glu Ser Ser 515 520 525 Ser Gly Ile Lys Gly
Ser Glu Lys Phe Asp Glu Asp Met Ser Gly Met 530 535 540 Pro Glu Pro
Pro Glu Gly Met Asp Gly Lys Met Pro Pro Gly Met Gly 545 550 555 560
Asn Met Asp Lys Gly Asp Met Asn Gly Lys Asn Gly Asn Met Asn Met 565
570 575 Asp Arg Asn Gln Asp Asn Pro Arg Glu Ala Gly Gly Phe Gly Asn
Arg 580 585 590 Gly Gly Gly Ser Val Ser Lys Thr Thr Thr Tyr Phe Lys
Leu Ile Leu 595 600 605 Gly Gly Ala Ser Met Ile Ile Met Ser Ile Met
Leu Val Gly Val Ser 610 615 620 Arg Val Lys Arg Arg Arg Phe Ile Lys
Ser Lys 625 630 635 2100PRTClostridium difficile 2Ser Lys Thr Ile
Lys Ser Gln Leu Ser Gly Glu Thr Ser Ser Thr Lys 1 5 10 15 Gln Asn
Ser Gly Asp Glu Ser Ser Ser Gly Ile Lys Gly Ser Glu Lys 20 25 30
Phe Asp Glu Asp Met Ser Gly Met Pro Glu Pro Pro Glu Gly Met Asp 35
40 45 Gly Lys Met Pro Pro Gly Met Gly Asn Met Asp Lys Gly Asp Met
Asn 50 55 60 Gly Lys Asn Gly Asn Met Asn Met Asp Arg Asn Gln Asp
Asn Pro Arg 65 70 75 80 Glu Ala Gly Gly Phe Gly Asn Arg Gly Gly Gly
Ser Val Ser Lys Thr 85 90 95 Thr Thr Tyr Phe 100 315PRTClostridium
difficile 3Glu Gly Ser Ser Leu Gln Tyr Lys Gly Asp Asp Pro Glu Ser
Tyr 1 5 10 15 415PRTClostridium difficile 4Leu Lys Asn Glu Thr Tyr
Lys Thr Lys Tyr His Lys Tyr Leu Glu 1 5 10 15 5677PRTClostridium
difficile 5Met Leu Ser Lys Glu Ile Asn Met Arg Arg Asn Thr Lys Leu
Leu Thr 1 5 10 15 Thr Gly Ile Leu Ser Met Ala Ile Val Ala Pro Thr
Met Ala Phe Ala 20 25 30 Thr Glu Ser Asn Ala Met Glu Asn Asn Ala
Asp Leu Asn Ile Asn Leu 35 40 45 Glu Lys Lys Ser Ile Val Leu Gly
Ser Lys Ser Lys Val Ser Val Lys 50 55 60 Phe Lys Glu Lys Pro Asp
Ala Asp Ser Ile Lys Leu Lys Tyr Lys Cys 65 70 75 80 Tyr Asp Met Pro
Leu Asn Thr Thr Leu Asn Tyr Asn Gln Ser Thr Gly 85 90 95 Ala Tyr
Glu Gly Ile Ile Asn Tyr Asn Lys Asp Pro Glu Tyr Leu Asn 100 105 110
Val Trp Glu Leu Gln Gly Ile Thr Ile Asn Ser Lys Thr Asn Pro Lys 115
120 125 Thr Leu Asn Arg Gln Asp Leu Glu Lys Met Gly Leu Asn Leu Lys
Asp 130 135 140 Tyr Asn Val Thr Gln Glu Cys Ile Ile Glu Asp Ile Thr
Ser Arg Lys 145 150 155 160 Asp Val Asn Lys Tyr Leu Arg Lys Thr Ser
Ser Pro Ile Thr Glu Leu 165 170 175 Thr Gly Ser Asp Arg Tyr Glu Thr
Ala Val Lys Ile Ser Lys Glu Gly 180 185 190 Trp Lys Asn Gly Ser Asp
Lys Val Val Ile Ile Asn Gly Asp Val Ser 195 200 205 Ile Asp Gly Ile
Ile Ser Thr Pro Leu Ala Thr Thr Tyr Asn Ala Pro 210 215 220 Ile Leu
Leu Val Glu Lys Asn Asn Val Pro Asn Ser Val Lys Ser Glu 225 230 235
240 Leu Lys Arg Leu Asn Pro Lys Asp Ile Ile Ile Ile Gly Asp Glu Asn
245 250 255 Ala Ile Ser Lys Thr Thr Ala Asn Gln Ile Lys Ser Thr Val
Asn Ala 260 265 270 Ser Gln Thr Arg Leu Asn Gly Ser Asn Arg Tyr Glu
Thr Ser Leu Leu 275 280 285 Ile Ala Lys Glu Ile Asp Lys Asn His Asp
Val Glu Lys Val Tyr Ile 290 295 300 Thr Asn Ala Asn Gly Gly Glu Val
Asp Ala Leu Thr Ile Ala Ala Lys 305 310 315 320 Ala Gly Gln Asp Lys
Gln Pro Ile Ile Leu Thr Asp Lys Asp Ser Ile 325 330 335 Thr Asp Asn
Thr Tyr Lys Trp Leu Lys Ser Glu Asp Leu Gln Asn Ala 340 345 350 Tyr
Phe Ile Gly Gly Pro Gln Met Ile Ser Thr Asn Val Ile Asn Lys 355 360
365 Val Asn Gly Ile Thr Lys Asp Ser Val Thr Asn Asn Arg Val Tyr Gly
370 375 380 Ala Asp Arg His Glu Thr Asn Ala Asn Val Ile Lys Lys Phe
Tyr Thr 385 390 395 400 Asp Asp Glu Leu Glu Ala Val Leu Val Ala Lys
Ser Asp Val Leu Val 405 410 415 Asp Ala Leu Ala Ala Gly Pro Leu Ala
Ala Asn Leu Lys Ser Pro Ile 420 425 430 Leu Ile Thr Pro Lys Thr Tyr
Val Ser Ala Tyr His Lys Asp Asn Leu 435 440 445 Glu Ala Lys Ser Ala
Asn Lys Val Tyr Lys Ile Gly Gly Gly Leu Thr 450 455 460 Ser Lys Val
Met Ser Ser Ile Ala Ser Ser Leu Ser Lys His Asn Thr 465 470 475 480
Thr Pro Thr Glu Pro Gly Asn Ser Gly Gly Lys Thr Val Met Ile Asp 485
490 495 Pro Gly His Gly Gly Ser Ala Pro Gly Asn Ser Ser Gly Gly Met
Ile 500 505 510 Glu Lys Asp Tyr Asn Leu Asn Thr Ser Leu Ala Thr Thr
Glu Tyr Leu 515 520 525 Arg Ser Lys Gly Phe Asn Val Ile Met Thr Arg
Asp Thr Asp Lys Thr 530 535 540 Leu Ser Leu Gly Asn Arg Thr Ala Leu
Ser Asn Ser Leu Lys Pro Asp 545 550 555 560 Leu Phe Thr Ser Ile His
Tyr Asn Gly Ser Thr Asn Lys Gln Gly His 565 570 575 Gly Val Glu Val
Phe Tyr Lys Leu Lys Asp Lys Asn Gly Gly Thr Thr 580 585 590 Lys Thr
Val Ala Thr Asn Ile Leu Asn Arg Ile Leu Glu Lys Phe Lys 595 600 605
Leu Thr Asn Arg Gly Ile Lys Thr Arg Val Leu Pro Ser Asp Ser Thr 610
615 620 Lys Asp Tyr Leu Tyr Val Leu Arg Ser Asn Asp Met Pro Ala Val
Leu 625 630 635 640 Val Glu Cys Ala Phe Leu Asp Asn Glu Asn Asp Met
Ser Leu Ile Asn 645 650 655 Ser Ser Ala Lys Val Lys Glu Met Gly Thr
Gln Ile Gly Lys Gly Ile 660 665 670 Glu Asp Ser Leu Lys 675
699PRTClostridium difficile 6Asp Lys Asn His Asp Val Glu Lys Val
Tyr Ile Thr Asn Ala Asn Gly 1 5 10 15 Gly Glu Val Asp Ala Leu Thr
Ile Ala Ala Lys Ala Gly Gln Asp Lys 20 25 30 Gln Pro Ile Ile Leu
Thr Asp Lys Asp Ser Ile Thr Asp Asn Tyr Lys 35 40 45 Trp Leu Lys
Ser Glu Asp Leu Gln Asn Ala Tyr Phe Ile Gly Gly Pro 50 55 60 Gln
Met Ile Ser Thr Asn Val Ile Asn Lys Val Asn Gly Ile Thr Lys 65 70
75 80 Asp Ser Val Thr Asn Asn Arg Val Tyr Gly Ala Asp Arg His Glu
Thr 85 90 95 Asn Ala Asn 715PRTClostridium difficile 7Tyr Lys Leu
Lys Asp Lys Asn Gly Gly Thr Thr Lys Thr Val Ala 1 5 10 15
815PRTClostridium difficile 8Lys Phe Lys Glu Lys Pro Asp Ala Asp
Ser Ile Lys Leu Lys Tyr 1 5 10 15 991PRTClostridium difficile 9Lys
Ser Val Asp Leu Tyr Ser Asp Val Tyr Ile Glu Lys Tyr Phe Asn 1 5 10
15 Arg Asp Lys Val Met Glu Val Asn Ile Glu Ile Asp Glu Ser Asp Leu
20 25 30 Lys Asp Met Asn Glu Asn Ala Ile Lys Glu Glu Phe Lys Val
Ala Lys 35 40 45 Val Thr Val Asp Gly Asp Thr Tyr Gly Asn Val Gly
Ile Arg Thr Lys 50 55 60 Gly Asn Ser Ser Leu Ile Ser Val Ala Asn
Ser Asp Ser Asp Arg Tyr 65 70 75 80 Ser Tyr Lys Ile Asn Phe Asp Lys
Tyr Asn Thr 85 90 10100PRTClostridium difficile 10Val Thr Gly Asp
Leu Tyr Lys Ser Asp Glu Gly Ser Ser Leu Gln Tyr 1 5 10 15 Lys Gly
Asp Asp Pro Glu Ser Tyr Ser Asn Leu Ile Val Glu Ser Asp 20 25 30
Lys Lys Thr Ala Asp Trp Ser Lys Ile Thr Lys Leu Leu Lys Ser Leu 35
40 45 Asp Thr Gly Glu Asp Ile Glu Lys Tyr Leu Asp Val Asp Ser Val
Leu 50 55 60 Lys Asn Ile Ala Ile Asn Thr Ala Leu Leu Asn Leu Asp
Ser Tyr Gln 65 70 75 80 Gly Ser Phe Ala His Asn Tyr Tyr Leu Tyr Glu
Gln Asp Gly Val Phe 85 90 95 Ser Met Leu Pro 100
11323PRTClostridium difficile 11Met Ile Ile Phe Leu Cys Ala Val Val
Gly Val Tyr Ser Thr Ser Ser 1 5 10 15 Asn Lys Ser Val Asp Leu Tyr
Ser Asp Val Tyr Ile Glu Lys Tyr Phe 20 25 30 Asn Arg Asp Lys Val
Met Glu Val Asn Ile Glu Ile Asp Glu Ser Asp 35 40 45 Leu Lys Asp
Met Asn Glu Asn Ala Ile Lys Glu Glu Phe Lys Val Ala 50 55 60 Lys
Val Thr Val Asp Gly Asp Thr Tyr Gly Asn Val Gly Ile Arg Thr 65 70
75 80 Lys Gly Asn Ser Ser Leu Thr Ser Val Ala Asn Ser Asp Ser Asp
Arg 85 90 95 Tyr Ser Tyr Lys Ile Asn Phe Asp Lys Tyr Asn Thr Ser
Gln Ser Met 100 105 110 Glu Gly Leu Thr Gln Leu Asn Leu Asn Asn Cys
Tyr Ser Asp Pro Ser 115 120 125 Tyr Met Arg Glu Phe Leu Thr Tyr Ser
Ile Cys Glu Glu Met Gly Leu 130 135 140 Ala Thr Pro Glu Phe Ala Tyr
Ala Lys Val Ser Ile Asn Gly Glu Tyr 145 150 155 160 His Gly Leu Tyr
Leu Ala Val Glu Gly Leu Lys Glu Ser Tyr Leu Glu 165 170 175 Asn Asn
Phe Gly Asn Val Thr Gly Asp Leu Tyr Lys Ser Asp Glu Gly 180 185 190
Ser Ser Leu Gln Tyr Lys Gly Asp Asp Pro Glu Ser Tyr Ser Asn Leu 195
200 205 Ile Val Glu Ser Asp Lys Lys Thr Ala Asp Trp Ser Lys Ile Thr
Lys 210 215 220 Leu Leu Lys Ser Leu Asp Thr Gly Glu Asp Ile Glu Lys
Tyr Leu Asp 225 230 235 240 Val Asp Ser Val Leu Lys Asn Ile Ala Ile
Asn Thr Ala Leu Leu Asn 245 250 255 Leu Asp Ser Tyr Gln Gly Ser Phe
Ala His Asn Tyr Tyr Leu Tyr Glu 260 265 270 Gln Asp Gly Val Phe Ser
Met Leu Pro Trp Asp Phe Asn Met Ser Phe 275 280 285 Gly Gly Phe Ser
Gly Phe Gly Gly Gly Ser Gln Ser Ile Ala Ile Asp 290 295 300 Glu Pro
Thr Thr Gly Asn Leu Glu Asp Arg Pro Leu Ile Ser Ser Leu 305 310 315
320 Leu Lys Lys 12274PRTClostridium difficile 12Met Thr Thr Lys Leu
His Asp Met Ile Ala Ser Tyr Val Lys Glu Asp 1 5 10 15 Pro Thr Ala
Phe Tyr Thr Tyr Glu Glu Phe Glu Lys Asn Ile Thr Ser 20 25 30 Ser
Ile Glu Asp Ser Ser Asp Asn Lys Gly Phe Gly Asn Lys Gly Phe 35 40
45 Asp Asn Asn Asn Ser Asn Asn Ser Asp Ser Asn Asn Asn Ser Asn Ser
50 55 60 Glu Asn Lys Arg Ser Gly Asn Gln Ser Asp Lys Lys Glu Val
Asn Ala 65 70 75 80 Glu Leu Thr Ser Ser Val Val Lys Thr Asn Thr Asp
Asn Glu Thr Glu 85 90 95 Asn Lys Thr Thr Asn Asp Ser Glu Ser Lys
Asn Asn Thr Asp Lys Asp 100 105 110 Lys Ser Gly Asn Asp Asn Asn Gln
Lys Leu Glu Gly Pro Arg Gly Lys 115 120 125 Gly Gly Lys Ser Ile Pro
Gly Val Leu Glu Val Ala Glu Asp Met Ser 130 135 140 Lys Thr Ile Lys
Ser Gln Leu Ser Gly Glu Thr Ser Ser Thr Lys Gln 145 150 155 160 Asn
Ser Gly Asp Glu Ser Ser Ser Gly Ile Lys Gly Ser Glu Lys Phe 165
170 175 Asp Glu Asp Met Ser Gly Met Pro Glu Pro Pro Glu Gly Met Asp
Gly 180 185 190 Lys Met Pro Pro Gly Met Gly Asn Met Asp Lys Gly Asp
Met Asn Gly 195 200 205 Lys Asn Gly Asn Met Asn Met Asp Arg Asn Gln
Asp Asn Pro Arg Glu 210 215 220 Ala Gly Gly Phe Gly Asn Arg Gly Gly
Gly Ser Val Ser Lys Thr Thr 225 230 235 240 Thr Tyr Phe Lys Leu Ile
Leu Gly Gly Ala Ser Met Ile Ile Met Ser 245 250 255 Ile Met Leu Val
Gly Val Ser Arg Val Lys Arg Arg Arg Phe Ile Lys 260 265 270 Ser Lys
13999DNAClostridium difficile 13aagataaaaa aatttaccct tcttatctct
attatgatta tatttttatg tgctgtagtt 60ggagtttata gtacatctag caacaaaagt
gttgatttat atagtgatgt atatattgaa 120aaatatttta acagagacaa
ggttatggaa gttaatatag agatagatga aagtgacttg 180aaggatatga
atgaaaatgc tataaaagaa gaatttaagg ttgcaaaagt aactgtagat
240ggagatacat atggaaacgt aggtataaga actaaaggaa attcaagtct
tacatctgta 300gcaaatagtg atagtgatag atacagctat aagattaatt
ttgataagta taatactagt 360caaagtatgg aagggcttac tcaattaaat
cttaataact gttactctga cccatcttat 420atgagagagt ttttaacata
tagtatttgc gaggaaatgg gattagcgac tccagaattt 480gcatatgcta
aagtctctat aaatggcgaa tatcatggtt tgtatttggc agtagaagga
540ttaaaagagt cttatcttga aaataatttt ggtaatgtaa ctggagactt
atataagtca 600gatgaaggaa gctcgttgca atataaagga gatgacccag
aaagttactc aaacttaatc 660gttgaaagtg ataaaaagac agctgattgg
tctaaaatta caaaactatt aaaatctttg 720gatacaggtg aagatattga
aaaatatctt gatgtagatt ctgtccttaa aaatatagca 780ataaatacag
ctttattaaa ccttgatagc tatcaaggca gttttgccca taactattat
840ttatatgagc aagatggagt attttctatg ttaccatggg attttaatat
gtcatttggt 900ggatttagtg gttttggtgg aggtagtcaa tctatagcaa
ttgatgaacc tacgacaggt 960aatttagaag acagacctct catatcctcg ttattaaaa
99914909DNAClostridium difficile 14aaaaatgaga cacacaaaac aaaataccat
aaatatctgg aagagatagt aacaaaatac 60ctagattcag actatttaga gaatatgaca
acaaaattgc atgacatgat agcatcatat 120gtaaaagaag acccaacagc
attttatact tatgaagaat ttgaaaaaaa tataacatct 180tcaattgaag
attctagtga taataaggga tttggtaata aagggtttga caacaataac
240tctaataaca gtgattctaa taataattct aatagtgaaa ataagcgctc
tggaaatcaa 300agtgataaaa aagaagttaa tgctgaatta acatcaagcg
tagtcaaaac taatacagat 360aatgaaactg aaaataaaac tacaaatgat
agcgaaagta agaataatac agataaagat 420aaaagtggaa atgataataa
tcaaaagcta gaaggtccta ggggtaaagg aggtaagtca 480ataccagggg
ttttggaagt tgcagaagat atgagtaaaa ctataaaatc tcaattaagt
540ggagaaactt cttcgacaaa gcaaaactct ggtgatgaaa gttcaagtgg
aattaaaggt 600agtgaaaagt ttgatgagga tatgagtggt atgccagaac
cacctgaggg aatggatggt 660aaaatgccac caggaatggg taatatggat
aagggagata tgaatggtaa aaatggcaat 720atgaatatgg atagaaatca
agataatcca agagaagctg gaggttttgg caatagagga 780ggaggctctg
tgagtaaaac aacaacatac ttcaaattaa ttttaggtgg agcttcaatg
840ataataatgt cgattatgtt agtaggtgta tcaagggtaa agagaagaag
atttataaag 900tcaaaataa 909151907DNAClostridium difficile
15atgaaagata aaaaatttac ccttcttatc tctattatga ttatattttt atgtgctgta
60ttggagttta tagtacatct agcaacaaaa gtgttgattt atatagtgat gtatatattg
120aaaaatattt taacagagac aaggttatgg aagttaatat agagatagat
gaaagtgact 180tgaaggatat gaatgaaaat gctataaaag aagaatttaa
ggttgcaaaa gtaactgtag 240atggagatac atatggaaac gtaggtataa
gaactaaagg aaattcaagt cttacatctg 300tagcaaatag tgatagtgat
agatacagct ataagattaa ttttgataag tataatacta 360gtcaaagtat
ggaagggctt actcaattaa atcttaataa ctgttactct gacccatctt
420atatgagaga gtttttaaca tatagtattt gcgaggaaat gggattagcg
actccagaat 480ttgcatatgc taaagtctct ataaatggcg aatatcatgg
tttgtatttg gcagtagaag 540gattaaaaga gtcttatctt gaaaataatt
ttggtaatgt aactggagac ttatataagt 600cagatgaagg aagctcgttg
caatataaag gagatgaccc agaaagttac tcaaacttaa 660tcgttgaaag
tgataaaaag acagctgatt ggtctaaaat tacaaaacta ttaaaatctt
720tggatacagg tgaagatatt gaaaaatatc ttgatgtaga ttctgtcctt
aaaaatatag 780caataaatac agctttatta aaccttgata gctatcaagg
cagttttgcc cataactatt 840atttatatga gcaagatgga gtattttcta
tgttaccatg ggattttaat atgtcatttg 900gtggatttag tggttttggt
ggaggtagtc aatctatagc aattgatgaa cctacgacag 960gtaatttaga
agacagacct ctcatatcct cgttattaaa aaatgagaca cacaaaacaa
1020aataccataa atatctggaa gagatagtaa caaaatacct agattcagac
tatttagaga 1080atatgacaac aaaattgcat gacatgatag catcatatgt
aaaagaagac ccaacagcat 1140tttatactta tgaagaattt gaaaaaaata
taacatcttc aattgaagat tctagtgata 1200ataagggatt tggtaataaa
gggtttgaca acaataactc taataacagt gattctaata 1260ataattctaa
tagtgaaaat aagcgctctg gaaatcaaag tgataaaaaa gaagttaatg
1320ctgaattaac atcaagcgta gtcaaaacta atacagataa tgaaactgaa
aataaaacta 1380caaatgatag cgaaagtaag aataatacag ataaagataa
aagtggaaat gataataatc 1440aaaagctaga aggtcctagg ggtaaaggag
gtaagtcaat accaggggtt ttggaagttg 1500cagaagatat gagtaaaact
ataaaatctc aattaagtgg agaaacttct tcgacaaagc 1560aaaactctgg
tgatgaaagt tcaagtggaa ttaaaggtag tgaaaagttt gatgaggata
1620tgagtggtat gccagaacca cctgagggaa tggatggtaa aatgccacca
ggaatgggta 1680atatggataa gggagatatg aatggtaaaa atggcaatat
gaatatggat agaaatcaag 1740ataatccaag agaagctgga ggttttggca
atagaggagg aggctctgtg agtaaaacaa 1800caacatactt caaattaatt
ttaggtggag cttcaatgat aataatgtcg attatgttag 1860taggtgtatc
aagggtaaag agaagaagat ttataaagtc aaaataa 190716679PRTClostridium
difficile 16Met Leu Ser Lys Glu Ile Asn Met Arg Arg Asn Thr Lys Leu
Leu Thr 1 5 10 15 Thr Gly Ile Leu Ser Met Ala Ile Val Thr Pro Thr
Met Ala Phe Ala 20 25 30 Thr Glu Ser Asn Ala Met Glu Asn Asn Ala
Asp Leu Asn Ile Asn Leu 35 40 45 Glu Lys Lys Ser Ile Val Leu Gly
Ser Thr Ser Lys Val Ser Val Lys 50 55 60 Phe Lys Glu Lys Pro Asp
Ala Asp Ser Ile Thr Leu Lys Tyr Lys Cys 65 70 75 80 Tyr Asp Met Pro
Leu Asp Thr Thr Leu Asn Tyr Asn Gln Ser Thr Glu 85 90 95 Ser Tyr
Glu Gly Thr Ile Asn Tyr Asn Lys Asp Pro Glu Tyr Leu Asn 100 105 110
Val Trp Glu Leu Gln Gly Ile Thr Ile Asn Ser Lys Asn Asn Pro Lys 115
120 125 Thr Leu Asn Lys Gln Glu Leu Glu Lys Met Gly Leu Asn Leu Lys
Asp 130 135 140 Tyr Asn Val Thr Gln Glu Cys Ile Ile Glu Asp Ile Thr
Ser Arg Lys 145 150 155 160 Asp Val Asn Lys Tyr Leu Arg Lys Thr Ser
Ala Pro Ile Thr Glu Leu 165 170 175 Thr Gly Ser Asp Arg Tyr Glu Thr
Ala Val Lys Ile Ser Lys Glu Gly 180 185 190 Trp Lys Asn Gly Ser Asp
Lys Val Val Ile Ile Asn Gly Asp Val Ser 195 200 205 Ile Asp Gly Ile
Ile Ser Thr Pro Leu Ala Thr Thr Tyr Asn Ala Pro 210 215 220 Ile Leu
Leu Val Glu Lys Asn Asn Val Pro Asn Ser Val Lys Ser Glu 225 230 235
240 Leu Lys Arg Leu Asn Pro Arg Asp Val Ile Ile Ile Gly Asp Glu Asn
245 250 255 Ala Ile Ser Lys Thr Thr Ala Asn Gln Ile Lys Ser Thr Val
Asn Ala 260 265 270 Ser Gln Thr Arg Leu Lys Gly Ser Asn Arg Tyr Glu
Thr Ser Leu Leu 275 280 285 Ile Ala Lys Glu Ile Asp Lys Asn His Asp
Val Glu Lys Val Tyr Ile 290 295 300 Thr Asn Ala Asn Gly Gly Glu Val
Asp Ala Leu Thr Ile Ala Ala Lys 305 310 315 320 Ala Gly Gln Asp Lys
Gln Pro Ile Ile Leu Thr Asp Lys Asn Ser Ile 325 330 335 Thr Asp Asn
Thr Tyr Lys Trp Leu Lys Ser Glu Asp Leu Gln Asn Ala 340 345 350 Tyr
Phe Ile Gly Gly Pro Gln Met Ile Ser Thr Asn Val Ile Asn Lys 355 360
365 Val Asn Asp Ile Thr Lys Asp Asn Val Thr Asn Asn Arg Val Tyr Gly
370 375 380 Ala Asp Arg His Glu Thr Asn Ala Asn Val Ile Lys Lys Phe
Tyr Thr 385 390 395 400 Asp Asp Glu Leu Glu Ala Val Leu Val Ala Lys
Ser Asp Val Leu Val 405 410 415 Asp Ala Leu Ala Ala Gly Pro Leu Ala
Ala Asn Leu Lys Ser Pro Ile 420 425 430 Leu Ile Thr Pro Lys Thr Tyr
Val Ser Ala Tyr His Lys Glu Asn Leu 435 440 445 Glu Ala Lys Ser Ala
Asn Lys Val Tyr Lys Ile Gly Gly Gly Leu Thr 450 455 460 Ser Lys Val
Met Ser Ser Ile Ala Ser Ser Leu Ser Lys His Asn Thr 465 470 475 480
Thr Pro Thr Glu Pro Gly Asn Ser Gly Gly Lys Thr Val Met Ile Asp 485
490 495 Pro Gly His Gly Gly Ser Asp Thr Gly Thr Thr Gly Lys Pro Leu
Gly 500 505 510 Gly Ile Arg Glu Lys Asp Tyr Thr Leu Asn Thr Ser Leu
Ala Thr Thr 515 520 525 Glu Tyr Leu Arg Ser Lys Gly Phe Asn Val Ile
Met Thr Arg Asp Thr 530 535 540 Asp Lys Thr Leu Ser Leu Gly Asn Arg
Thr Ala Leu Ser Asn Ser Leu 545 550 555 560 Arg Pro Asp Leu Phe Thr
Ser Ile His Tyr Asn Ala Ser Asp Thr Thr 565 570 575 Gly Asn Gly Val
Glu Val Phe Tyr Lys Leu Lys Asp Lys Asp Gly Gly 580 585 590 Thr Thr
Lys Thr Val Ala Thr Asn Ile Leu Asn Arg Ile Leu Glu Lys 595 600 605
Phe Asn Leu Lys Asn Arg Gly Ala Lys Thr Arg Thr Leu Ser Thr Asp 610
615 620 Pro Thr Lys Asp Tyr Leu Tyr Val Leu Arg Asn Asn Asp Met Pro
Ala 625 630 635 640 Val Leu Val Glu Cys Ala Phe Leu Asp Asn Glu Lys
Asp Met Ser Leu 645 650 655 Leu Asn Thr Ser Asn Lys Val Lys Glu Met
Gly Thr Gln Ile Gly Lys 660 665 670 Gly Ile Glu Asp Ser Leu Lys 675
17675PRTClostridium difficile 17Met Met Lys Lys Thr Thr Lys Leu Leu
Ala Thr Gly Met Leu Ser Val 1 5 10 15 Ala Met Val Ala Pro Asn Val
Ala Leu Ala Ala Glu Asn Thr Thr Ala 20 25 30 Asn Thr Glu Ser Asn
Ser Asp Ile Asn Ile Asn Leu Gln Arg Lys Ser 35 40 45 Val Val Leu
Gly Ser Lys Ser Asn Ala Ser Val Lys Phe Lys Glu Lys 50 55 60 Leu
Asn Ala Asp Ser Ile Thr Leu Asn Phe Met Cys Tyr Asp Met Pro 65 70
75 80 Leu Glu Ala Thr Leu Asn Tyr Asn Glu Lys Thr Asp Ser Tyr Glu
Gly 85 90 95 Val Ile Asn Tyr Asn Lys Asp Pro Glu Tyr Leu Asn Val
Trp Glu Leu 100 105 110 Gln Ser Ile Lys Ile Asn Gly Lys Asp Glu Gln
Lys Val Leu Asn Lys 115 120 125 Glu Asp Leu Glu Ser Met Gly Leu Asn
Leu Lys Asp Tyr Asp Val Thr 130 135 140 Gln Glu Phe Ile Ile Ser Asp
Ala Asn Ser Thr Lys Ala Val Asn Glu 145 150 155 160 Tyr Met Arg Lys
Thr Ser Ala Pro Val Lys Lys Leu Ala Gly Ala Thr 165 170 175 Arg Phe
Glu Thr Ala Val Glu Ile Ser Lys Gln Gly Trp Lys Asp Gly 180 185 190
Ser Ser Lys Val Val Ile Val Asn Gly Glu Leu Ala Ala Asp Gly Ile 195
200 205 Thr Ala Thr Pro Leu Ala Ser Thr Tyr Asp Ala Pro Ile Leu Leu
Ala 210 215 220 Asn Lys Asp Asp Ile Pro Glu Ser Thr Lys Ala Glu Leu
Lys Arg Leu 225 230 235 240 Asn Pro Ser Asp Val Ile Ile Ile Gly Asp
Asp Gly Ser Val Ser Gln 245 250 255 Lys Ala Val Ser Gln Ile Lys Ser
Ala Val Asn Val Asn Val Thr Arg 260 265 270 Ile Gly Gly Val Asp Arg
His Glu Thr Ser Leu Leu Ile Ala Lys Glu 275 280 285 Ile Asp Lys Tyr
His Asp Val Asn Lys Ile Tyr Ile Ala Asn Gly Tyr 290 295 300 Ala Gly
Glu Tyr Asp Ala Leu Asn Ile Ser Ser Lys Ala Gly Glu Asp 305 310 315
320 Gln Gln Pro Ile Ile Leu Ala Asn Lys Asp Ser Val Pro Gln Gly Thr
325 330 335 Tyr Asn Trp Leu Ser Ser Gln Gly Leu Glu Glu Ala Tyr Tyr
Ile Gly 340 345 350 Gly Ser Gln Ser Leu Ser Ser Lys Ile Ile Asp Gln
Ile Ser Lys Ile 355 360 365 Ala Lys Asn Gly Thr Ser Lys Asn Arg Val
Ser Gly Ala Asp Arg His 370 375 380 Glu Thr Asn Ala Asn Val Ile Lys
Thr Phe Tyr Pro Asp Lys Glu Leu 385 390 395 400 Ser Ala Met Leu Val
Ala Lys Ser Asp Ile Ile Val Asp Ser Ile Thr 405 410 415 Ala Gly Pro
Leu Ala Ala Lys Leu Lys Ala Pro Ile Leu Ile Thr Pro 420 425 430 Lys
Thr Tyr Val Ser Ala Tyr His Ser Thr Asn Leu Ser Glu Lys Thr 435 440
445 Ala Glu Thr Val Tyr Gln Ile Gly Asp Gly Met Lys Asp Ser Val Ile
450 455 460 Asn Ser Ile Ala Ser Ser Leu Ser Lys His Asn Ala Pro Thr
Glu Pro 465 470 475 480 Asp Asn Ser Gly Ser Ala Ala Gly Lys Thr Val
Val Ile Asp Pro Gly 485 490 495 His Gly Gly Ser Asp Ser Gly Ala Thr
Ser Gly Leu Asn Gly Gly Ala 500 505 510 Gln Glu Lys Lys Tyr Thr Leu
Asn Thr Ala Leu Ala Thr Thr Glu Tyr 515 520 525 Leu Arg Ser Lys Gly
Ile Asn Val Val Met Thr Arg Asp Thr Asp Lys 530 535 540 Thr Met Ala
Leu Gly Glu Arg Thr Ala Leu Ser Asn Thr Ile Lys Pro 545 550 555 560
Asp Leu Phe Thr Ser Ile His Tyr Asn Ala Ser Asn Gly Ser Gly Asn 565
570 575 Gly Val Glu Ile Tyr Tyr Lys Val Lys Asp Lys Asn Gly Gly Thr
Thr 580 585 590 Lys Thr Ala Ala Ser Asn Ile Leu Lys Arg Ile Leu Glu
Lys Phe Asn 595 600 605 Met Lys Asn Arg Gly Ile Lys Thr Arg Thr Leu
Asp Asn Gly Lys Asp 610 615 620 Tyr Leu Tyr Val Leu Arg Asn Asn Asn
Tyr Pro Ala Ile Leu Val Glu 625 630 635 640 Cys Ala Phe Ile Asp Asn
Lys Ser Asp Met Asp Lys Leu Asn Thr Ala 645 650 655 Glu Lys Val Lys
Thr Met Gly Thr Gln Ile Gly Ile Gly Ile Glu Asp 660 665 670 Thr Val
Lys 675 18618PRTClostridium difficile 18Met Phe Arg Phe Lys Glu Lys
Pro Asp Ala Asp Ser Ile Thr Leu Lys 1 5 10 15 Tyr Lys Cys Tyr Asp
Met Pro Leu Asp Thr Thr Leu Asn Tyr Asn Gln 20 25 30 Ser Thr Glu
Ser Tyr Glu Gly Thr Ile Asn Tyr Asn Lys Asp Pro Glu 35 40 45 Tyr
Leu Asn Val Trp Glu Leu Gln Gly Ile Thr Ile Asn Ser Lys Asn 50 55
60 Asn Pro Lys Thr Leu Asn Lys Gln Glu Leu Glu Lys Met Gly Leu Asn
65 70 75 80 Leu Lys Asp Tyr Asn Val Thr Gln Glu Cys Ile Ile Glu Asp
Ile Thr 85 90 95 Ser Arg Lys Asp Val Asn Lys Tyr Leu Arg Lys Thr
Ser Ala Pro Ile 100 105 110 Thr Glu Leu Thr Gly Ser Asp Arg Tyr Glu
Thr Ala Val Lys Ile Ser 115 120 125 Lys Glu Gly Trp Lys Asn Gly Ser
Asp Lys Val Val Ile Ile Asn Gly 130 135 140 Asp Val Ser Ile Asp Gly
Ile Ile Ser Thr Pro Leu Ala Thr Thr Tyr 145 150 155 160 Asn Ala Pro
Ile Leu Leu Val Glu Lys Asn Asn Val Pro Asn Ser Val 165 170 175 Lys
Ser Glu Leu Lys Arg Leu Asn Pro Arg Asp Val Ile Ile Ile Gly 180 185
190 Asp Glu Asn Ala Ile Ser Lys Thr Thr Ala Asn Gln Ile Lys Ser Thr
195 200 205 Val Asn Ala Ser Gln Thr Arg Leu Lys Gly Ser Asn Arg Tyr
Glu Thr 210 215 220 Ser Leu Leu Ile
Ala Lys Glu Ile Asp Lys Asn His Asp Val Glu Lys 225 230 235 240 Val
Tyr Ile Thr Asn Ala Asn Gly Gly Glu Val Asp Ala Leu Thr Ile 245 250
255 Ala Ala Lys Ala Gly Gln Asp Lys Gln Pro Ile Ile Leu Thr Asp Lys
260 265 270 Asn Ser Ile Thr Asp Asn Thr Tyr Lys Trp Leu Lys Ser Glu
Asp Leu 275 280 285 Gln Asn Ala Tyr Phe Ile Gly Gly Pro Gln Met Ile
Ser Thr Asn Val 290 295 300 Ile Asn Lys Val Asn Asp Ile Thr Lys Asp
Asn Val Thr Asn Asn Arg 305 310 315 320 Val Tyr Gly Ala Asp Arg His
Glu Thr Asn Ala Asn Val Ile Lys Lys 325 330 335 Phe Tyr Thr Asp Asp
Glu Leu Glu Ala Val Leu Val Ala Lys Ser Asp 340 345 350 Val Leu Val
Asp Ala Leu Ala Ala Gly Pro Leu Ala Ala Asn Leu Lys 355 360 365 Ser
Pro Ile Leu Ile Thr Pro Lys Thr Tyr Val Ser Ala Tyr His Lys 370 375
380 Asp Asn Leu Glu Ala Lys Ser Ala Asn Lys Val Tyr Lys Ile Gly Gly
385 390 395 400 Gly Leu Thr Ser Lys Val Met Asn Ser Ile Ala Ser Ser
Leu Ser Lys 405 410 415 His Asn Thr Thr Pro Thr Glu Pro Gly Asn Ser
Gly Gly Lys Thr Val 420 425 430 Met Ile Asp Pro Gly His Gly Gly Ser
Asp Thr Gly Thr Thr Gly Lys 435 440 445 Pro Leu Gly Gly Ile Lys Glu
Lys Asp Tyr Thr Leu Asn Thr Ser Leu 450 455 460 Ala Thr Thr Glu Tyr
Leu Arg Ser Lys Gly Phe Asn Val Ile Met Thr 465 470 475 480 Arg Asp
Thr Asp Lys Thr Leu Ser Leu Gly Asn Arg Thr Ala Leu Ser 485 490 495
Asn Ser Leu Arg Pro Asp Leu Phe Thr Ser Ile His Tyr Asn Ala Ser 500
505 510 Asp Thr Thr Gly Asn Gly Val Glu Val Phe Tyr Lys Leu Lys Asp
Lys 515 520 525 Asp Gly Gly Thr Thr Lys Thr Val Ala Thr Asn Ile Leu
Asn Arg Ile 530 535 540 Leu Glu Lys Phe Asn Leu Lys Asn Arg Gly Ala
Lys Thr Arg Thr Leu 545 550 555 560 Ser Thr Asp Pro Thr Lys Asp Tyr
Leu Tyr Val Leu Arg Asn Asn Asp 565 570 575 Met Pro Ala Val Leu Val
Glu Cys Ala Phe Leu Asp Asn Glu Lys Asp 580 585 590 Met Ser Leu Leu
Asn Thr Ser Asn Lys Val Lys Glu Met Gly Thr Gln 595 600 605 Ile Gly
Lys Gly Ile Glu Asp Ser Leu Lys 610 615 19552PRTClostridium
difficile 19Met Leu Ser Lys Glu Ile Asn Met Arg Arg Asn Thr Lys Leu
Leu Thr 1 5 10 15 Thr Gly Ile Leu Ser Met Ala Ile Val Ala Pro Thr
Met Ala Phe Ala 20 25 30 Thr Glu Ser Asn Ala Met Glu Asn Asn Ala
Asp Leu Asn Ile Asn Leu 35 40 45 Glu Lys Lys Ser Ile Val Leu Gly
Ser Lys Ser Lys Val Ser Val Lys 50 55 60 Phe Lys Glu Lys Pro Asp
Ala Asp Ser Ile Thr Leu Lys Tyr Lys Cys 65 70 75 80 Tyr Asp Met Pro
Leu Asp Thr Thr Leu Asn Tyr Asn Gln Ser Thr Gly 85 90 95 Ala Tyr
Glu Gly Thr Ile Asn Tyr Asn Gln Asp Pro Glu Tyr Leu Asn 100 105 110
Val Trp Glu Leu Gln Gly Ile Thr Ile Asn Ser Lys Asn Asn Pro Lys 115
120 125 Thr Leu Asn Gly Gln Asp Leu Glu Lys Met Gly Leu Asn Leu Lys
Asp 130 135 140 Tyr Asn Val Thr Gln Glu Cys Ile Ile Glu Asp Ile Thr
Ser Arg Lys 145 150 155 160 Asp Val Asn Lys Tyr Leu Arg Lys Thr Ser
Ala Pro Ile Thr Glu Leu 165 170 175 Thr Gly Ser Asp Arg Tyr Glu Thr
Ala Val Lys Ile Ser Lys Glu Gly 180 185 190 Trp Lys Asn Gly Ser Asp
Lys Val Val Ile Ile Asn Gly Asp Val Ser 195 200 205 Ile Asp Gly Ile
Ile Ser Thr Pro Leu Ala Thr Thr Tyr Asn Ala Pro 210 215 220 Ile Leu
Leu Val Glu Lys Asn Asn Val Pro Asn Ser Val Lys Ser Glu 225 230 235
240 Leu Lys Arg Leu Asn Pro Lys Asp Ile Ile Ile Ile Gly Asp Glu Asn
245 250 255 Ala Ile Ser Lys Thr Thr Ala Asn Gln Ile Lys Ser Thr Val
Asn Ala 260 265 270 Ser Gln Thr Arg Leu Asn Gly Ser Asn Arg Tyr Glu
Thr Ser Leu Leu 275 280 285 Ile Ala Lys Glu Ile Asp Lys Asn His Asp
Val Glu Lys Val Tyr Ile 290 295 300 Thr Asn Ala Asn Gly Gly Glu Val
Asp Ala Leu Thr Ile Ala Ala Lys 305 310 315 320 Ala Gly Gln Asp Lys
Gln Pro Ile Ile Leu Thr Asp Lys Asp Ser Ile 325 330 335 Thr Asp Asn
Thr Tyr Lys Trp Leu Lys Ser Glu Asp Leu Gln Asn Ala 340 345 350 Tyr
Phe Ile Gly Gly Pro Gln Met Ile Ser Thr Asn Val Ile Asn Lys 355 360
365 Val Asn Gly Ile Thr Lys Asp Ser Val Thr Asn Asn Arg Val Tyr Gly
370 375 380 Ala Asp Arg His Glu Thr Asn Ala Asn Val Ile Lys Lys Phe
Tyr Thr 385 390 395 400 Glu Asp Glu Ile Glu Ala Val Leu Val Ala Lys
Ser Asp Val Leu Val 405 410 415 Asp Ala Leu Ala Ala Gly Pro Leu Ala
Ala Asn Leu Lys Ser Pro Ile 420 425 430 Leu Ile Thr Pro Lys Thr Tyr
Val Ser Ala Tyr His Lys Asp Asn Leu 435 440 445 Glu Ala Lys Ser Ala
Asn Lys Val Tyr Lys Ile Gly Gly Gly Leu Thr 450 455 460 Ser Lys Val
Met Ser Ser Ile Ala Ser Ser Leu Ser Lys His Asn Thr 465 470 475 480
Thr Pro Thr Glu Pro Gly Asn Ser Gly Gly Lys Thr Val Met Ile Asp 485
490 495 Pro Gly His Gly Gly Ser Ala Pro Gly Asn Ser Ser Gly Gly Met
Ile 500 505 510 Glu Lys Asp Tyr Asn Leu Asn Thr Ser Leu Ala Thr Thr
Glu Tyr Leu 515 520 525 Arg Ser Lys Gly Phe Asn Val Ile Met Thr Arg
Asp Thr Asp Lys Thr 530 535 540 Leu Ser Leu Gly Asn Arg Thr Ala 545
550 20675PRTClostridium difficile 20Met Met Lys Lys Thr Thr Lys Leu
Leu Ala Thr Gly Met Leu Ser Val 1 5 10 15 Ala Met Val Ala Pro Asn
Val Ala Leu Ala Ala Glu Asn Thr Thr Ala 20 25 30 Asn Thr Glu Ser
Asn Ser Asp Ile Asn Ile Asn Leu Gln Arg Lys Ser 35 40 45 Val Val
Leu Gly Ser Lys Ser Asn Ala Ser Val Lys Phe Lys Glu Lys 50 55 60
Leu Asn Ala Asp Ser Ile Thr Leu Asn Phe Met Cys Tyr Asp Met Pro 65
70 75 80 Leu Glu Ala Thr Leu Asn Tyr Asn Glu Lys Thr Asp Ser Tyr
Glu Gly 85 90 95 Val Ile Asn Tyr Asn Lys Asp Pro Glu Tyr Leu Asn
Val Trp Glu Leu 100 105 110 Gln Ser Ile Lys Ile Asn Gly Lys Asp Glu
Gln Lys Val Leu Asn Lys 115 120 125 Glu Asp Leu Glu Ser Met Gly Leu
Asn Leu Lys Asp Tyr Asp Val Thr 130 135 140 Gln Glu Phe Ile Ile Ser
Asp Ala Asn Ser Thr Lys Ala Val Asn Glu 145 150 155 160 Tyr Met Arg
Lys Thr Ser Ala Pro Val Lys Lys Leu Ala Gly Ala Thr 165 170 175 Arg
Phe Glu Thr Ala Val Glu Ile Ser Lys Gln Gly Trp Lys Asp Gly 180 185
190 Ser Ser Lys Val Val Ile Val Asn Gly Glu Leu Ala Ala Asp Gly Ile
195 200 205 Thr Ala Thr Pro Leu Ala Ser Thr Tyr Asp Ala Pro Ile Leu
Leu Ala 210 215 220 Asn Lys Asp Asp Ile Pro Glu Ser Thr Lys Ala Glu
Leu Lys Arg Leu 225 230 235 240 Asn Pro Ser Asp Val Ile Ile Ile Gly
Asp Asp Gly Ser Val Ser Gln 245 250 255 Lys Ala Val Ser Gln Ile Lys
Ser Ala Val Asn Val Asn Val Thr Arg 260 265 270 Ile Gly Gly Val Asp
Arg His Glu Thr Ser Leu Leu Ile Ala Lys Glu 275 280 285 Ile Asp Lys
Tyr His Asp Val Asn Lys Ile Tyr Ile Ala Asn Gly Tyr 290 295 300 Ala
Gly Glu Tyr Asp Ala Leu Asn Ile Ser Ser Lys Ala Gly Glu Asp 305 310
315 320 Gln Gln Pro Ile Ile Leu Ala Asn Lys Asp Ser Val Pro Gln Gly
Thr 325 330 335 Tyr Asn Trp Leu Ser Ser Gln Gly Leu Glu Glu Ala Tyr
Tyr Ile Gly 340 345 350 Gly Ser Gln Ser Leu Ser Ser Lys Ile Ile Asp
Gln Ile Ser Lys Ile 355 360 365 Ala Lys Asn Gly Thr Ser Lys Asn Arg
Val Ser Gly Ala Asp Arg His 370 375 380 Glu Thr Asn Ala Asn Val Ile
Lys Thr Phe Tyr Pro Asp Lys Glu Leu 385 390 395 400 Ser Ala Met Leu
Val Ala Lys Ser Asp Ile Ile Val Asp Ser Ile Thr 405 410 415 Ala Gly
Pro Leu Ala Ala Lys Leu Lys Ala Pro Ile Leu Ile Thr Pro 420 425 430
Lys Thr Tyr Val Ser Ala Tyr His Ser Thr Asn Leu Ser Glu Lys Thr 435
440 445 Ala Gly Thr Val Tyr Gln Ile Gly Asp Gly Met Lys Asp Ser Val
Ile 450 455 460 Asn Ser Ile Ala Ser Ser Leu Ser Lys His Asn Ala Pro
Thr Glu Pro 465 470 475 480 Asp Asn Ser Gly Ser Ala Ala Gly Lys Thr
Val Val Ile Asp Pro Gly 485 490 495 His Gly Gly Ser Asp Ser Gly Ala
Thr Ser Gly Leu Asn Gly Gly Ala 500 505 510 Gln Glu Lys Lys Tyr Thr
Leu Asn Thr Ala Leu Ala Thr Thr Glu Tyr 515 520 525 Leu Arg Ser Lys
Gly Ile Asn Val Val Met Thr Arg Asp Thr Asp Lys 530 535 540 Thr Met
Ala Leu Gly Glu Arg Thr Ala Leu Ser Asn Thr Ile Lys Pro 545 550 555
560 Asp Leu Phe Thr Ser Ile His Tyr Asn Ala Ser Asn Gly Ala Gly Asn
565 570 575 Gly Val Glu Ile Tyr Tyr Lys Val Lys Asp Lys Asn Gly Gly
Thr Thr 580 585 590 Lys Thr Ala Ala Ser Asn Ile Leu Lys Arg Ile Leu
Glu Lys Phe Asn 595 600 605 Met Lys Asn Arg Gly Ile Lys Thr Arg Thr
Leu Asp Asn Gly Lys Asp 610 615 620 Tyr Leu Tyr Val Leu Arg Asn Asn
Asn Tyr Pro Ala Ile Leu Val Glu 625 630 635 640 Cys Ala Phe Ile Asp
Asn Lys Ser Asp Met Asp Lys Leu Asn Thr Ala 645 650 655 Glu Lys Val
Lys Thr Met Gly Thr Gln Ile Gly Ile Gly Ile Glu Asp 660 665 670 Thr
Val Lys 675 21333PRTClostridium difficile 21Lys Ile Lys Lys Phe Thr
Leu Leu Ile Ser Ile Met Ile Ile Phe Leu 1 5 10 15 Cys Ala Val Val
Gly Val Tyr Ser Thr Ser Ser Asn Lys Ser Val Asp 20 25 30 Leu Tyr
Ser Asp Val Tyr Ile Glu Lys Tyr Phe Asn Arg Asp Lys Val 35 40 45
Met Glu Val Asn Ile Glu Ile Asp Glu Ser Asp Leu Lys Asp Met Asn 50
55 60 Glu Asn Ala Ile Lys Glu Glu Phe Lys Val Ala Lys Val Thr Val
Asp 65 70 75 80 Gly Asp Thr Tyr Gly Asn Val Gly Ile Arg Thr Lys Gly
Asn Ser Ser 85 90 95 Leu Thr Ser Val Ala Asn Ser Asp Ser Asp Arg
Tyr Ser Tyr Lys Ile 100 105 110 Asn Phe Asp Lys Tyr Asn Thr Ser Gln
Ser Met Glu Gly Leu Thr Gln 115 120 125 Leu Asn Leu Asn Asn Cys Tyr
Ser Asp Pro Ser Tyr Met Arg Glu Phe 130 135 140 Leu Thr Tyr Ser Ile
Cys Glu Glu Met Gly Leu Ala Thr Pro Glu Phe 145 150 155 160 Ala Tyr
Ala Lys Val Ser Ile Asn Gly Glu Tyr His Gly Leu Tyr Leu 165 170 175
Ala Val Glu Gly Leu Lys Glu Ser Tyr Leu Glu Asn Asn Phe Gly Asn 180
185 190 Val Thr Gly Asp Leu Tyr Lys Ser Asp Glu Gly Ser Ser Leu Gln
Tyr 195 200 205 Lys Gly Asp Asp Pro Glu Ser Tyr Ser Asn Leu Ile Val
Glu Ser Asp 210 215 220 Lys Lys Thr Ala Asp Trp Ser Lys Ile Thr Lys
Leu Leu Lys Ser Leu 225 230 235 240 Asp Thr Gly Glu Asp Ile Glu Lys
Tyr Leu Asp Val Asp Ser Val Leu 245 250 255 Lys Asn Ile Ala Ile Asn
Thr Ala Leu Leu Asn Leu Asp Ser Tyr Gln 260 265 270 Gly Ser Phe Ala
His Asn Tyr Tyr Leu Tyr Glu Gln Asp Gly Val Phe 275 280 285 Ser Met
Leu Pro Trp Asp Phe Asn Met Ser Phe Gly Gly Phe Ser Gly 290 295 300
Phe Gly Gly Gly Ser Gln Ser Ile Ala Ile Asp Glu Pro Thr Thr Gly 305
310 315 320 Asn Leu Glu Asp Arg Pro Leu Ile Ser Ser Leu Leu Lys 325
330 22302PRTClostridium difficile 22Lys Asn Glu Thr His Lys Thr Lys
Tyr His Lys Tyr Leu Glu Glu Ile 1 5 10 15 Val Thr Lys Tyr Leu Asp
Ser Asp Tyr Leu Glu Asn Met Thr Thr Lys 20 25 30 Leu His Asp Met
Ile Ala Ser Tyr Val Lys Glu Asp Pro Thr Ala Phe 35 40 45 Tyr Thr
Tyr Glu Glu Phe Glu Lys Asn Ile Thr Ser Ser Ile Glu Asp 50 55 60
Ser Ser Asp Asn Lys Gly Phe Gly Asn Lys Gly Phe Asp Asn Asn Asn 65
70 75 80 Ser Asn Asn Ser Asp Ser Asn Asn Asn Ser Asn Ser Glu Asn
Lys Arg 85 90 95 Ser Gly Asn Gln Ser Asp Lys Lys Glu Val Asn Ala
Glu Leu Thr Ser 100 105 110 Ser Val Val Lys Thr Asn Thr Asp Asn Glu
Thr Glu Asn Lys Thr Thr 115 120 125 Asn Asp Ser Glu Ser Lys Asn Asn
Thr Asp Lys Asp Lys Ser Gly Asn 130 135 140 Asp Asn Asn Gln Lys Leu
Glu Gly Pro Arg Gly Lys Gly Gly Lys Ser 145 150 155 160 Ile Pro Gly
Val Leu Glu Val Ala Glu Asp Met Ser Lys Thr Ile Lys 165 170 175 Ser
Gln Leu Ser Gly Glu Thr Ser Ser Thr Lys Gln Asn Ser Gly Asp 180 185
190 Glu Ser Ser Ser Gly Ile Lys Gly Ser Glu Lys Phe Asp Glu Asp Met
195 200 205 Ser Gly Met Pro Glu Pro Pro Glu Gly Met Asp Gly Lys Met
Pro Pro 210 215 220 Gly Met Gly Asn Met Asp Lys Gly Asp Met Asn Gly
Lys Asn Gly Asn 225 230 235 240 Met Asn Met Asp Arg Asn Gln Asp Asn
Pro Arg Glu Ala Gly Gly Phe 245 250 255 Gly Asn Arg Gly Gly Gly Ser
Val Ser Lys Thr Thr Thr Tyr Phe Lys 260 265 270 Leu Ile Leu Gly Gly
Ala Ser Met Ile Ile Met Ser Ile Met Leu Val 275 280 285 Gly Val Ser
Arg Val Lys Arg Arg Arg Phe Ile Lys Ser Lys 290 295 300
23635PRTClostridium difficile 23Met Lys Asp Lys Lys Phe Thr Leu Leu
Ile Ser Ile Met Ile Ile Phe 1 5 10 15 Leu Cys Ala Val Val Gly Val
Tyr Ser Thr Ser Ser Asn Lys Ser Val 20 25
30 Asp Leu Tyr Ser Asp Val Tyr Ile Glu Lys Tyr Phe Asn Arg Asp Lys
35 40 45 Val Met Glu Val Asn Ile Glu Ile Asp Glu Ser Asp Leu Lys
Asp Met 50 55 60 Asn Glu Asn Ala Ile Lys Glu Glu Phe Lys Val Ala
Lys Val Thr Val 65 70 75 80 Asp Gly Asp Thr Tyr Gly Asn Val Gly Ile
Arg Thr Lys Gly Asn Ser 85 90 95 Ser Leu Thr Ser Val Ala Asn Ser
Asp Ser Asp Arg Tyr Ser Tyr Lys 100 105 110 Ile Asn Phe Asp Lys Tyr
Asn Thr Ser Gln Ser Met Glu Gly Leu Thr 115 120 125 Gln Leu Asn Leu
Asn Asn Cys Tyr Ser Asp Pro Ser Tyr Met Arg Glu 130 135 140 Phe Leu
Thr Tyr Ser Ile Cys Glu Glu Met Gly Leu Ala Thr Pro Glu 145 150 155
160 Phe Ala Tyr Ala Lys Val Ser Ile Asn Gly Glu Tyr His Gly Leu Tyr
165 170 175 Leu Ala Val Glu Gly Leu Lys Glu Ser Tyr Leu Glu Asn Asn
Phe Gly 180 185 190 Asn Val Thr Gly Asp Leu Tyr Lys Ser Asp Glu Gly
Ser Ser Leu Gln 195 200 205 Tyr Lys Gly Asp Asp Pro Glu Ser Tyr Ser
Asn Leu Ile Val Glu Ser 210 215 220 Asp Lys Lys Thr Ala Asp Trp Ser
Lys Ile Thr Lys Leu Leu Lys Ser 225 230 235 240 Leu Asp Thr Gly Glu
Asp Ile Glu Lys Tyr Leu Asp Val Asp Ser Val 245 250 255 Leu Lys Asn
Ile Ala Ile Asn Thr Ala Leu Leu Asn Leu Asp Ser Tyr 260 265 270 Gln
Gly Ser Phe Ala His Asn Tyr Tyr Leu Tyr Glu Gln Asp Gly Val 275 280
285 Phe Ser Met Leu Pro Trp Asp Phe Asn Met Ser Phe Gly Gly Phe Ser
290 295 300 Gly Phe Gly Gly Gly Ser Gln Ser Ile Ala Ile Asp Glu Pro
Thr Thr 305 310 315 320 Gly Asn Leu Glu Asp Arg Pro Leu Ile Ser Ser
Leu Leu Lys Asn Glu 325 330 335 Thr His Lys Thr Lys Tyr His Lys Tyr
Leu Glu Glu Ile Val Thr Lys 340 345 350 Tyr Leu Asp Ser Asp Tyr Leu
Glu Asn Met Thr Thr Lys Leu His Asp 355 360 365 Met Ile Ala Ser Tyr
Val Lys Glu Asp Pro Thr Ala Phe Tyr Thr Tyr 370 375 380 Glu Glu Phe
Glu Lys Asn Ile Thr Ser Ser Ile Glu Asp Ser Ser Asp 385 390 395 400
Asn Lys Gly Phe Gly Asn Lys Gly Phe Asp Asn Asn Asn Ser Asn Asn 405
410 415 Ser Asp Ser Asn Asn Asn Ser Asn Ser Glu Asn Lys Arg Ser Gly
Asn 420 425 430 Gln Ser Asp Lys Lys Glu Val Asn Ala Glu Leu Thr Ser
Ser Val Val 435 440 445 Lys Thr Asn Thr Asp Asn Glu Thr Glu Asn Lys
Thr Thr Asn Asp Ser 450 455 460 Glu Ser Lys Asn Asn Thr Asp Lys Asp
Lys Ser Gly Asn Asp Asn Asn 465 470 475 480 Gln Lys Leu Glu Gly Pro
Arg Gly Lys Gly Gly Lys Ser Ile Pro Gly 485 490 495 Val Leu Glu Val
Ala Glu Asp Met Ser Lys Thr Ile Lys Ser Gln Leu 500 505 510 Ser Gly
Glu Thr Ser Ser Thr Lys Gln Asn Ser Gly Asp Glu Ser Ser 515 520 525
Ser Gly Ile Lys Gly Ser Glu Lys Phe Asp Glu Asp Met Ser Gly Met 530
535 540 Pro Glu Pro Pro Glu Gly Met Asp Gly Lys Met Pro Pro Gly Met
Gly 545 550 555 560 Asn Met Asp Lys Gly Asp Met Asn Gly Lys Asn Gly
Asn Met Asn Met 565 570 575 Asp Arg Asn Gln Asp Asn Pro Arg Glu Ala
Gly Gly Phe Gly Asn Arg 580 585 590 Gly Gly Gly Ser Val Ser Lys Thr
Thr Thr Tyr Phe Lys Leu Ile Leu 595 600 605 Gly Gly Ala Ser Met Ile
Ile Met Ser Ile Met Leu Val Gly Val Ser 610 615 620 Arg Val Lys Arg
Arg Arg Phe Ile Lys Ser Lys 625 630 635 2430DNAArtificial
SequenceChemically-synthesized CD1021 primer + restriction site
24taagctagca tgaaagataa aaaatttacc 302530DNAArtificial
SequenceChemically-synthesized CD1021 primer + restriction site
25ttactcgagt tttgacttta taaatcttct 302633DNAArtificial
SequenceChemically-synthesized CD1021 primer + restriction site
26acagctagca tgaaaagtgt tgatttatat agt 332731DNAArtificial
SequenceChemically-synthesized CD1021 primer + restriction site
27actctcgaga gtattatact tatcaaaatt a 312836DNAArtificial
SequenceChemically-synthesized CD1021 primer + restriction site
28aatgctagca tggtaactgg agacttatat aagtca 362932DNAArtificial
SequenceChemically-synthesized CD1021 primer + restriction site
29aaactcgagt ggtaacatag aaaatactcc at 323033DNAArtificial
SequenceChemically-synthesized CD1021 primer + restriction site
30gcagctagca tgagtaaaac tataaaatct caa 333133DNAArtificial
SequenceChemically-synthesized CD1021 primer + restriction site
31aatctcgagg aagtatgttg ttgttttact cac 333217DNAArtificial
SequenceCommercial M13 forward sequencing primer 32gtaaaacgac
ggccagt 173317DNAArtificial SequenceCommercial M13 reverse
sequencing primer 33caggaaacag ctatgac 173429DNAArtificial
SequenceChemically-synthesized CD1036 + restriction site
34aatccatggt aagtaaggag attaatatg 293530DNAArtificial
SequenceChemically-synthesized CD1036 primer + restriction site
35ttcctcgagt tttaatgaat cttctattcc 303629DNAArtificial
SequenceChemically-synthesized CD1036 primer + restriction site
36aggccatgga taaaaatcat gatgtggaa 293730DNAArtificial
SequenceChemically-synthesized CD1036 primer + restriction site
37tttctcgagg tttgcatttg tttcgtgtct 3038635PRTArtificial
SequenceComputer program-generated consensus protein sequence for
CD1021 38Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Met Ile
Xaa Phe 1 5 10 15 Leu Cys Ala Val Val Gly Val Tyr Ser Thr Ser Ser
Asn Lys Ser Val 20 25 30 Asp Leu Tyr Ser Asp Val Tyr Ile Glu Lys
Tyr Phe Asn Arg Asp Lys 35 40 45 Val Met Glu Val Asn Ile Glu Ile
Asp Glu Ser Asp Leu Lys Asp Met 50 55 60 Asn Glu Asn Ala Ile Lys
Glu Glu Phe Lys Val Ala Lys Val Thr Val 65 70 75 80 Asp Gly Asp Thr
Tyr Gly Asn Val Gly Ile Arg Thr Lys Gly Asn Ser 85 90 95 Ser Leu
Xaa Ser Val Ala Asn Ser Asp Ser Asp Arg Tyr Ser Tyr Lys 100 105 110
Ile Asn Phe Asp Lys Tyr Asn Thr Ser Gln Ser Met Glu Gly Leu Thr 115
120 125 Gln Leu Asn Leu Asn Asn Cys Tyr Ser Asp Pro Ser Tyr Met Arg
Glu 130 135 140 Phe Leu Thr Tyr Ser Ile Cys Glu Glu Met Gly Leu Ala
Thr Pro Glu 145 150 155 160 Phe Ala Tyr Ala Lys Val Ser Ile Asn Gly
Glu Tyr His Gly Leu Tyr 165 170 175 Leu Ala Val Glu Gly Leu Lys Glu
Ser Tyr Leu Glu Asn Asn Phe Gly 180 185 190 Asn Val Thr Gly Asp Leu
Tyr Lys Ser Asp Glu Gly Ser Ser Leu Gln 195 200 205 Tyr Lys Gly Asp
Asp Pro Glu Ser Tyr Ser Asn Leu Ile Val Glu Ser 210 215 220 Asp Lys
Lys Thr Ala Asp Trp Ser Lys Ile Thr Lys Leu Leu Lys Ser 225 230 235
240 Leu Asp Thr Gly Glu Asp Ile Glu Lys Tyr Leu Asp Val Asp Ser Val
245 250 255 Leu Lys Asn Ile Ala Ile Asn Thr Ala Leu Leu Asn Leu Asp
Ser Tyr 260 265 270 Gln Gly Ser Phe Ala His Asn Tyr Tyr Leu Tyr Glu
Gln Asp Gly Val 275 280 285 Phe Ser Met Leu Pro Trp Asp Phe Asn Met
Ser Phe Gly Gly Phe Ser 290 295 300 Gly Phe Gly Gly Gly Ser Gln Ser
Ile Ala Ile Asp Glu Pro Thr Thr 305 310 315 320 Gly Asn Leu Glu Asp
Arg Pro Leu Ile Ser Ser Leu Leu Lys Xaa Xaa 325 330 335 Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 340 345 350 Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Met Thr Thr Lys Leu His Asp 355 360
365 Met Ile Ala Ser Tyr Val Lys Glu Asp Pro Thr Ala Phe Tyr Thr Tyr
370 375 380 Glu Glu Phe Glu Lys Asn Ile Thr Ser Ser Ile Glu Asp Ser
Ser Asp 385 390 395 400 Asn Lys Gly Phe Gly Asn Lys Gly Phe Asp Asn
Asn Asn Ser Asn Asn 405 410 415 Ser Asp Ser Asn Asn Asn Ser Asn Ser
Glu Asn Lys Arg Ser Gly Asn 420 425 430 Gln Ser Asp Lys Lys Glu Val
Asn Ala Glu Leu Thr Ser Ser Val Val 435 440 445 Lys Thr Asn Thr Asp
Asn Glu Thr Glu Asn Lys Thr Thr Asn Asp Ser 450 455 460 Glu Ser Lys
Asn Asn Thr Asp Lys Asp Lys Ser Gly Asn Asp Asn Asn 465 470 475 480
Gln Lys Leu Glu Gly Pro Arg Gly Lys Gly Gly Lys Ser Ile Pro Gly 485
490 495 Val Leu Glu Val Ala Glu Asp Met Ser Lys Thr Ile Lys Ser Gln
Leu 500 505 510 Ser Gly Glu Thr Ser Ser Thr Lys Gln Asn Ser Gly Asp
Glu Ser Ser 515 520 525 Ser Gly Ile Lys Gly Ser Glu Lys Phe Asp Glu
Asp Met Ser Gly Met 530 535 540 Pro Glu Pro Pro Glu Gly Met Asp Gly
Lys Met Pro Pro Gly Met Gly 545 550 555 560 Asn Met Asp Lys Gly Asp
Met Asn Gly Lys Asn Gly Asn Met Asn Met 565 570 575 Asp Arg Asn Gln
Asp Asn Pro Arg Glu Ala Gly Gly Phe Gly Asn Arg 580 585 590 Gly Gly
Gly Ser Val Ser Lys Thr Thr Thr Tyr Phe Lys Leu Ile Leu 595 600 605
Gly Gly Ala Ser Met Ile Ile Met Ser Ile Met Leu Val Gly Val Ser 610
615 620 Arg Val Lys Arg Arg Arg Phe Ile Lys Ser Lys 625 630 635
391905DNAClostridium difficile 39atgaaagata aaaaatttac ccttcttatc
tcgattatga ttatattttt atgtgctgta 60gttggagttt atagtacatc tagcaacaaa
agtgttgatt tatatagtga tgtatatatt 120gaaaaatatt ttaacagaga
caaggttatg gaagttaata tagagataga tgaaagtgac 180ttgaaggata
tgaatgaaaa tgctataaaa gaagaattta aggttgcaaa agtaactgta
240gatggagata catatggaaa cgtaggtata agaactaaag gaaattcaag
tcttatatct 300gtagcaaata gtgatagtga tagatacagc tataagatta
attttgataa gtataatact 360agtcaaagta tggaagggct tactcaatta
aatcttaata actgttactc tgacccatct 420tatatgagag agtttttaac
atatagtatt tgcgaggaaa tgggattagc gactccagaa 480tttgcatatg
ctaaagtctc tataaatggc gaatatcatg gtttgtattt ggcagtagaa
540ggattaaaag agtcttatct tgaaaataat tttggtaatg taactggaga
cttatataag 600tcagatgaag gaagctcgtt gcaatataaa ggagatgacc
cagaaagtta ctcaaactta 660atcgttgaaa gtgataaaaa gacagctgat
tggtctaaaa tcacaaaact attaaaatct 720ttggatacag gtgaagatat
tgaaaaatat cttgatgtag attctgtcct taaaaatata 780gcaataaata
cagctttatt aaaccttgat agctatcaag ggagttttgc ccataactat
840tatttatatg agcaagatgg agtattttct atgttaccat gggattttaa
tatgtcattt 900ggtggattta gtggttttgg tggaggtagt caatctatag
caattgatga acctacgaca 960ggtaatttag aagacagacc tctcatatcc
tcgttattaa aaaatgagac atacaaaaca 1020aaataccata aatatctgga
agagatagta acaaaatacc tagattcaga ctatttagag 1080aatatgacaa
caaaattgca tgacatgata gcatcatatg taaaagaaga cccaacagca
1140ttttatactt atgaagaatt tgaaaaaaat ataacatctt caattgaaga
ttctagtgat 1200aataagggat ttggtaataa agggtttgac aacaataact
ctaataacag tgattctaat 1260aataattcta atagtgaaaa taagcgctct
ggaaatcaaa gtgatgaaaa agaagttaat 1320gctgaattaa catcaagcgt
agtcaaagct aatacagata atgaaactaa aaataaaact 1380acaaatgata
gtgaaagtaa gaataataca gataaagata aaagtggaaa tgataataat
1440caaaagctag aaggtcctat gggtaaagga ggtaagtcaa taccaggggt
tttggaagtt 1500gcagaagata tgagtaaaac tataaaatct caattaagtg
gagaaacttc ttcgacaaag 1560caaaactctg gtgatgaaag ttcaagtgga
attaaaggta gtgaaaagtt tgatgaggat 1620atgagtggta tgccagaacc
acctgaggga atggatggta aaatgccacc aggaatgggt 1680aatatggata
agggagatat gaatggtaaa aatggcaata tgaatatgga tagaaatcaa
1740gataatccaa gagaagctgg aggttttggc aatagaggag gaggctctgt
gagtaaaaca 1800acaacatact tcaaattaat tttaggtgga gcttcaatga
taataatgtc gattatgtta 1860gttggtgtat caagggtaaa gagaagaaga
tttataaagt caaaa 190540276DNAClostridium difficile 40atgaaaagtg
ttgatttata tagtgatgta tatattgaaa aatattttaa cagagacaag 60gttatggaag
ttaatataga gatagatgaa agtgacttga aggatatgaa tgaaaatgct
120ataaaagaag aatttaaggt tgcaaaagta actgtagatg gagatacata
tggaaacgta 180ggtataagaa ctaaaggaaa ttcaagtctt atatctgtag
caaatagtga tagtgataga 240tacagctata agattaattt tgataagtat aatact
27641303DNAClostridium difficile 41atggtaactg gagacttata taagtcagat
gaaggaagct cgttgcaata taaaggagat 60gacccagaaa gttactcaaa cttaatcgtt
gaaagtgata aaaagacagc tgattggtct 120aaaatcacaa aactattaaa
atctttggat acaggtgaag atattgaaaa atatcttgat 180gtagattctg
tccttaaaaa tatagcaata aatacagctt tattaaacct tgatagctat
240caagggagtt ttgcccataa ctattattta tatgagcaag atggagtatt
ttctatgtta 300cca 30342303DNAClostridium difficile 42atgagtaaaa
ctataaaatc tcaattaagt ggagaaactt cttcgacaaa gcaaaactct 60ggtgatgaaa
gttcaagtgg aattaaaggt agtgaaaagt ttgatgagga tatgagtggt
120atgccagaac cacctgaggg aatggatggt aaaatgccac caggaatggg
taatatggat 180aagggagata tgaatggtaa aaatggcaat atgaatatgg
atagaaatca agataatcca 240agagaagctg gaggttttgg caatagagga
ggaggctctg tgagtaaaac aacaacatac 300ttc 303432031DNAClostridium
difficile 43atggtaagta aggagattaa tatgagaaga aatacaaaat tattaacaac
agggattctt 60tcaatggcaa tcgtcgcacc tacaatggca tttgctactg aatctaatgc
tatggaaaat 120aacgctgatt taaatataaa cttagagaaa aaaagtatcg
ttttaggtag caaatcaaaa 180gttagtgtca aatttaaaga aaaaccagat
gcagatagca ttacattaaa gtataaatgc 240tatgacatgc cattgaatac
aactctaaat tacaatcaat caactggggc atatgaagga 300actatcaatt
ataaccaaga cccagaatat ctaaatgttt gggaactaca agggataaca
360ataaacagca aaaataatca taaaacttta aacagacaag acctagaaaa
gctgggatta 420aatttaaaag actataatgt aacacaggaa tgtataattg
aagatataac ttctagaaaa 480gatgtaaata aatatttgag aaaaacttct
tcacctatta cagaacttac aggaagtgat 540agatatgaaa cagcagttaa
aataagtaaa gagggctgga aaaatggttc agataaggta 600gttataataa
atggggatgt aagtatagat ggcattatat caactccact ggcaaccaca
660tataatgcac caatactttt ggttgaaaaa aacaatgtac ctaatagtgt
aaaatcagaa 720ttaaagcgcc taaaccctaa agatataatt ataattggag
atgagaatgc tatttctaaa 780actactgcta atcaaattaa atcaactgta
aatgctagtc aaacacgttt aaatggttct 840aatagatatg agacatcttt
attgatagca aaggaaatag ataaaaatca tgatgtggaa 900aaagtataca
taacaaatgc taatggcgga gaagtggatg cacttactat agcagcaaaa
960gcaggtcaag acaagcaacc aattatatta actgataaag atagtattac
agacaataca 1020tataaatggt taaagagtga ggatttacaa aatgcttatt
ttataggtgg tcctcaaatg 1080atatcaacaa atgttataaa taaggtaaat
ggaataacta aagatagtgt tactaataat 1140agagtatacg gagcagatag
acacgaaaca aatgcaaacg taataaaaaa attctataca 1200gatgatgagt
tagaggctgt tttagtagct aaatcagatg tacttgttga tgctttagca
1260gcaggtccat tggctgcgaa cttaaaatct ccaatactta taacaccaaa
gacgtatgta 1320tctgcatacc ataaagataa tttagaagct aaatcagcta
ataaggtata caaaatagga 1380ggaggattga cttctaaggt aatgagctct
atagcatcat cattatctaa acacaatacg 1440actccaacag aaccaggaaa
tagtgggggc aagacagtta tgattgaccc agggcatggt 1500ggttcagcac
ctggaaattc atctggagga atgattgaaa aagattacaa tttaaatact
1560tcacttgcaa caactgaata tttacgttca aagggattca atgtaataat
gacaagagac 1620acagataaga ctttatctct tggaaataga actgctctat
ctaattcatt gaaaccagat 1680ttatttacaa gtatacatta taatggctca
actaataaac aaggtcatgg tgtagaagta 1740ttttataagc ttaaagataa
aaatggaggg actactaaaa ctgtagctac caatatatta 1800aatagaattt
tagagaaatt taaacttaca aatagaggta taaaaacaag agtacttcct
1860agtgattcta caaaagatta tttatacgtt
ttaagaagta atgatatgcc agctgtactt 1920gtagaatgtg catttttgga
taatgaaaat gatatgagtt taataaactc atctgcaaaa 1980gtaaaagaaa
tgggtacaca aataggtaaa ggaatagaag attcattaaa a
203144303DNAClostridium difficile 44atggataaaa atcatgatgt
ggaaaaagta tacataacaa atgctaatgg cggagaagtg 60gatgcactta ctatagcagc
aaaagcaggt caagacaagc aaccaattat attaactgat 120aaagatagta
ttacagacaa tacatataaa tggttaaaga gtgaggattt acaaaatgct
180tattttatag gtggtcctca aatgatatca acaaatgtta taaataaggt
aaatggaata 240actaaagata gtgttactaa taatagagta tacggagcag
atagacacga aacaaatgca 300aac 30345635PRTClostridium difficile 45Met
Lys Asp Lys Lys Phe Thr Leu Leu Ile Ser Ile Met Ile Ile Phe 1 5 10
15 Leu Cys Ala Val Val Gly Val Tyr Ser Thr Ser Ser Asn Lys Ser Val
20 25 30 Asp Leu Tyr Ser Asp Val Tyr Ile Glu Lys Tyr Phe Asn Arg
Asp Lys 35 40 45 Val Met Glu Val Asn Ile Glu Ile Asp Glu Ser Asp
Leu Lys Asp Met 50 55 60 Asn Glu Asn Ala Ile Lys Glu Glu Phe Lys
Val Ala Lys Val Thr Val 65 70 75 80 Asp Gly Asp Thr Tyr Gly Asn Val
Gly Ile Arg Thr Lys Gly Asn Ser 85 90 95 Ser Leu Ile Ser Val Ala
Asn Ser Asp Ser Asp Arg Tyr Ser Tyr Lys 100 105 110 Ile Asn Phe Asp
Lys Tyr Asn Thr Ser Gln Ser Met Glu Gly Leu Thr 115 120 125 Gln Leu
Asn Leu Asn Asn Cys Tyr Ser Asp Pro Ser Tyr Met Arg Glu 130 135 140
Phe Leu Thr Tyr Ser Ile Cys Glu Glu Met Gly Leu Ala Thr Pro Glu 145
150 155 160 Phe Ala Tyr Ala Lys Val Ser Ile Asn Gly Glu Tyr His Gly
Leu Tyr 165 170 175 Leu Ala Val Glu Gly Leu Lys Glu Ser Tyr Leu Glu
Asn Asn Phe Gly 180 185 190 Asn Val Thr Gly Asp Leu Tyr Lys Ser Asp
Glu Gly Ser Ser Leu Gln 195 200 205 Tyr Lys Gly Asp Asp Pro Glu Ser
Tyr Ser Asn Leu Ile Val Glu Ser 210 215 220 Asp Lys Lys Thr Ala Asp
Trp Ser Lys Ile Thr Lys Leu Leu Lys Ser 225 230 235 240 Leu Asp Thr
Gly Glu Asp Ile Glu Lys Tyr Leu Asp Val Asp Ser Val 245 250 255 Leu
Lys Asn Ile Ala Ile Asn Thr Ala Leu Leu Asn Leu Asp Ser Tyr 260 265
270 Gln Gly Ser Phe Ala His Asn Tyr Tyr Leu Tyr Glu Gln Asp Gly Val
275 280 285 Phe Ser Met Leu Pro Trp Asp Phe Asn Met Ser Phe Gly Gly
Phe Ser 290 295 300 Gly Phe Gly Gly Gly Ser Gln Ser Ile Ala Ile Asp
Glu Pro Thr Thr 305 310 315 320 Gly Asn Leu Glu Asp Arg Pro Leu Ile
Ser Ser Leu Leu Lys Asn Glu 325 330 335 Thr Tyr Lys Thr Lys Tyr His
Lys Tyr Leu Glu Glu Ile Val Thr Lys 340 345 350 Tyr Leu Asp Ser Asp
Tyr Leu Glu Asn Met Thr Thr Lys Leu His Asp 355 360 365 Met Ile Ala
Ser Tyr Val Lys Glu Asp Pro Thr Ala Phe Tyr Thr Tyr 370 375 380 Glu
Glu Phe Glu Lys Asn Ile Thr Ser Ser Ile Glu Asp Ser Ser Asp 385 390
395 400 Asn Lys Gly Phe Gly Asn Lys Gly Phe Asp Asn Asn Asn Ser Asn
Asn 405 410 415 Ser Asp Ser Asn Asn Asn Ser Asn Ser Glu Asn Lys Arg
Ser Gly Asn 420 425 430 Gln Ser Asp Glu Lys Glu Val Asn Ala Glu Leu
Thr Ser Ser Val Val 435 440 445 Lys Ala Asn Thr Asp Asn Glu Thr Lys
Asn Lys Thr Thr Asn Asp Ser 450 455 460 Glu Ser Lys Asn Asn Thr Asp
Lys Asp Lys Ser Gly Asn Asp Asn Asn 465 470 475 480 Gln Lys Leu Glu
Gly Pro Met Gly Lys Gly Gly Lys Ser Ile Pro Gly 485 490 495 Val Leu
Glu Val Ala Glu Asp Met Ser Lys Thr Ile Lys Ser Gln Leu 500 505 510
Ser Gly Glu Thr Ser Ser Thr Lys Gln Asn Ser Gly Asp Glu Ser Ser 515
520 525 Ser Gly Ile Lys Gly Ser Glu Lys Phe Asp Glu Asp Met Ser Gly
Met 530 535 540 Pro Glu Pro Pro Glu Gly Met Asp Gly Lys Met Pro Pro
Gly Met Gly 545 550 555 560 Asn Met Asp Lys Gly Asp Met Asn Gly Lys
Asn Gly Asn Met Asn Met 565 570 575 Asp Arg Asn Gln Asp Asn Pro Arg
Glu Ala Gly Gly Phe Gly Asn Arg 580 585 590 Gly Gly Gly Ser Val Ser
Lys Thr Thr Thr Tyr Phe Lys Leu Ile Leu 595 600 605 Gly Gly Ala Ser
Met Ile Ile Met Ser Ile Met Leu Val Gly Val Ser 610 615 620 Arg Val
Lys Arg Arg Arg Phe Ile Lys Ser Lys 625 630 635 4692PRTClostridium
difficile 46Met Lys Ser Val Asp Leu Tyr Ser Asp Val Tyr Ile Glu Lys
Tyr Phe 1 5 10 15 Asn Arg Asp Lys Val Met Glu Val Asn Ile Glu Ile
Asp Glu Ser Asp 20 25 30 Leu Lys Asp Met Asn Glu Asn Ala Ile Lys
Glu Glu Phe Lys Val Ala 35 40 45 Lys Val Thr Val Asp Gly Asp Thr
Tyr Gly Asn Val Gly Ile Arg Thr 50 55 60 Lys Gly Asn Ser Ser Leu
Ile Ser Val Ala Asn Ser Asp Ser Asp Arg 65 70 75 80 Tyr Ser Tyr Lys
Ile Asn Phe Asp Lys Tyr Asn Thr 85 90 47101PRTClostridium difficile
47Met Val Thr Gly Asp Leu Tyr Lys Ser Asp Glu Gly Ser Ser Leu Gln 1
5 10 15 Tyr Lys Gly Asp Asp Pro Glu Ser Tyr Ser Asn Leu Ile Val Glu
Ser 20 25 30 Asp Lys Lys Thr Ala Asp Trp Ser Lys Ile Thr Lys Leu
Leu Lys Ser 35 40 45 Leu Asp Thr Gly Glu Asp Ile Glu Lys Tyr Leu
Asp Val Asp Ser Val 50 55 60 Leu Lys Asn Ile Ala Ile Asn Thr Ala
Leu Leu Asn Leu Asp Ser Tyr 65 70 75 80 Gln Gly Ser Phe Ala His Asn
Tyr Tyr Leu Tyr Glu Gln Asp Gly Val 85 90 95 Phe Ser Met Leu Pro
100 48101PRTClostridium difficile 48Met Ser Lys Thr Ile Lys Ser Gln
Leu Ser Gly Glu Thr Ser Ser Thr 1 5 10 15 Lys Gln Asn Ser Gly Asp
Glu Ser Ser Ser Gly Ile Lys Gly Ser Glu 20 25 30 Lys Phe Asp Glu
Asp Met Ser Gly Met Pro Glu Pro Pro Glu Gly Met 35 40 45 Asp Gly
Lys Met Pro Pro Gly Met Gly Asn Met Asp Lys Gly Asp Met 50 55 60
Asn Gly Lys Asn Gly Asn Met Asn Met Asp Arg Asn Gln Asp Asn Pro 65
70 75 80 Arg Glu Ala Gly Gly Phe Gly Asn Arg Gly Gly Gly Ser Val
Ser Lys 85 90 95 Thr Thr Thr Tyr Phe 100 49677PRTClostridium
difficile 49Met Val Ser Lys Glu Ile Asn Met Arg Arg Asn Thr Lys Leu
Leu Thr 1 5 10 15 Thr Gly Ile Leu Ser Met Ala Ile Val Ala Pro Thr
Met Ala Phe Ala 20 25 30 Thr Glu Ser Asn Ala Met Glu Asn Asn Ala
Asp Leu Asn Ile Asn Leu 35 40 45 Glu Lys Lys Ser Ile Val Leu Gly
Ser Lys Ser Lys Val Ser Val Lys 50 55 60 Phe Lys Glu Lys Pro Asp
Ala Asp Ser Ile Thr Leu Lys Tyr Lys Cys 65 70 75 80 Tyr Asp Met Pro
Leu Asn Thr Thr Leu Asn Tyr Asn Gln Ser Thr Gly 85 90 95 Ala Tyr
Glu Gly Thr Ile Asn Tyr Asn Gln Asp Pro Glu Tyr Leu Asn 100 105 110
Val Trp Glu Leu Gln Gly Ile Thr Ile Asn Ser Lys Asn Asn His Lys 115
120 125 Thr Leu Asn Arg Gln Asp Leu Glu Lys Leu Gly Leu Asn Leu Lys
Asp 130 135 140 Tyr Asn Val Thr Gln Glu Cys Ile Ile Glu Asp Ile Thr
Ser Arg Lys 145 150 155 160 Asp Val Asn Lys Tyr Leu Arg Lys Thr Ser
Ser Pro Ile Thr Glu Leu 165 170 175 Thr Gly Ser Asp Arg Tyr Glu Thr
Ala Val Lys Ile Ser Lys Glu Gly 180 185 190 Trp Lys Asn Gly Ser Asp
Lys Val Val Ile Ile Asn Gly Asp Val Ser 195 200 205 Ile Asp Gly Ile
Ile Ser Thr Pro Leu Ala Thr Thr Tyr Asn Ala Pro 210 215 220 Ile Leu
Leu Val Glu Lys Asn Asn Val Pro Asn Ser Val Lys Ser Glu 225 230 235
240 Leu Lys Arg Leu Asn Pro Lys Asp Ile Ile Ile Ile Gly Asp Glu Asn
245 250 255 Ala Ile Ser Lys Thr Thr Ala Asn Gln Ile Lys Ser Thr Val
Asn Ala 260 265 270 Ser Gln Thr Arg Leu Asn Gly Ser Asn Arg Tyr Glu
Thr Ser Leu Leu 275 280 285 Ile Ala Lys Glu Ile Asp Lys Asn His Asp
Val Glu Lys Val Tyr Ile 290 295 300 Thr Asn Ala Asn Gly Gly Glu Val
Asp Ala Leu Thr Ile Ala Ala Lys 305 310 315 320 Ala Gly Gln Asp Lys
Gln Pro Ile Ile Leu Thr Asp Lys Asp Ser Ile 325 330 335 Thr Asp Asn
Thr Tyr Lys Trp Leu Lys Ser Glu Asp Leu Gln Asn Ala 340 345 350 Tyr
Phe Ile Gly Gly Pro Gln Met Ile Ser Thr Asn Val Ile Asn Lys 355 360
365 Val Asn Gly Ile Thr Lys Asp Ser Val Thr Asn Asn Arg Val Tyr Gly
370 375 380 Ala Asp Arg His Glu Thr Asn Ala Asn Val Ile Lys Lys Phe
Tyr Thr 385 390 395 400 Asp Asp Glu Leu Glu Ala Val Leu Val Ala Lys
Ser Asp Val Leu Val 405 410 415 Asp Ala Leu Ala Ala Gly Pro Leu Ala
Ala Asn Leu Lys Ser Pro Ile 420 425 430 Leu Ile Thr Pro Lys Thr Tyr
Val Ser Ala Tyr His Lys Asp Asn Leu 435 440 445 Glu Ala Lys Ser Ala
Asn Lys Val Tyr Lys Ile Gly Gly Gly Leu Thr 450 455 460 Ser Lys Val
Met Ser Ser Ile Ala Ser Ser Leu Ser Lys His Asn Thr 465 470 475 480
Thr Pro Thr Glu Pro Gly Asn Ser Gly Gly Lys Thr Val Met Ile Asp 485
490 495 Pro Gly His Gly Gly Ser Ala Pro Gly Asn Ser Ser Gly Gly Met
Ile 500 505 510 Glu Lys Asp Tyr Asn Leu Asn Thr Ser Leu Ala Thr Thr
Glu Tyr Leu 515 520 525 Arg Ser Lys Gly Phe Asn Val Ile Met Thr Arg
Asp Thr Asp Lys Thr 530 535 540 Leu Ser Leu Gly Asn Arg Thr Ala Leu
Ser Asn Ser Leu Lys Pro Asp 545 550 555 560 Leu Phe Thr Ser Ile His
Tyr Asn Gly Ser Thr Asn Lys Gln Gly His 565 570 575 Gly Val Glu Val
Phe Tyr Lys Leu Lys Asp Lys Asn Gly Gly Thr Thr 580 585 590 Lys Thr
Val Ala Thr Asn Ile Leu Asn Arg Ile Leu Glu Lys Phe Lys 595 600 605
Leu Thr Asn Arg Gly Ile Lys Thr Arg Val Leu Pro Ser Asp Ser Thr 610
615 620 Lys Asp Tyr Leu Tyr Val Leu Arg Ser Asn Asp Met Pro Ala Val
Leu 625 630 635 640 Val Glu Cys Ala Phe Leu Asp Asn Glu Asn Asp Met
Ser Leu Ile Asn 645 650 655 Ser Ser Ala Lys Val Lys Glu Met Gly Thr
Gln Ile Gly Lys Gly Ile 660 665 670 Glu Asp Ser Leu Lys 675
50101PRTClostridium difficile 50Met Asp Lys Asn His Asp Val Glu Lys
Val Tyr Ile Thr Asn Ala Asn 1 5 10 15 Gly Gly Glu Val Asp Ala Leu
Thr Ile Ala Ala Lys Ala Gly Gln Asp 20 25 30 Lys Gln Pro Ile Ile
Leu Thr Asp Lys Asp Ser Ile Thr Asp Asn Thr 35 40 45 Tyr Lys Trp
Leu Lys Ser Glu Asp Leu Gln Asn Ala Tyr Phe Ile Gly 50 55 60 Gly
Pro Gln Met Ile Ser Thr Asn Val Ile Asn Lys Val Asn Gly Ile 65 70
75 80 Thr Lys Asp Ser Val Thr Asn Asn Arg Val Tyr Gly Ala Asp Arg
His 85 90 95 Glu Thr Asn Ala Asn 100
* * * * *