U.S. patent application number 10/555059 was filed with the patent office on 2007-06-07 for method of diagnosis of foot and mouth disease and the diagnostic kit.
This patent application is currently assigned to Republic of Korea ( National Veterinary Research and Quarantine Service). Invention is credited to Soo-Hwan An, In-Soo Cho, Suh-Ha Hwang, Bang-Hun Hyun, Ki-Yong Jang, Yi-Seok Joo, Je-Mo Kang, Chang-Ho Kim, Hee-Jeong Kim, In-Joong Kim, Ok-Kyung Kim, Song-Woo Ko, Young-Joon Ko, Bok-Kyung Ku, Soo-Jeong Kye, Kwang-Nyeong Lee, Jae-Ku Oem, Nam-Kyu Shin.
Application Number | 20070128587 10/555059 |
Document ID | / |
Family ID | 36584422 |
Filed Date | 2007-06-07 |
United States Patent
Application |
20070128587 |
Kind Code |
A1 |
Cho; In-Soo ; et
al. |
June 7, 2007 |
Method of diagnosis of foot and mouth disease and the diagnostic
kit
Abstract
The present invention provides a method for diagnosing
foot-and-mouth disease virus infection, comprising the steps of
applying a predetermined amount of a test sample to a loading
region of a strip; coupling a detection reagent including a given
labeling reagent to an analyte of interest in the test sample to
form a complex therebetween; developing the complex onto a wicking
membrane; and observing changes in appearance of a reactivity zone
having at least more than one immobilized phase selected from
antigen, antibody or hapten on the predetermined region of the
wicking membrane, derived from FMDV or obtainable from FMDV through
an immunological reaction to determine the presence or absence of
foot-and-mouth disease virus infection. It also provides a kit for
diagnosing foot-and-mouth disease virus infection comprising a
strip 1 including a reactivity zone 13 containing at least more
than one immobilized phase selected from antigen, antibody or
hapten thereon, derived from FMDV or obtainable from FMDV through
an immunological reaction, and a control zone 14 for confining
normal operation of the kit, provided on a predetermined region of
a wicking membrane 9; and a housing 20 protecting the strip 1 from
a variety of contaminants, and including at least a test sample
application port 2 and an indicia window 4 for observing results of
reaction in the reactivity zone 13 and the control zone 14 on the
strip.
Inventors: |
Cho; In-Soo; (Kyunggi-do,
KR) ; Hyun; Bang-Hun; (Kangdong-gu, KR) ; Lee;
Kwang-Nyeong; (Kyunggi-do, KR) ; Oem; Jae-Ku;
(Seoul, KR) ; Kye; Soo-Jeong; (Seoul, KR) ;
Ko; Young-Joon; (Kyunggi-do, KR) ; Ku; Bok-Kyung;
(Kyungsangnam-do, KR) ; Joo; Yi-Seok; (Seoul,
KR) ; An; Soo-Hwan; (Seoul, KR) ; Kim;
In-Joong; (Seoul, KR) ; Kim; Ok-Kyung; (Seoul,
KR) ; Kim; Hee-Jeong; (Seoul, KR) ; Jang;
Ki-Yong; (Kyungsangbuk-do, KR) ; Shin; Nam-Kyu;
(Incheon, KR) ; Hwang; Suh-Ha; (Kyunggi-do,
KR) ; Kang; Je-Mo; (Princeton, NJ) ; Kim;
Chang-Ho; (Seoul, KR) ; Ko; Song-Woo; (Seoul,
KR) |
Correspondence
Address: |
STERNE, KESSLER, GOLDSTEIN & FOX P.L.L.C.
1100 NEW YORK AVENUE, N.W.
WASHINGTON
DC
20005
US
|
Assignee: |
Republic of Korea ( National
Veterinary Research and Quarantine Service)
480 Anyang 6-dong, Manan-gu
Kyonggi-do
NJ
430-824
Princeton Biomeditech East, Inc.
5th Fl., Jungmin Bldg., 786-19, Yeoksam-Dong
Seoul
135-080
Princeton Biomeditech Corporation
P.O. Box 7139
Princeton
08543
|
Family ID: |
36584422 |
Appl. No.: |
10/555059 |
Filed: |
May 6, 2003 |
PCT Filed: |
May 6, 2003 |
PCT NO: |
PCT/KR03/00896 |
371 Date: |
September 15, 2006 |
Current U.S.
Class: |
435/5 |
Current CPC
Class: |
Y10S 436/811 20130101;
G01N 33/56983 20130101; G01N 33/558 20130101; G01N 33/526 20130101;
G01N 2333/09 20130101 |
Class at
Publication: |
435/005 |
International
Class: |
C12Q 1/70 20060101
C12Q001/70 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 28, 2003 |
KR |
10 2003 0026809 |
Claims
1. A method for diagnosing foot-and-mouth disease virus infection,
comprising the steps of: (1) applying a predetermined amount of a
test sample to a loading region of a strip; (2) coupling a
detection reagent including a given labeling reagent to an analyte
of interest in the test sample to form a complex therebetween; (3)
developing the complex onto a wicking membrane; and (4) observing
changes in appearance of a reactivity zone having at least more
than one immobilized phase selected from antigen, antibody or
hapten on the predetermined region of the wicking membrane, derived
from FMDV or obtainable from FMDV through an immunological reaction
to determine the presence or absence of foot-and-mouth disease
virus infection.
2. The method as set forth in claim 1, wherein the method is a
sandwich method or a competition method.
3. The method as set forth in claim 1, wherein the immobilized
phase contains at least more than one protein selected from
FMDV-derived non-structural proteins and/or structural
proteins.
4. The method as set forth in claim 1 or 3, wherein the immobilized
phase is the structural proteins VP1-VP4 polypeptide or an
inactivated FMDV virus disrupted material.
5. The method as set forth in claim 1 or 3, wherein the immobilized
phase contains at least more than one polypeptide selected from the
group consisting of non-structural proteins leader peptide (Lb),
2B, 2C, 3D, 3A, 3AB and 3ABC.
6. The method as set forth in claim 1, wherein the reactivity zone
includes a first reactivity zone containing at least one selected
from the structural protein and 3D protein, these being capable of
vaccine production; and a second reactivity zone containing at
least one selected from the non-structural protein, except for
3D.
7. The method as set forth in claim 1, wherein the detection
reagent is provided at any site on the strip.
8. The method as set forth in claim 7, wherein the detection
reagent includes at least more than one material selected from
labeled antigens, antibodies or haptens.
9. The method as set forth in claim 7, wherein the detection
reagent includes at least more than one material selected from the
group of antibodies capable of binding to labeled protein G,
protein A, protein G/A, or species-specific IgG and IgM.
10. The method as set forth in claim 1, wherein the test sample is
blood, serum, plasma, urine, tears, saliva or milk.
11. A kit for diagnosing foot-and-mouth disease virus infection
comprising: a strip including a reactivity zone containing at least
more than one immobilized phase selected from antigen, antibody or
hapten thereon, derived from FMDV or obtainable from FMDV through
an immunological reaction, and a control zone for confirming normal
operation of the kit, provided on a predetermined region of a
wicking membrane; and a housing receiving the strip, and including
at least a test sample application port and an indicia window for
observing results of reaction in the reactivity zone and the
control zone on the strip.
12. The kit as set forth in claim 11, wherein the immobilized phase
contains at least more than one protein selected from FMDV-derived
non-structural proteins and/or structural proteins.
13. The kit as set forth in claim 11 or 12, wherein the immobilized
phase is the structural protein VP1-VP4 polypeptide or an
inactivated FMDV virus disrupted material.
14. The kit as set forth in claim 8, wherein the immobilized phase
contains at least more than one polypeptide selected from the group
consisting of non-structural proteins leader peptide (Lb), 2B, 2C,
3D, 3A, 3AB and 3ABC.
15. The kit as set forth in claim 11, wherein the reactivity zone
includes a first reactivity zone containing at least one selected
from the structural protein and 3D protein which can be used in
vaccine production; and a second reactivity zone containing at
least one selected from the non-structural proteins, except for
3D.
16. The kit as set forth in claim 11, wherein the detection reagent
is provided at any site on the strip.
17. The kit as set forth in claim 16, wherein the detection reagent
includes at least more than one material selected from a group of
antibodies capable of binding to labeled protein G, protein A,
protein G/A, or species-specific IgG and IgM.
18. The kit as set forth in claim 11, wherein the strip has a
configuration in which a reservoir pad, a filter pad in contact
with one end of the reservoir pad for filtering the test sample, a
wicking membrane in contact with one end of the filter pad for
moving the filtered sample by capillary action, and an absorbent
pad in contact with one end of the wicking membrane are connected
sequentially on a base member.
19. The kit as set forth in claim 18, wherein the detection reagent
is provided at any site on the strip.
20. The kit as set forth in claim 18, wherein the base member is
made of plastic or glass material.
21. The kit as set forth in claim 18, wherein the detection reagent
contains a predetermined label such that an analyte of interest can
be confirmed by naked eye or other instrumentation from the
outside.
22. The kit as set forth in claim 18, wherein the detection reagent
contains at least one label selected from the group consisting of
catalyst, enzyme, substrate for enzyme, fluorescent compound,
chemoluminescent compound, radioactive element, metal sol,
non-metal sol, dye sol, color indicator, color matter contained in
liposome, latex particle, and immunochemical label.
23. The kit as set forth in claim 11, wherein the analyte in the
test sample is antigen, antibody or hapten and any combinations
thereof.
24. The kit as set forth in claim 23, wherein the antibody is
monoclonal or polyclonal antibody.
25. The kit as set forth in claim 18, wherein the strip further
comprises another filter pad between the reservoir pad and the
filter pad.
26. The kit as set forth in claim 11, wherein material for the
wicking membrane is at least more than one material selected from
the group consisting of nylon, polyester, cellulose, polysulfone,
polyvinylidenedifluoride, cellulose acetate, polyurethane, glass
fiber and nitrocellulose.
27. The kit as set forth in claim 11, wherein the immobilized phase
constituting a control zone is one selected from avidin, biotin,
FITC, anti-FITC antibody, mouse immunoglobulin and anti-mouse
immunoglobulin antibody.
28. The kit as set forth in claim 11, wherein the control reagent
is selected from labeled protein, antigen and antibody which
specifically bind to the immobilized phase constituting the control
zone on the wicking membrane.
Description
TECHNICAL FIELD
[0001] The present invention relates to a method and kit for
diagnosing an infection of the subject with foot-and-mouth disease
virus (FMDV), and more particularly, a method and kit for rapidly
detecting infection of the subjects with FMDV by observing a change
in appearance of a reactivity zone containing at least more than
one immobilized phase selected from antigens, antibodies or haptens
derived from FMDV, or obtainable from FMDV via an immunological
reaction, which is immunologically reactive with the test sample
from animals as a target.
BACKGROUND ART
[0002] Foot and mouth disease (FMD) is a devastating disease of
livestock and an Office International des Epizooties list A
disease. All species of cloven-hoofed animals (cattle, pigs, sheep
and goats) are susceptible and the disease is extremely contagious.
Financial losses as a result of FMD can be significant. There are
direct losses due to deaths in young animals, loss of milk, loss of
meat and a decrease in productive performance. The costs associated
with eradication or control can be high and, in addition, there are
indirect losses due to the imposition of trade restrictions.
[0003] The causative agent is FMDV, anaphthovirus of the
Picornaviridae family (Bittle et al., 1982 and Fross et al., 1984).
The FMDV genome consists of a single RNA positive strand of
approximately 8,000 nucleotide bases. The RNA is initially
translated as a single polypeptide which is subsequently cleaved by
viral-encoded proteases to produce four capsid proteins (VP1-VP4)
and non-structural polypeptides (2C, 3A, 3ABC and 3D) in infected
cells. The coding region for structural and nonstructural proteins
is shown schematically in FIG. 3.
[0004] FMD virus is antigenically heterogeneous. Seven distinct
serotypes have been recognized, O, A, C, ASIA1, SAT1, SAT2 and SAT3
(SAT=Southern African Territories). Each serotype of FMDV is
antigenically distinct from the other six serotypes. Serotype A
viruses are the most variable, having more than 30 subtypes.
Furthermore, each serotype can be subdivided into antigenically
distinct multiple subtypes. The serotypes of FMD virus were
originally identified by cross-immunity experiments in animals. An
animal recovered from infection with one serotype being resistant
to challenge by the same serotype but remaining susceptible to
infection by any other serotype. The different serotypes of FMDV
have different geological distributions. In Asia, serotypes A, O,
and Asia are most common. In Europe and South America, serotypes A,
O, and C are found. In Africa, serotypes A, O, and SAT are
prevalent. Some countries in Africa, Asia and South America are
endemic area.
[0005] Primary diagnosis of FMD commonly involves recognition of
typical clinical signs in affected animals. Clinical signs of FMD
are essentially similar in all species although the severity may
vary considerably. The principal signs are pyrexia followed by
vesicle formation in the mouth and feet resulting in salivation and
lameness. Serological diagnosis is determined by the presence of
FMDV-specific antigens or antibodies in the suspected animals and
can be usually performed by ELISA and Virus neutralization
test.
[0006] After animals have been infected with FMDV, specific
antibodies against structural proteins (SPs) and non-structural
proteins (NSPs) begin to appear and titers increase and remain
long. Thus, the presence of specific FMD virus antibody in a serum
indicates that the animal from which the sample was collected has
had contact with FMD virus or antigen.
[0007] The detection of antibody to FMD virus in serum has several
usefulness. The antibody detection evidences previous infection in
animals from which vesicular material is not available. Diagnosis
of FMD by clinical signs may be difficult, especially for sheep and
goats, in which clinical signs are often mild (Barnett, P. V et
al., 1999 and Callens, M., K. et al., 1998). Furthermore, several
other vesicular virus infections, including those caused by swine
vesicular disease (SVD) virus, vesicular stomatitis virus, and
others, cannot be distinguished from FMDV infection by the clinical
findings. FMDV can establish a persistent or carrier stage in
ruminants and they show no signs of FMD. Such carrier animals can
become the source of new outbreaks of the disease. Because of these
problems, a rapid serological method is needed to identify infected
and/or asymptomatic carrier animals and distinguish them from
vaccinated animals. This antibody detection method also can be used
in epidemiological surveys and to measure the effectiveness of
vaccination.
[0008] Both vaccination and infection induce antibodies to the
structural capsid proteins. Therefore, if the capsid protein alone
used in the diagnostic assays, it will detect both vaccinated and
infected animal based on the detection of antibody to structural
protein. For this reason, the antibody test to structural protein
can be used only in vaccine-free region, such as the USA or the UK,
but not in regions where vaccination practice is established. But
even in areas where animals are vaccinated, the FMD occurs
frequenctly. In such a region, diagnostic tests that can
differentiate the infection from the vaccination are required.
Several studies reported that using nonstructural proteins of FMDV
such as 2C and 3ABC, animals which have been infected with FMDV
could be differentiated from the vaccinated animals on the basis of
the detection of antibody to one or more non-structural proteins of
the virus (Rodriguez A et al, Mackey D k et al., Sorensen K J et
al.).
[0009] Another method to detect FMDV is PCR (polymerase chain
reaction). To detect specific RNA sequence from FMDV, RT-PCR
(reverse transcription-polymerase chain reaction) assay also has
been developed (Munez et al). This technique can provide specific
and highly sensitive, and so it can detect FMD viral RNA in poorly
preserved samples when insufficient virus is present to initiate
infection in tissue culture. But this method requires equipments
for PCR and electricity, which make it impractical in field assay,
and if inhibitory substances to PCR reaction are present in some
samples, some samples that contain virus or viral genome will not
give a positive result by PCR.
DISCLOSURE OF THE INVENTION
[0010] Therefore, the present invention has been made in view of
the above problems, and it is an object of the present invention to
provide a method for diagnosing infection of the subject animal
with foot-and-mouth disease virus, making it possible to simply and
rapidly identify whether the animal is infected with that virus or
not, upon using biological samples obtained from the animal.
[0011] It is another object of the present invention to provide a
diagnostic kit for realizing the above-mentioned method.
[0012] In accordance with the present invention, the above and
other objects can be accomplished by the provision of a method for
diagnosing foot-and-mouth disease virus infection, comprising the
steps of:
[0013] (1) applying a predetermined amount of a test sample to a
loading region of a strip; (2) coupling a detection reagent
including a given labeling reagent to an analyte of interest in the
test sample to form a complex therebetween; (3) developing the
complex onto a wicking membrane; and (4) observing changes in
appearance of a reactivity zone having at least more than one
immobilized phase selected from antigen, antibody or hapten on the
predetermined region of the wicking membrane, derived from FMDV or
obtainable from FMDV through an immunological reaction to determine
the presence or absence of foot-and-mouth disease virus
infection.
[0014] The diagnostic method according to the present invention
includes a sandwich assay or a competition assay.
[0015] As a diagnostic kit in order to realize the diagnostic
method as described above, the present invention provides a kit for
diagnosing foot-and-mouth disease virus infection comprising:
[0016] a strip 1 including a reactivity zone 13 containing at least
more than one immobilized phase selected from antigen, antibody or
hapten thereon, derived from FMDV or obtainable from FMDV through
an immunological reaction, and a control zone 14 for confirming
normal operation of the kit, provided on a predetermined region of
a wicking membrane 9; and
[0017] a housing 20 protecting the strip 1 from a variety of
contaminants, and including at least a test sample application port
2 and an indicia window 4 for observing results of reaction in the
reactivity zone 13 and the control zone 14 on the strip 1.
[0018] The test sample is preferably a body fluid which is secreted
out of the body and includes blood, serum, plasma, urine, tears,
saliva, milk, etc.
[0019] Further, the analyte of interest which is contained in the
test sample to be analyzed may include any substances containing
specific-binding members which may be naturally formed or
artificially imparted, including antigen-presenting substances,
antibodies (including monoclonal and polyclonal antibody), haptens,
and combinations thereof, for example.
[0020] In addition, an immobilized phase (or, capture reagent) is
an unlabeled specific bonding member which specifically binds to
the analyte, an indicator reagent, an auxiliary specific-binding
member, or the like and then captures the analyte, and is
immobilized directly or indirectly on the wicking membrane 9 of the
strip 1.
[0021] The detection reagent may bind diffusibly or non-diffusibly
to a pad and includes a labeled reagent, the auxiliary
specific-binding member and/or a component of a signal generating
system. The signal generating system includes at least a catalytic
member and solute. The solute may be catalyzed by the catalytic
member to induce a reaction, and generates a signal recognizable
from membrane surface or inside. The catalytic member may be enzyme
or non-enzyme. The solute may carry out a reaction which is
catalyzed by the catalytic member. Such a reaction produces a large
amount of signal-generating compound which may be directly or
indirectly detectable. The signal detectable by these components
includes spectrophotometric, visible signal, electrochemical
signal, and other electrically detectable signals.
BEST MODE FOR CARRYING OUT THE INVENTION
[0022] Now, the present invention will be described in detail with
reference to the accompanying drawings.
[0023] FIGS. 1a-b are, respectively, separate perspective views of
exemplified diagnostic kits as a preferred embodiment used in the
present invention.
[0024] The diagnostic kit includes a strip 1 and a housing 20. The
housing is required for spotting a test sample from a test sample
application port 2 and a developing reagent application port 3 on a
filter pad (or a dye pad: it refers to a pad containing a detection
reagent). The kit also includes a main body 7 having a cover 5
comprising an indicia widow 4 for showing test results, and a
strip-mounting member 6 for placing and fixing the strip 1 in place
therein.
[0025] The cover 5 and main body 7 are interconnected via a
fastening member 8. They are required for fixing the strip 1, and
for preventing contact with a reactivity zone or contamination
thereof, and are preferably made of a non-reactive material which
does not react with any other reagents used in the test, such as
plastics or the like.
[0026] As shown in a configuration of FIG. 1a in accordance with
the first aspect of the present invention, where the test sample is
spotted through the application port 2 on the filter pad 11 (also
serving as a dye pad), a separate developing reagent application
port 3 is required. The developing reagent application port 3 is
configured to have a curvature in a cup-like shape so as to receive
a predetermined amount of the developing reagent. Further, the
lower part thereof is preferably in close contact with a reservoir
pad 10 so as to prevent the developing reagent from flowing out
into other regions of the test device.
[0027] Differently from the configuration in FIG. 1a, where a test
sample application port 2' is positioned over an immediately upper
part of a reservoir pad 10' constituting the strip so as to
immediately apply the test sample to the reservoir pad 10' (in this
case, the reservoir pad also has a function of the filter pad as in
following example below, thereby no separate developing reagent
application port is required), as shown in a configuration of FIG.
1b in accordance with the second aspect of the present invention,
the application port 2' is configured to have a curvature in a
cup-like shape so as to receive a predetermined amount of the test
sample. Further, the lower part thereof is preferably in close
contact with a reservoir pad so as to prevent the test sample from
flowing out into other regions of the test device. In this case, a
pad 11' is a dye pad containing the detection reagent, or a second
filter pad, or may be eliminated.
[0028] An indicia window 4 is designed for externally observing
changes occurring in a reactivity zone 13 and a control zone 14 on
the wicking membrane 9 constituting the strip 1, and is provided on
the housing cover 5 so as to be positioned immediately over the
reactivity zone 13 and control zone 14.
[0029] Further, the housing cover 5 may be provided with given
discrimination symbols, for example `Date` for test date, `It 0000`
for the subject, `C` for the control zone, `T` (Test) for the
reactivity zone, `S` (Sample) for the test sample application port,
`D` for the developing reagent, and the like, such that the test
date, subject, test sample application port, developing reagent
application port, indicia window for showing test result, etc. may
be easily distinguished. Those symbols may be any letter, number,
icon, or the like and any combinations thereof different from the
foregoing.
[0030] Now, as the preferred embodiment, a structure of a strip
constituting the diagnostic device in accordance with the present
invention will be described with reference to FIGS. 1a-b and FIG.
2.
[0031] FIG. 1a shows one embodiment in accordance with a first
aspect of the present invention. The inventive strip 1 includes a
wicking membrane 9, a reservoir pad 10, a filter pad 11 (also,
serving as a dye pad), an absorbent pad 12, and a reactivity zone
13 and a control zone 14 provided on the wicking membrane 9. To the
back surface of the strip 1 is attached a base member 15 for fixing
the strip 1 on a mounting member 6 of the main housing body 7. The
base member 15 is preferably made of plastic or a glass plate.
[0032] FIG. 1b shows another embodiment in accordance with the
second aspect of the present invention. The inventive strip 1' has
the same configuration as in the strip shown in FIG. 1a, except for
a reservoir pad 10', and a filter pad 11' (which may also serve as
a dye pad). The detailed description of respective configuration
thereof will be based on the first aspect of the present invention
shown in FIG. 1a, but only the difference therebetween will be
mentioned.
[0033] A filter pad 11 is in contact with a back surface of a
wicking membrane 9 for chromatography to form a connection passage
for fluid flow into the wicking membrane 9. The back surface of the
filter pad 11 is in contact with the reservoir pad 10 to form one
connection passage for fluid flow therebetween. The absorbent pad
12 is attached to the upper part of the wicking membrane 9. On the
predetermined region of the wicking membrane 9 are spaced apart a
reactivity zone 13 containing at least more than one immobilized
phase which specifically binds to an analyte to be detected, a
labeled reagent, an auxiliary specific-binding member, or the like,
and a control zone 14 for determining whether the kit is normally
operating.
[0034] The reservoir pad 10 absorbs the test sample, or a solution
necessary for other tests, for example, a developing reagent, and
the like and includes a capillary membrane to transfer analyte to
the filter pad or wicking membrane. The reservoir pad 10 is
required to have voids and volume sufficient to receive the test
sample or developing reagent. Material suitable for the reservoir
pad is preferably low molecular weight protein binding substances,
including cellulose, polyester, polyurethane, glass fiber having a
pore size of 0.45 to 60 .mu.m, etc.
[0035] The filter pad 11 filters unnecessary components in the test
sample and may contain a detection reagent (in this case, the
filter pad may also function as a dye pad). Where the detection
reagent is contained in the filter pad, there is an advantage of
eliminating the step of premixing the detection reagent with the
test sample for the test. As material suitable for the filter pad
11, there may be mentioned polyester, polyurethane, polyacetate,
cellulose, glass fiber, nylon having a pore size of 0.45 to 60
.mu.m, etc. Where appropriate, the reservoir pad 10 and filter pad
11 may be made of the same material, and in this case, the
detection reagent may be contained within the bottom of one long
filter pad 11.
[0036] The detection reagent is provided with a labeled reagent,
auxiliary specific-binding member, and/or a constitutional
component of a signal generating system, which enable it to
identify the presence of analyte of interest by naked eye or other
instrumentation from the outside. Labeled detection reagents are
well known to those skilled in the art. Examples of such labels
include catalysts, enzymes (for example, phosphatase, peroxydase,
etc., and more specifically, alkaline phosphatase and horseradish
peroxidase, or the like, which is used in combination with a
substrate for an enzyme), substrate for enzyme (for example,
nitrobluetetrazolium, 3,5',5,5' tetranitrobenzidine,
4-methoxy-1-naphthol, 4-chloro-1-naphthol,
5-bromo-4-chloro-3-indolylphosphate, chemoluminescent substrates
for enzymes, for example, dioxethane, and derivatives and analogs
thereof), fluorescent compounds (for example, fluorescein,
phycobiliprotein, rhodamine, derivatives and analogs thereof),
chemoluminescent compounds, radioactive elements, and the like. In
addition to those, metal sol, dye sol, particulate latex, color
indicator, color matter contained in liposome, carbon sol and
non-metal sol such as selenium may be mentioned as disclosed in
U.S. Pat. No. 5,728,587 as well. Further, this patent, from columns
8-10, discloses a large number of immunochemical labels as labels
usable in the diagnostic method of the present invention.
[0037] The above-mentioned labeling reagents may form a conjugate
with a given auxiliary specific-binding member having a property of
easily binding to an analyte of interest. An auxiliary
specific-binding member is not particularly limited and includes
antigen, antibody, hapten, or the like, for example, protein G,
protein A, protein G/A, known as material binding well to an
antibody in case the analyte is antibody and various antibodies
known as binding well to other antibodies IgG and IgM. These
materials are presently commercially available as recombinants from
Sigma, etc.
[0038] From the foregoing, the detection reagent needs not
necessarily be included in the filter pad 11. The detection reagent
may be provided at any point between the reactivity zone 13 on the
wicking membrane showing a detection result and the test sample
application port. This detection reagent may be applied to upper or
inside of any point of the filter pad 11 or wicking membrane 9, in
the dried or freeze-dried state.
[0039] Then, if desired, in order to enhance sensitivity of the
test, reactivity, etc., a variety of auxiliary agents may be added,
such as buffer, detergent, anti-coagulating stock solution, or the
like, for example. In addition, the strip 1 may further include a
given control reagent to determine whether the kit is normally
operating or not. Similar to the detection reagent, the control
reagent may also be provided at any point between the filter pad 11
or the reactivity zone 13 on the wicking membrane 9 and the test
sample application port. The control reagent may be selected from
labeled protein, antigen, antibody, and the like which specifically
bind to an immobilized phase (for example, protein, antigen,
antibody, or the like) forming a control zone (or a control band)
on the wick membrane 9. These immobilized phase and control reagent
are well known to those skilled in the art. As the labeling reagent
which may be included in the control reagent, those as described in
the detection reagent may be applied. The auxiliary
specific-binding member is not particularly limited and includes
one species selected from avidin, biotin, FITC, anti-FITC mouse
antibody, mouse immunoglobulin, or anti-mouse immunoglobulin
antibody, for example.
[0040] The wicking membrane 9 should have sufficient voids, and be
able to absorb substantial portions of test sample which has passed
through the filter pad 11. As an example of material suitable for
such a wicking membrane, there may be mentioned at least more than
one material selected from nylon, polyester, cellulose,
polysulfone, polyvinylidene difluoride, cellulose acetate,
polyurethane, glass fiber, nitrocellulose, or the like.
[0041] Where a developing reagent is employed, an example of
material suitable for the developing reagent may include phosphate
buffer, saline, Tris-HCl, water, etc. The developing reagent is
required where the test sample application port 2 is positioned
over the immediately upper part of the filter pad 11 so as to spot
the test sample. Therefore, as in the embodiment in accordance with
the second aspect of the FIG. 1b, the developing reagent is not
particularly required where the test sample application port 2' is
positioned on the reservoir pad 10' so as to load a predetermined
amount of the test sample thereon.
[0042] Where the complex labeled with the detection reagent
contains the analyte to be detected, it binds to the immobilized
phase located on the reactivity zone 13 of the wicking membrane 9
and then results in externally discernable change. Material which
may be used as such an immobilized phase may include at least more
than one selected from antigen, antibody or hapten, which
constitutes foot-and-mouse disease virus, or may be derived
therefrom through an immunological reaction.
[0043] Material which may be used as the antigen includes
non-structural and/or structural proteins. Structural proteins
include inactivated FMDV disrupted material or constituents
thereof, VP1-VP4 polypeptide. Non-structural proteins may include
at least more than one polypeptide selected from leader peptide
(Lb), 2B, 2C, 3A, 3D, 3AB and 3ABC. FIG. 3 shows a map of a
polyprotein precursor comprising the structural and non-structural
proteins.
[0044] The structural proteins with the same name may also exhibit
some difference in constitutional amino acids among them, depending
on serological classification of FMDV. Preferably, the structural
proteins which may be used in the present invention are not
particularly limited, so long as they may specifically react with
antibodies formed against all the sero-types. An example of these
structural proteins includes, but is not limited to, VP1
represented by SEQ ID NO: 118.
[0045] However, even when the above-described structural protein
alone is used as an immobilized phase for constructing the
diagnostic kit, there is a case in which it is possible to
precisely diagnose whether or not cattle are infected with
foot-and-mouth disease virus, for non-vaccinated cattle, but it is
difficult to distinguish infected cattle from vaccinated cattle. An
example of antigen employed in the vaccine production includes, in
addition to the structural protein, a non-structural protein 3D.
Thus, even when the non-structural protein 3D is used as an
immobilized phase, the same limitations as described above exist.
An example of the non-structural protein 3D is shown in SEQ ID NO:
121.
[0046] The non-structural proteins (except for 3D) are proteins
that have not been used in conventional vaccine production and an
antibody to those proteins is observable only in a virus-infected
animal. Thus, when this protein is applied as an immobilized phase,
it will be possible to make an exact diagnosis for the infected
animal. Of course, to such a non-structural protein also has some
difference in constituent amino acids thereof, depending on
respective sero-type of FMDV. Usable non-structural proteins are
not particularly limited, as long as preferably, they may
specifically react with antibodies produced against all sero-types.
Examples of these structural proteins include 2C represented by SEQ
ID NO: 119 and 3ABC represented by SEQ ID NO: 120.
[0047] FIG. 4 shows an example to which the non-structural protein
was applied as a single immobilized phase. T represents a
reactivity zone in which the non-structural protein (2C or 3ABC)
was immobilized, which has never been used in vaccine production up
to now. C represents a control zone. The kit having both the
discolored T and C (FIG. 4a) represents the infected animal,
whereas the kit having only the discolored C (FIG. 4b) represents a
negative animal prior to vaccination or a vaccinated animal. If C
was not discolored in any case, it means that the kit of interest
was not normally operated with the result thus obtained being
unreliable.
[0048] Where only the non-structural protein as described above is
employed as an immobilized phase, it is difficult to distinguish
between the negative animal prior to vaccination and the vaccinated
animal, in any case. Therefore, the most preferred embodiment of
the present invention provides a diagnostic method and kit which
make it possible to distinguish an FMDV-infected animal as well as
a vaccinated animal. For this purpose, it is preferable to have a
first reactivity zone in which as an immobilized phase, an antigen
usable in vaccine production known until now was immobilized at a
particular site on a wicking membrane, and a second reactivity zone
in which as an immobilized phase, a non-structural protein that was
known as never used before in vaccine production was spaced and
immobilized at a particular site different from the first
reactivity zone on the wicking membrane. Therefore, it is possible
to diagnose whether animal was vaccinated, virus-infected or
negative prior to vaccination, through change in appearance
produced from a binding reaction between these first and second
reactivity zones and a labeled complex.
[0049] The immobilized phase bonding to a control reagent forms a
control zone at a different site spaced from the reactivity zones.
As such an immobilized phase, various reagents and immobilized
phases which are applied in other commercially available diagnostic
kits may be used. Details on that will be described in examples as
follows.
[0050] Now, a method for diagnosing FMDV infection using the kit
having a configuration as described above will be described with
reference to preferred embodiments. A test sample such as animal
serum (or plasma, whole blood) is spotted on the test sample
application port 2 formed on the housing cover. The filter pad 10
constituting the strip is positioned at the lower end of the
application port 2. The filter pad 10 also contains a protein
G-gold conjugate as a detection reagent. The protein G-gold
conjugate can form complexes with all the antibodies present in the
test sample. A predetermined amount of a developing reagent is
loaded on the developing reagent application port 3 in which the
reservoir pad 10 constituting the strip is positioned on the lower
end thereof. Application of the developing reagent results in
formation of a complex between the labeled conjugate and an
antibody in the test sample. Then, this complex is chromatographed
along the longitudinal axis of the wicking membrane 9 (preferably,
nitrocellulose membrane). FMDV recombinant antigen (construction
thereof will be described in detail with reference to the following
examples) was previously applied and immobilized on the reactivity
zone 13 of the wicking membrane 9, and thus if the complex contains
a specific antibody to the recombinant antigen, it will undergo
reaction and then show discoloration in the form of a red line.
[0051] If the recombinant antigen includes both the structural and
non-structural proteins, there is described for example, a method
to diagnose whether the animal was vaccinated, virus-infected or
negative prior to vaccination.
[0052] FIG. 5 shows this example. T1 represents a line on which an
antigen (structural protein or non-structural protein 3D) which had
been introduced in vaccine production up to now was immobilized. T2
represents a line on which a non-structural protein (2C or 3ABC)
which has never been used before in vaccine production was
immobilized. C represents a control line. First, the kit (FIG. 5a)
in which all the T1, T2 and C were discolored represents a
virus-infected animal. Secondly, a kit (FIG. 5b) in which only the
T1 and C were discolored represents a vaccinated animal. Finally, a
kit (FIG. 5c) means a negative animal prior to vaccination, as only
the C was discolored. If C was not discolored in any case, it means
that the kit was not normally operated with the result thus
obtained being unreliable.
[0053] As described above, the diagnostic device usable in the
present invention can be made of various configurations and
modifications as disclosed in U.S. Pat. No. 5,728,587, and the
particular disclosure of the strip construction (i.e., arrangement
of the pads) included in this separate device does not constitute
the essence of the present invention. Thus, the arrangement of the
pad is not limited to those described above, and other arrangement
such as a reservoir pad/a first filter pad/a second filter pad/a
wicking membrane/an absorbent pad may be considered. In this case,
the first filter pad or the second filter pad serves to filter and
separate blood cells from blood, or filter and remove foreign
materials unnecessary for sample test.
[0054] Although the present invention was conveniently explained by
way of example of structural and non-structural proteins
constituting foot-and-mouth disease virus as an immobilized phase
contained in the reactivity zone, the scope and sprit of the
present invention should be construed to encompass any material
which had been already supplied as antigen for vaccine production
at the time of filing the present invention or would be supplied as
antigen in the near future and a certain material capable of
inducing an antibody in vivo (including hapten), or various
antibodies obtainable from FMDV through an immunological
reaction.
BRIEF DESCRIPTION OF THE DRAWINGS
[0055] FIG. 1a shows a separate perspective view of a device (a
rapid kit) for diagnosing infection of the subject animal with
foot-and-mouth disease virus according to a first aspect of the
present invention.
[0056] FIG. 1b shows a separate perspective view of a device (a
rapid kit) for diagnosing infection of the subject animal with
foot-and-mouth disease virus according to a second aspect of the
present invention;
[0057] FIG. 2 shows a configuration of a strip constituting a
diagnostic device according to the present invention;
[0058] FIG. 3 shows a structure of a polyprotein expressed from
foot-and-mouth disease virus;
[0059] FIG. 4 shows an exemplified test result for the one-line
test kit;
[0060] FIG. 5 shows an exemplified test result for the two-line
test kit;
[0061] FIG. 6 shows a map of plasmid pBM-VPITw97F;
[0062] FIG. 7 shows a map of plasmid pBM-2CTw97F;
[0063] FIG. 8 shows a map of plasmid pBM-3ABCTw97F; and
[0064] FIG. 9 shows a map of plasmid pBM-3DTw97F.
EXAMPLES
[0065] Now, the present invention will be described in more detail
with reference to following examples. These examples are provided
only for illustrating the present invention and should not be
construed as limiting the scope and spirit of the present
invention.
[0066] Materials
[0067] Oligonucleotides for gene construction and sequencing were
synthesized at ResGen (Huntsville, Ala.). Unless otherwise
indicated, DNA sequencing was also performed at ResGen.
[0068] For polymerase chain reaction (PCR), Vent DNA polymerase and
buffer were purchased from New England Biolabs, Inc. (Beverly,
Mass.) and a mixture of dNTPs was purchased from Amersham-Pharmacia
(Piscataway, N.J.) and used according to the manufacturer's
specifications unless otherwise indicated. PCR amplifications were
performed on a GeneAmp 2400 thermal cycler from Perkin-Elmer
Corporation (Foster City, Calif.). The PCR product was purified
using Qiagen PCR spin column (Qiagen Inc., Chatsworth, Calif.) as
recommended by the manufacturer. Unless indicated otherwise,
restriction enzymes were purchased from New England BioLabs, and
DNA fragments were isolated on agarose (Sigma-Aldrich) gels,
treated with restriction enzymes and then used for cloning.
[0069] Desired fragment was excised and the DNA was extracted with
a QIAEX II gel extraction kit as recommended by the manufacturer.
DNA was resuspended in H.sub.2O or TE (1 mM
ethylenediaminetetraacetic acid (EDTA; pH 8.0; Sigma-Aldrich), 10
mM tris(hydroxymethyl)aminomethane-hydrochloride (Tris-HCl; pH 8.0;
Sigma-Aldrich)). Ligations were performed using DNA ligase
(Boehringer Mannheim Corporation, Indianapolis, Ind.) as
recommended by the manufacturer. Ligation reaction was incubated at
16.degree. C. overnight. Bacterial transformations were performed
using E. coli XL1-Blue competent cells. Unless indicated otherwise,
transformations and bacterial restreaks were plated on LB agar
(Lennox) plates supplemented with 100 ug/ml ampicillin. All
bacterial incubations (plates and liquid cultures) were conducted
overnight (16 hours) at 37.degree. C.
[0070] Screening of transformants to identify desired clones was
accomplished by restriction enzyme digestion of miniprep DNA and/or
by colony PCR. Miniprep DNA was prepared according to Molecular
Cloning: A Laboratory Manual, unless otherwise indicated. Colonies
containing desired clones were propagated from the transfer plate
or stocked in glycerol at -70.degree. C.
[0071] Antigen Production
Example 1
[0072] Preparation of Recombinant FMDV VP1 Antigen
[0073] A. Construction of FMDV VP1 Expression Vectors
[0074] (i) Construction of Synthetic VP1 Gene
[0075] VP1 protein of Foot and Mouth Disease virus Taiwan Type O 97
sequence was retrieved from NCBI GenBank data and oligonucleotides
for syntheictc gene were synthesized at ResGen (Huntsville, Ala.).
In the synthetic oligonucleotides, the native FMDV codons were
altered to conform to E. coli codon bias in an effort to increase
expression levels of the recombinant protein in E. coli. See, for
example, M. Gouy and C. Gautier, Nucleic Acids Research 10:7055
(1982); H. Grosjean and W. Fiers, Gene 18:199 (1982); J. Watson et
al. (eds.), Molecular Biology of the Gene, 4th Ed., Benjamin
Kumming Publishing Co., pp. 440 (1987). The recursive PCR method
was used to assemble the oligonucleotides into full VP1 gene. The
gene construction strategy involved synthesis of a series of
overlapping oligonucleotides with complementary ends. When
annealed, the ends served as primers for the extension of the
complementary strand. The fragments then were amplified by
excessive outside primers.
[0076] Oligonucleotide was designed to contain a BamHI restriction
site for cloning into the expression vector pGEX-4T-1.
[0077] Reverse oligonucleotide contains a translation stop codon
(TAA) and EcoRI restriction site. When external primer TW97-1 (SEQ
ID NO: 1) and TW97-16 (SEQ ID NO: 16) were used, whole VP1 (213
amino acids) gene was synthesized (SEQ ID NO: 118).
[0078] These steps for recursive PCR are detailed hereinbelow.
[0079] PCR reaction (100 ul volume) was set up as follows:
[0080] Vent DNA polymerase (1U) and 1.times. buffer, along with 25
uM of each dNTP (dATP, dCTP, dGTP, and dTTP), 50 pmol each of
oligonucleotides TW97-1 (SEQ ID NO: 1) and TW97-16 (SEQ ID NO: 1),
and 0.25 pmol each of oligonucleotides TW97-2 (SEQ ID NO: 2)
through TW97-15 (SEQ ID NO: 15).
[0081] The reaction was incubated at 95.degree. C. for 5 minutes,
and then amplified with 30 cycles of 95.degree. C. for 15 seconds,
58 CC for 15 seconds and 72.degree. C. for 60 seconds, followed by
incubation at 72.degree. C. for 5 minutes. PCR-derived product was
purified using Qiagen PCR spin column.
[0082] (ii) Cloning of the PCR Product.
[0083] The PCR product amplified as described hereinabove was
digested with the restriction endonucleases Bam HI+Eco RI and
ligated into the vector pGEX-4T-1 that had been digested with Bam
HI+Eco RI and gel-isolated. The ligation product was used to
transform XL-1 Blue competent cells. The transformed cells were
plated on LB plates supplemented with 100 ug/ml ampicillin.
Miniprep DNAs were prepared from overnight cultures of colonies and
digested with Bam HI+Eco RI to screen the desired clones. The clone
with right insert was designated as pBM-VPITw97F (FIG. 6).
[0084] The pBM-VPITw97F clone was sequenced with the
oligonucleotide primers pGEX5 (SEQ ID NO: 116) and pGEX3 (SEQ ID
NO: 117).
[0085] B. Growth and Induction of E. coli Strains with VP1
Plasmids
[0086] Overnight seed cultures of each E. coli clones were prepared
in 500 ml sterile LB supplemented with 100 ug/ml ampicillin, and
placed in a shaking orbital incubator at 37.degree. C.
[0087] 50 ml inoculums from seed cultures were transferred
[0088] to flasks containing 0.5 liter sterile LB supplemented with
100 ug/ml ampicillin. Cultures were incubated at 37.degree. C.
until the cultures reached mid-logarithmic growth and then induced
with 1 mM ITPG (isopropylthiogalactoside) for 3 hours at 37.degree.
C. After the induction period, cells were pelleted by
centrifugation and harvested following standard procedures.
Pelleted cells were stored at -70.degree. C. until further
processed.
[0089] C. Preparation of VP1 Antigen
[0090] Frozen cells obtained from Example B were resuspended in PBS
with 1 mM PMSF.
[0091] The cells were disrupted by ultrasonication (Branson).
Inclusion bodies were separated from soluble proteins by
centrifugation. Pelletized inclusion bodies were washed
sequentially in (1) PBS; and (2) water. The washed inclusion bodies
were resuspended in a solution of PBS and 5 M urea with brief
sonication. Once again, the centrifugally pelleted inclusion bodies
were fully solubilized in 7M guanidine-HCl. The solubilized
recombinant antigens were clarified by centrifugation, and passed
through a 0.2 um filter.
[0092] Guanidine-HCl solubilized fusion protein was denatured by
diluting in water and the denatured protein was precipitated by
centrifugation. The pellet was washed with water and suspended in
water. 2M NaOH solution was added to solubilize the denatured
protein completely and then was added to neutralize the pH of
protein solution.
Example 2
[0093] Preparation of Recombinant FMDV 2C Antigen
[0094] A. Construction of FMDV 2C Expression Vector
[0095] The genome sequence of FMDV 2C protein was retrieved from
NCBI GenBank data (GI: 5921457, O strain Chu-Pei) and
oligonucleotides for the synthesis of whole 2C gene and sequencing
were synthesized at ResGen (Huntsville, Ala.). The coding DNA
sequence is 954 nucleotides long, which encodes 318 amino acids
(SEQ ID NO: 119).
[0096] (i) Construction of Synthetic Full-Length 2C Gene
[0097] To obtain the 2C gene of FMD virus, 24 oligonucleotide
primers were synthesized, each with complementary ends, at
Resgen.
[0098] We used the recursive PCR method to assemble the
oligonucleotides into full 2C gene. The gene construction strategy
involved synthesis of a series of overlapping oligonucleotides with
complementary ends. When annealed, the ends served as primers for
the extension of the complementary strand. The fragments then were
amplified by excessive outside primers. Because of the large size
of 2C gene to be synthesized, the oligonucleotides were divided
into three groups and respective recursive PCRs were performed. The
produced DNAs were designated as A, B and C fragment. B and C
fragment were joined with PCR and then the B-C fragment was joined
with A fragment to produce full 2C gene. One of the
oligonucleotides was designed to contain a BamHI restriction site
for cloning into the expression vector pGEX-4T-1.
[0099] The reaction was incubated at 95.degree. C. for 5 minutes,
and then amplified with 35 cycles of 95.degree. C. for 30 seconds,
53.degree. C. for 30 seconds and 73.degree. C. for 100 seconds,
followed by incubation at 73.degree. C. for 5 minutes. Aliquot of
the reaction mixture was analyzed by electrophoresis on agarose
mini-gel.
[0100] (ii) Cloning of the PCR Product.
[0101] The PCR product amplified as described herein above was
digested with the restriction endonucleases Bam HI+Hind III and
ligated into the vector pGEX-4T-1 that had been digested with Bam
HI+Hind III previously. The ligation product was used to transform
E. coli XL-1 Blue competent cells. The transformed cells were
plated on LB plates supplemented with 100 ug/ml ampicillin.
Miniprep DNAs were prepared from overnight cultures of transformed
colonies using QIAprep plasmid DNA mini-preparation kit and
digested with Bam HI+Hind III to screen the desired clones. The
clone with right insert was designated as pGEX-2CTw97F (FIG.
7).
[0102] The pGEX-2CTw97F clone was sequenced with the
oligonucleotide primers pGEX5 (SEQ ID NO: 116), pGEX3 (SEQ ID NO:
117), 2C-25 (SEQ ID NO: 41) and 2C-26 (SEQ ID NO: 42).
[0103] B. Growth and Induction of E. coli Strains with 2C
Plasmid
[0104] Overnight seed cultures of pGEX-2CTw97F were prepared in 500
ml sterile LB supplemented with 100 ug/ml ampicillin, and placed in
a shaking orbital incubator at 37.degree. C. 50 ml inoculum from
seed cultures was transferred to flask containing 0.5 liter sterile
LB supplemented with 100 ug/ml ampicillin. Cultures were incubated
at 37.degree. C. until it reached mid-logarithmic growth and then
induced with 1 mM ITPG (isopropylthiogalactoside) for 3 hours at
37.degree. C. After the induction period, cells were pelleted by
centrifugation and harvested following standard procedures.
Pelleted cells were stored at -70.degree. C. until further
process.
[0105] C. Preparation of FMDV 2C Antigen
[0106] Frozen cells obtained from Example 2B were resuspended in
PBS with 1 mM PMSF and Triton X-100 detergent, and then disrupted
by ultrasonication (Branson). Inclusion bodies were separated from
soluble proteins by centrifugation. Protein fraction enriched with
2C was obtained through 3-4 rounds of washing off the contaminants
and solubilization of cell lysate pellet in urea or Guanidin-HCl.
Recombinant 2C was purified through size exclusion chromatography
(FPLC, Sephacryl S 200 HR) under denaturing condition (5N GuHCl,
PBS (pH7.4)) and eluted fraction containing 2C was identified by
SDS-PAGE and dialyzed against 20 mM phosphate buffer (pH 9.0).
Protein solution was stored refrigerated after adding sodium azide
to 0.05%. For longer storage (over 1 month), protein solution was
aliquoted and frozen at -70.degree. C.
Example 3
[0107] Preparation of Recombinant FMDV 3ABC Antigen
[0108] A. Construction of FMDV 3ABC Expression Vector
[0109] The genome sequence of FMDV 3ABC protein was retrieved from
NCBI GenBank data (GI: 5921457, O strain Chu-Pei) and
oligonucleotides for the synthesis of whole 3ABC gene and
sequencing were synthesized at ResGen (Huntsville, Ala.). The
coding DNA sequence is 1281 nucleotides long, which encodes 427
amino acids (SEQ ID NO: 120).
[0110] (i) Construction of Synthetic Full-Length 3ABC Genes
[0111] To obtain the 3ABC gene of FMD virus, 33 oligonucleotide
primers were synthesized, each with complementary ends, at
Resgen.
[0112] We used the recursive PCR method to assemble the
oligonucleotides into full 3ABC gene. The gene construction
strategy involved synthesis of a series of overlapping
oligonucleotides with complementary ends. When annealed, the ends
served as primers for the extension of the complementary strand.
The fragments then were amplified by excessive outside primers.
[0113] Because of the large size of 3ABC gene to be synthesized,
the oligonucleotides were divided into four groups and respective
recursive PCRs were performed. The produced DNAs were designated as
A, B, C and D fragment. A and B fragment were joined and C and D
fragment were joined through PCR. Then A-B fragment was joined with
C-D fragment to produce full 3ABC gene.
[0114] One of the oligonucleotide was designed to contain a BamHI
restriction site for cloning into the expression vector pGEX-4T-1.
The anti-sense oligonucleotide contains a translational termination
codon (TAA) and an EcoRI restriction site. When N- and C-terminal
primers, 3ABC-1 (SEQ ID NO: 43) and 3ABC-33 (SEQ ID NO: 75), were
used, a full-length 3ABC (427 amino acids) gene was
synthesized.
[0115] PCR reaction (100 ul volume) was set up as follows:
[0116] Vent DNA polymerase (1U) and 1.times. buffer, along with 25
uM of each dNTP (dATP, dCTP, dGTP, and dTTP), 4 ul 100 mM
MgSO.sub.4 and 100 pmol of each oligonucleotide. The template was
mixture of A-B fragment and C-D fragment described above.
[0117] The reaction was incubated at 95.degree. C. for 5 minutes,
and then amplified with 35 cycles of 95.degree. C. for 30 seconds,
60.degree. C. for 30 seconds and 73.degree. C. for 120 seconds,
followed by incubation at 73.degree. C. for 5 minutes. PCR-derived
product was run on the agarose gel and the DNA band was excised and
eluted from the gel using Quigen gel extraction kit.
[0118] (ii) Cloning of the PCR Product.
[0119] The PCR product amplified as described herein above was
digested with the restriction endonucleases Bam HI+Hind III and
ligated into the vector pGEX-4T-1 that had been digested with Bam
HI+Hind III previously. The ligation product was used to transform
E. coli XL-1 Blue competent cells. The transformed cells were
plated on LB plates supplemented with 100 ug/ml ampicillin.
Miniprep DNAs were prepared from overnight cultures of transformed
colonies using QIAprep plasmid DNA mini-preparation kit and
digested with Bam HI+Hind III to screen the desired clones. The
clone with right insert was designated as pBM-3ABCTw97F (FIG.
8).
[0120] The pBM-3ABCTw97F clone was sequenced with the
oligonucleotide primers pGEX5 (SEQ ID NO: 116), pGEX3 (SEQ ID NO:
117), 3ABC-36 (SEQ ID NO: 78) and 3ABC-37 (SEQ ID NO: 79).
[0121] B. Growth and Induction of E. coli Strains with E3ABC
Plasmid.
[0122] Overnight seed cultures of pGEX-3ABCTw97F were prepared in
500 ml sterile LB supplemented with 100 ug/ml ampicillin, and
placed in a shaking orbital incubator at 37.degree. C. 50 ml
inoculum from seed cultures was transferred to flask containing 0.5
liter sterile LB supplemented with 100 ug/ml ampicillin. Cultures
were incubated at 37.degree. C. until it reached mid-logarithmic
growth and then induced with 1 mM ITPG (isopropylthiogalactoside)
for 3 hours at 37.degree. C. After the induction period, cells were
pelleted by centrifugation and harvested following standard
procedures. Pelleted cells were stored at -70.degree. C. until
further process.
[0123] C. Preparation of FMDV 3ABC Antigen
[0124] Frozen cells obtained from Example 3B were resuspended in
PBS with 1 mM PMSF and Triton X-100 detergent and disrupted by
ultrasonication (Branson). Inclusion bodies were separated from
soluble proteins by centrifugation. Protein fraction enriched with
3ABC was obtained through 3-4 rounds of washing off the
contaminants and solubilization of cell lysate pellet in urea.
Recombinant 3ABC was run through ion-exchange chromatography (FPLC,
Q-Sepharose FF) under denaturing condition (8M urea, 10 mM DTT, 20
mM potassium phosphate, pH 7.0) and eluted by NaCl gradient. The
eluted fraction was dialyzed against 20 mM phosphate buffer (pH
9.0). After measuring the protein concentration by Bradford method
and adding sodium azide to 0.05%, protein solution was stored
refrigerated. For longer storage (over 1 month), protein solution
was aliquoted and frozen at -70.degree. C.
Example 4
[0125] Preparation of Recombinant FMDV 3D Antigen
[0126] A. Construction of FMDV 3D Expression Vector
[0127] (i) Construction of Synthetic Full-Length 3D Genes
[0128] To obtain the 3D gene of FMD virus, 36 oligonucleotides were
synthesized, each with complementary ends, at Resgen. We used the
recursive PCR method to assemble the oligonucleotides into full 3D
gene (SEQ ID NO: 121). The gene construction strategy involved
synthesis of a series of overlapping oligonucleotides with
complementary ends. When annealed, the ends served as primers for
the extension of the complementary strand. The fragments then were
amplified by excessive outside primers.
[0129] Because of the large size of 3D gene to be synthesized, the
oligonucleotides were divided into three groups and recursive PCRs
were performed. The produced DNAs were designated as A, B and C
fragment. B and C fragments were joined with PCR and then the B-C
fragment was joined with A fragment to produce full 3D gene.
[0130] Oligonucleotide was designed to contain a BamHI restriction
site for cloning into the expression vector pGEX-4T-1.
[0131] The anti-sense oligonucleotide contains a translational
termination codons (TAA) and an EcoRI restriction site. When N- and
C-terminal primers, 3d-1A (SEQ ID NO: 80) and 3d-36A (SEQ ID NO:
115), were used, a full-length 3D (470 amino acids) gene was
synthesized.
[0132] These steps are detailed herein below.
[0133] 1. 3DA Fragment PCR
[0134] PCR reaction (100 ul volume) was set up as follows:
[0135] Vent DNA polymerase (1U) and 1.times. buffer, along with 25
uM of each dNTP (dATP, dCTP, dGTP, and dTTP), 4 ul 100 mM
MgSO.sub.4, 100 pmol each of oligonucleotides 3d-1A (SEQ ID NO: 80)
and 3d-14 (SEQ ID NO: 93). The template was mixture of 0.83 pmol of
each oligonucleotides 3d-1A to 3d-14.
[0136] The reaction was incubated at 95.degree. C. for 5 minutes,
and then amplified with 35 cycles of 95.degree. C. for 30 seconds,
53.degree. C. for 30 seconds and 73.degree. C. for 100 seconds,
followed by incubation at 73.degree. C. for 5 minutes. Aliquot of
the reaction mixture was analyzed by electrophoresis on agarose
mini-gel.
[0137] 2. 3 DB Fragment PCR
[0138] PCR reaction (100 ul volume) was set up as follows:
[0139] Vent DNA polymerase (1U) and 1.times. buffer, along with 25
uM of each dNTP (dATP, dCTP, dGTP, and dTTP), 4 ul 100 mM
MgSO.sub.4, 100 pmol each of oligonucleotides 3d-13 (SEQ ID NO: 92)
and 3d-24 (SEQ ID NO: 103). The template was mixture of 0.83 pmol
of each oligonucleotides 3d-13 to 3d-24.
[0140] The reaction was incubated at 95.degree. C. for 5 minutes,
and then amplified with 35 cycles of 95.degree. C. for 30 seconds,
55.degree. C. for 30 seconds and 72.degree. C. for 90 seconds,
followed by incubation at 72.degree. C. for 5 minutes. Aliquot of
the reaction mixture was analyzed by electrophoresis on agarose
mini-gel.
[0141] 3. 3DC Fragment PCR
[0142] PCR reaction (100 ul volume) was set up as follows:
[0143] Vent DNA polymerase (1U) and 1.times. buffer, along with 25
uM of each dNTP (dATP, dCTP, dGTP, and dTTP), 4 ul 100 mM
MgSO.sub.4, 100 pmol each of oligonucleotides 3d-25 (SEQ ID NO:
104) and 3d-36A (SEQ ID NO: 115). The template was mixture of 0.83
pmol of each oligonucleotides 3d-25 to 3d-36A.
[0144] The reaction was incubated at 95.degree. C. for 5 minutes,
and then amplified with 35 cycles of 95.degree. C. for 30 seconds,
53.degree. C. for 30 seconds and 73.degree. C. for 100 seconds,
followed by incubation at 73.degree. C. for 5 minutes. Aliquot of
the reaction mixture was analyzed by electrophoresis on agarose
mini-gel.
[0145] 4. 3DB-C Fragment PCR
[0146] PCR reaction (100 ul volume) was set up as follows:
[0147] Vent DNA polymerase (1U) and 1.times. buffer, along with 25
uM of each dNTP (dATP, dCTP, dGTP, and dTTP), 4 ul 100 mM
MgSO.sub.4, 100 pmol each of oligonucleotides 3d-13 (SEQ ID NO: 92)
and 3d-36A (SEQ ID NO: 115). The template was mixture of B and C
fragments described above.
[0148] The reaction was incubated at 95.degree. C. for 5 minutes,
and then amplified with 35 cycles of 95.degree. C. for 30 seconds,
55.degree. C. for 30 seconds and 73.degree. C. for 90 seconds,
followed by incubation at 73.degree. C. for 5 minutes. Aliquot of
the reaction mixture was analyzed by electrophoresis on agarose
mini-gel.
[0149] 5. Full-Length 3D (ABC) PCR
[0150] PCR reaction (100 ul volume) was set up as follows:
[0151] Vent DNA polymerase (1U) and 1.times. buffer, along with 25
uM of each dNTP (dATP, dCTP, dGTP, and dTTP), 4 ul 100 mM
MgSO.sub.4, 100 pmol each of oligonucleotides 3d-1A (SEQ ID NO: 80)
and 3d-36A (SEQ ID NO: 115). The template was mixture of A, B and C
fragments described above.
[0152] The reaction was incubated at 95.degree. C. for 5 minutes,
and then amplified with 35 cycles of 95.degree. C. for 30 seconds,
60.degree. C. for 30 seconds and 73.degree. C. for 120 seconds,
followed by incubation at 73.degree. C. for 5 minutes. PCR-derived
product was run on the agarose gel and the DNA band was cut from
the gel and then the DNA was eluted using Quigen gel extraction
kit.
[0153] (ii) Cloning of the PCR Product.
[0154] The PCR product amplified as described hereinabove was
digested with the restriction endonucleases Bam HI+Eco RI and
ligated into the vector pGEX-4T-1 that had been digested with Bam
HI+Eco RI and gel-isolated. The ligation product was used to
transform XL-1 Blue competent cells. The transformed cells were
plated on LB plates supplemented with 100 ug/ml ampicillin.
Miniprep DNAs were prepared from overnight cultures of colonies and
digested with Bam HI+Eco RI to screen the desired clones. The clone
with right insert was designated as pGEX-3Df (FIG. 9).
[0155] B. Growth and Induction of E. coli Strains with pGEX-3Df
[0156] To expressed recombinant GST-3D protein, pGEX-3Df plasmid
was transformed into E. coli BL21(DE3) and transformants were
spreaded on LB-agar plate supplemented with 100 ug/ml
ampicillin.
[0157] Overnight seed cultures of pGEX-3Df clone were prepared in
500 ml sterile LB supplemented with 100 ug/ml ampicillin, and
placed in a shaking orbital incubator at 37.degree. C. 50 ml
inoculums from seed cultures were transferred to flasks containing
0.5 liter sterile LB supplemented with 100 ug/ml ampicillin.
Cultures were incubated at 37.degree. C. until the cultures reached
mid-logarithmic growth and then induced with 1 mM ITPG
(isopropylthiogalactoside) for 3 hours at 37.degree. C. After the
induction period, cells were pelleted by centrifugation and
harvested following standard procedures. Pelleted cells were stored
at -70.degree. C. until further processed.
[0158] C. Preparation of GST-3D Protein
[0159] Frozen cells obtained from Example were resuspended in PBS
with 1 mM PMSF.
[0160] The cells were lysed by sonication (Branson, model S-125).
Soluble crude lysate was prepared by centrifugation of the
cell-lysate (10,000 rpm, 30 min) and filtered with 0.45 um syringe
filter (Sartorius).
[0161] Glutathione affinity chromatography was carried out to
purify rGST-3D protein, Soluble cell lysate was loaded onto
glutathione sepharose 4B (Pharmacia) column equilibrated with PBS.
After washing the column with three bed volume of PBS, GST-3D was
eluted with 10 mM reduced glutathione, 50 mM Tris-HCl, pH 8.0
buffer solution. The elution fractions were analyzed on the 8%
SDS-PAGE. The fractions which contained the fusion protein were
dialyzed in PBS overnight.
[0162] Kit Assay
Example 5
[0163] FMDV Antibody Detection Kit Formulation
[0164] A. Preparation of Antigen Printed Membrane
[0165] From the stock solution, recombinant 2C and 3ABC were
adjusted and mixed to 0.5 mg/ml and filtered through 0.22 .mu.m
filter unit Millex-GV (Millipore). Avidin solution in PBS (pH 7.4)
was used as internal control after filtered. The antigen mixture
and control solution were applied to nitrocellulose membrane using
Bio-Dot equipment (Bio-Dot). After dried in the low humidity room
overnight, the membrane was blocked with 3% BSA in PBS for 20 min
and then dried on a fan at least for 2 hours. The processed
membrane plates must be stored in an enclosed container with
desiccant or low humidity room.
[0166] B. Preparation of Protein G-Gold Conjugate
[0167] Recombinant Protein G engineered to eliminate
non-specific-binding with serum albumin was purchased from Sigma
and was made to a concentration of 1 mg/ml. Protein G was added
dropwise to gold solution while stirring to make a final
concentration of 10 .mu.g/ml and the solution was kept stirring for
15 min. Then 15% BSA solution was added to gold particle suspension
used. After stirring for another 15 min, coupled gold solution was
centrifuged and supernatant was discarded in order to remove
unbound Protein G. To the coupled gold solution, 2% BSA was added
and sonicated in sonic bath (Branson model #2200 or equivalent) in
order to resuspend the pellet. The suspension was centrifuged again
and the final pellet was suspended in 2% BSA and stored in
refrigerator.
[0168] C. Preparation of Biotin-BSA-Gold Conjugate: Control
Indicator
[0169] Biotinylated BSA purchased from Pierce was used for gold
coupling. The conjugation procedures were basically the same as
described above as for Protein G. 10 .mu.g of biotinylated BSA per
every ml of gold particle suspension was added to gold solution
with vigorous stirring. At the end of the coupling reaction, 15%
BSA solution was added per ml of gold particle suspension. After
stirring for another 15 min, Biotin-BSA coupled gold conjugate
suspension was centrifuged to discard supernatant to remove unbound
Biotin-BSA. To the pellet of coupled gold solution, 2% BSA (10 mM
Sodium phosphate, pH 7.5) was added and suspension was centrifuged
a gain to wash. The pellet was resuspended in 2% BSA and stored in
refrigerator.
[0170] D. Preparation of Filter Pad (Also Serving as Dye Pad)
[0171] Protein G coupled gold solution was diluted using dye
dilution buffer (1% casein, 100 mM sodium phosphate, pH 7.0).
Biotin-BSA coupled gold solution was added for generation of the
control line which binds to avidin on the membrane. The diluted
gold solution was spread onto the Lydall pad strip (microglass
paper) and dried in lyophilizer. The Lydall pad was stored in low
humidity room until use
[0172] E. Preparation of Reservoir Pad
[0173] Cellulose filter paper was presoaked in pretreatment buffer
(100 mM sodium phosphate, pH 7.0) and dried on a fan after blotting
off excessive liquid. The prepared reservoir pad was stored in a
low humidity room.
[0174] F. Device Assembly
[0175] Absorbent pad was attached along the long axis of the plate
after protective sheet from the tape at the top was peeled off.
Filter pad was attached beneath test membrane area along the long
axis of the plate after protective sheet from the tape at the
bottom of the plate was peeled off. The dye pad should overlap the
bottom of the test membrane. Then reservoir pad was attached to the
plate to cover the bottom of filter pad. The dressed membrane plate
was cut into a strip having a width so as to fit into housing.
[0176] Result
[0177] A total of 1540 identified cattle, swine, goat and sheep
sera were used. A test serum consists of the negative animal prior
to vaccination, the uninfected and vaccinated animal and the
infected animal. 3ABC ELISA (Italy and USDA, USA) was used as a
reference test, for each test cattle. Overall, relative
sensitivity, specificity and overall accuracy were 98.6% ( 69/70),
98.6% ( 1449/1470) and 98.6% ( 1518/1540), respectively.
[0178] The Inventive Test vs. Reference Test TABLE-US-00001 BioSign
.TM. FMDV Positive Negative Total Reference Infected(+) 69 1 70
Test Naive (-) 11 1236 1247 Vaccinated(-) Single 4 149 153 Multi 6
64 70 Total 90 1450 1540
INDUSTRIAL APPLICABILITY
[0179] In accordance with the present invention, it is possible to
rapidly and accurately diagnose whether an animal is infected with
foot-and-mouth disease virus (FMDV), based on test samples obtained
from the animal. Further, where appropriate, it is also possible to
distinguish between FMV vaccinated animals and an infected animal
with only a small volume of test sample. Thus, it allows easy and
rapid determination of whether FMV-susceptible animal was infected
with FMV regardless of FMV vaccination.
[0180] Although the preferred embodiments of the preset invention
have been disclosed for illustrative purpose, those skilled in the
art will appreciate that various modifications, additions and
substitutions are possible, without departing from the scope and
sprit of the invention as disclosed in the accompanying claims.
Therefore, embodiments of the present invention were illustrated by
way of FMDV antigen only, but the scope and sprit of the present
invention, of course, can also be applied to detection of FMDV
antibody, PRRSV (Porcine Respiratory and Reproductive Symptom
Virus) antigen and antibody, FeLV (Feline Leukemia Virus) antigen,
FIV (Feline Immunodeficiency Virus) antibody, diagnostic marker for
hydrophobia, CSF (Classical Swine Fever) antigen and antibody, B.
canis (Brucellosis canis) antigen and antibody, Johnes antibody,
BVDV (Bovine Viral Diarrhea Virus) antigen and antibody.
[0181] Further, the present invention is applicable to a variety of
disease markers in organisms, such as cancer diagnostic markers,
hormones, enzymes, drugs and various antigens in the test sample.
Sequence CWU 1
1
121 1 63 DNA Artificial Sequence Synthetic Oligonucleotide for
VP1(TW97-1) 1 atccaaggat ccaccacctc tgcgggtgag tctgcggacc
cggtgactgc caccgttgag 60 aac 63 2 61 DNA Artificial Sequence
Synthetic Oligonucleotide for VP1(TW97-2) 2 ccgtgtgctg gcgacgctga
acttgggtct caccaccgta gttctcaacg gtggcagtca 60 c 61 3 60 DNA
Artificial Sequence Synthetic Oligonucleotide for VP1(TW97-3) 3
tcagcgtcgc cagcacacgg acagcgcgtt catcttggac cgtttcgtga aagttaagcc
60 4 62 DNA Artificial Sequence Synthetic Oligonucleotide for
VP1(TW97-4) 4 cagggatctg catcaggtcc aacacattaa cttgttcctt
tggcttaact ttcacgaaac 60 gg 62 5 63 DNA Artificial Sequence
Synthetic Oligonucleotide for VP1(TW97-5) 5 tggacctgat gcagatccct
gcccacacct tggtaggtgc gctcctgcgt acggccacct 60 act 63 6 60 DNA
Artificial Sequence Synthetic Oligonucleotide for VP1(TW97-6) 6
tcgccctcgt gcttaacggc cagctccagg tcagagaagt agtaggtggc cgtacgcagg
60 7 62 DNA Artificial Sequence Synthetic Oligonucleotide for
VP1(TW97-7) 7 gccgttaagc acgagggcga tctcacctgg gttccaaacg
gcgcccctga gaccgcactg 60 ga 62 8 60 DNA Artificial Sequence
Synthetic Oligonucleotide for VP1(TW97-8) 8 gagcggttcc ttgtggtaag
cggttgggtt ggtagtgttg tccagtgcgg tctcaggggc 60 9 64 DNA Artificial
Sequence Synthetic Oligonucleotide for VP1(TW97-9) 9 cttaccacaa
ggaaccgctc acccgtctgg cgctgcctta cacggctcca caccgtgttt 60 tagc 64
10 62 DNA Artificial Sequence Synthetic Oligonucleotide for
VP1(TW97-10) 10 tgctggtgtc accgtacttg ctgctaccgt tgtaaacggt
cgctaaaaca cggtgtggag 60 cc 62 11 62 DNA Artificial Sequence
Synthetic Oligonucleotide for VP1(TW97-11) 11 caagtacggt gacaccagca
ctaacaacgt gcgtggtgac ctgcaagtgt tagctcagaa 60 gg 62 12 65 DNA
Artificial Sequence Synthetic Oligonucleotide for VP1(TW97-12) 12
gatggcaccg aagttgaagg aggtaggcag agtacgttct gccttctgag ctaacacttg
60 caggt 65 13 62 DNA Artificial Sequence Synthetic Oligonucleotide
for VP1(TW97-13) 13 tccttcaact tcggtgccat caaggcaact cgtgttactg
aactgctcta ccgtatgaag 60 cg 62 14 60 DNA Artificial Sequence
Synthetic Oligonucleotide for VP1(TW97-14) 14 ttgaatggcg agcagcggac
gcggacagta ggtctcggca cgcttcatac ggtagagcag 60 15 60 DNA Artificial
Sequence Synthetic Oligonucleotide for VP1(TW97-15) 15 gtccgctgct
cgccattcaa ccgagcgacg ctcgtcacaa gcagcgtatt gtggcaccgg 60 16 50 DNA
Artificial Sequence Synthetic Oligonucleotide for VP1(TW97-16) 16
gcctatgaat tcttacagca gctgttttgc cggtgccaca atacgctgct 50 17 62 DNA
Artificial Sequence Synthetic Oligonucleotide for 2C(2C-1) 17
gcaggatccg acgacgacga caaactcaaa gcacgtgaca tcaacgacat atttgccgtt
60 ct 62 18 60 DNA Artificial Sequence Synthetic Oligonucleotide
for 2C(2C-2) 18 ttgctgtata aacggcaaga attcttgcca ctcaccgacc
agtttgacta ggaccggtag 60 19 60 DNA Artificial Sequence Synthetic
Oligonucleotide for 2C(2C-3) 19 tcaaactgat cctggccatc cgcgactgga
ttaaggcatg gatcgcctca gaagagaagt 60 20 60 DNA Artificial Sequence
Synthetic Oligonucleotide for 2C(2C-4) 20 ctagcggagt cttctcttca
aacagtggta ctgtctggac cacggaccgt aggaactttc 60 21 60 DNA Artificial
Sequence Synthetic Oligonucleotide for 2C(2C-5) 21 gtgcctggca
tccttgaaag tcaacgggat ctcaatgacc ccggcaaata caaggaggcc 60 22 60 DNA
Artificial Sequence Synthetic Oligonucleotide for 2C(2C-6) 22
ggccgtttat gttcctccgg ttccttaccg acctgttgcg cgcagttcgc acaaacttct
60 23 60 DNA Artificial Sequence Synthetic Oligonucleotide for
2C(2C-7) 23 gcgtcaagcg tgtttgaaga gcgggaacgt gcacattgcc aatctgtgta
aagtggtcgc 60 24 60 DNA Artificial Sequence Synthetic
Oligonucleotide for 2C(2C-8) 24 ttagacacat ttcaccagcg aggccgcggg
tcgttcagct ctgggcttgg tcaccagcac 60 25 59 DNA Artificial Sequence
Synthetic Oligonucleotide for 2C(2C-9) 25 gacccgaacc agtggtcgtg
tgccttcgcg gcaaatccgg cacaaggaaa agcatcctc 59 26 60 DNA Artificial
Sequence Synthetic Oligonucleotide for 2C(2C-10) 26 gtgttccttt
tcgtaggagc gcttgcacga gcgcgtccgt taaaggtgtg tgaagtgacc 60 27 60 DNA
Artificial Sequence Synthetic Oligonucleotide for 2C(2C-11) 27
atttccacac acttcactgg taggaccgac tcggtctggt actgcccgcc cgaccctgac
60 28 60 DNA Artificial Sequence Synthetic Oligonucleotide for
2C(2C-12) 28 tgacgggcgg gctgggactg gtgaaactgc caatgttagt cgtctggcag
cagcactacc 60 29 60 DNA Artificial Sequence Synthetic
Oligonucleotide for 2C(2C-13) 29 gcagaccgtc gtcgtgatgg acgacttggg
ccaaaaccca gacggcaaag acttcaagta 60 30 60 DNA Artificial Sequence
Synthetic Oligonucleotide for 2C(2C-14) 30 ctgccgtttc tgaagttcat
gaaacgggtt taccagaggt ggtgccccaa gtagggcgga 60 31 60 DNA Artificial
Sequence Synthetic Oligonucleotide for 2C(2C-15) 31 ccacggggtt
catcccgcct atggcctcgc tcgaggataa gggtaaaccc ttcaacagca 60 32 60 DNA
Artificial Sequence Synthetic Oligonucleotide for 2C(2C-16) 32
cccatttggg aagttgtcgt tccagtatta tcgatgttgg ttggacatga gccctaagtg
60 33 60 DNA Artificial Sequence Synthetic Oligonucleotide for
2C(2C-17) 33 aacctgtact cgggattcac cccaaagacc atggtgtgcc ccgatgcgct
taaccggagg 60 34 60 DNA Artificial Sequence Synthetic
Oligonucleotide for 2C(2C-18) 34 ggctacgcga attggcctcc aaagtgaaac
tgtagctgca ctcgcggttt ctgcccatgt 60 35 60 DNA Artificial Sequence
Synthetic Oligonucleotide for 2C(2C-19) 35 gagcgccaaa gacgggtaca
agatcaacaa caaactggac atagtcaaag cacttgaaga 60 36 60 DNA Artificial
Sequence Synthetic Oligonucleotide for 2C(2C-20) 36 tatcagtttc
gtgaacttct gtgggtgcga ttgggccacc gctacaaggt tatgctgacg 60 37 60 DNA
Artificial Sequence Synthetic Oligonucleotide for 2C(2C-21) 37
cgatgttcca atacgactgc gctcttctca acggaatggc cgttgaaatg aagagaatgc
60 38 60 DNA Artificial Sequence Synthetic Oligonucleotide for
2C(2C-22) 38 gcaactttac ttctcttacg tcgttctgta caagttcgga gttggtggga
aggtcttgta 60 39 60 DNA Artificial Sequence Synthetic
Oligonucleotide for 2C(2C-23) 39 caaccaccct tccagaacat ctaccagctc
gttcaggagg tgattgagcg ggtggaacta 60 40 56 DNA Artificial Sequence
Synthetic Oligonucleotide for 2C(2C-24) 40 actaactcgc ccaccttgat
gtgcttttcc acagctcggt gggctataaa tttgtc 56 41 30 DNA Artificial
Sequence Synthetic Oligonucleotide for 2C(2C-25) 41 gtcgagaccc
gaaccagtgg tcgtgtgcct 30 42 30 DNA Artificial Sequence Synthetic
Oligonucleotide for 2C(2C-26) 42 aggcacacga ccactggttc gggtctcgac
30 43 58 DNA Artificial Sequence Synthetic Oligonucleotide for
3ABC(3ABC-1) 43 gcaggatccg acgacgacga caaaatttca atcccttccc
agaagtccgt gttgtact 58 44 60 DNA Artificial Sequence Synthetic
Oligonucleotide for 3ABC(3ABC-2) 44 ggtcttcagg cacaacatga
aggagtaact cttcccagtc gtgcttcgtc gctagctcaa 60 45 60 DNA Artificial
Sequence Synthetic Oligonucleotide for 3ABC(3ABC-3) 45 cacgaagcag
cgatcgagtt cttcgagggg atggtccacg attccatcaa agaggaactc 60 46 60 DNA
Artificial Sequence Synthetic Oligonucleotide for 3ABC(3ABC-4) 46
taaggtagtt tctccttgag gctggggagt aagtcgtctg gagcaagcat tttgcgcgga
60 47 60 DNA Artificial Sequence Synthetic Oligonucleotide for
3ABC(3ABC-5) 47 ctcgttcgta aaacgcgcct tcaagcgcct gaaagagaac
tttgaagttg tagccctgtg 60 48 60 DNA Artificial Sequence Synthetic
Oligonucleotide for 3ABC(3ABC-6) 48 aaacttcaac atcgggacac
aaactgggag aaccgtttgt atcactaata cgaggcggtt 60 49 60 DNA Artificial
Sequence Synthetic Oligonucleotide for 3ABC(3ABC-7) 49 tagtgattat
gctccgccaa gcgcgcaaga ggtaccaatc ggtggatgac ccactggacg 60 50 60 DNA
Artificial Sequence Synthetic Oligonucleotide for 3ABC(3ABC-8) 50
ccacctactg ggtgacctgc cgctgcatcg agaaccgctg cgccttttct tgggagacct
60 51 60 DNA Artificial Sequence Synthetic Oligonucleotide for
3ABC(3ABC-9) 51 gcggaaaaga accctctgga gacgagtgcc gctagcgctg
tcggtttcag agagagatcc 60 52 60 DNA Artificial Sequence Synthetic
Oligonucleotide for 3ABC(3ABC-10) 52 agccaaagtc tctctctagg
gggtggctcg ttccctgcgc gcttctgcgc ttgcgactcg 60 53 60 DNA Artificial
Sequence Synthetic Oligonucleotide for 3ABC(3ABC-11) 53 cgaagacgcg
aacgctgagc ccgtcgtgtt cggtagggaa caaccgcgag ctgaaggacc 60 54 60 DNA
Artificial Sequence Synthetic Oligonucleotide for 3ABC(3ABC-12) 54
gttggcgctc atgcggccgg gttacctctc tgtctttggc gatttccact ttcgttttcg
60 55 60 DNA Artificial Sequence Synthetic Oligonucleotide for
3ABC(3ABC-13) 55 gtcagaaacc tcttaaagtg aaagccgagc tgccacaaca
ggagggacca tacgccggcc 60 56 60 DNA Artificial Sequence Synthetic
Oligonucleotide for 3ABC(3ABC-14) 56 gcttttgctt tcacctttag
cggtttctgt ctctccattg ggccggcgta tggtccctcc 60 57 60 DNA Artificial
Sequence Synthetic Oligonucleotide for 3ABC(3ABC-15) 57 ctaaaggtga
aagcaaaagc ccccgtcgtg aaggaaggac cttacgaggg accggtgaag 60 58 60 DNA
Artificial Sequence Synthetic Oligonucleotide for 3ABC(3ABC-16) 58
gaatgctccc tggccacttc tttggacagc gaaattttca ctttcgtttc ttgaactatc
60 59 60 DNA Artificial Sequence Synthetic Oligonucleotide for
3ABC(3ABC-17) 59 gaaagcaaag aacttgatag tcactgagag tggtgcgcca
ccgaccgact tgcaaaagat 60 60 60 DNA Artificial Sequence Synthetic
Oligonucleotide for 3ABC(3ABC-18) 60 ggctggctga acgttttcta
ccagtacccg ttgtgattcg gtcagctcga gtaggagctg 60 61 60 DNA Artificial
Sequence Synthetic Oligonucleotide for 3ABC(3ABC-19) 61 cagtcgagct
catcctcgac ggcaagacgg tagccatttg ctgtgctacc ggagtgttcg 60 62 60 DNA
Artificial Sequence Synthetic Oligonucleotide for 3ABC(3ABC-20) 62
gacacgatgg cctcacaagc cgtgacggat ggagcacgga gcagtagaga agcgcctttt
60 63 60 DNA Artificial Sequence Synthetic Oligonucleotide for
3ABC(3ABC-21) 63 cgtcatctct tcgcggaaaa gtacgacaag atcatgttgg
acggcagagc cttgacagac 60 64 60 DNA Artificial Sequence Synthetic
Oligonucleotide for 3ABC(3ABC-22) 64 tgccgtctcg gaactgtctg
tcactgatgt ctcacaaact caaactctaa tttcattttc 60 65 60 DNA Artificial
Sequence Synthetic Oligonucleotide for 3ABC(3ABC-23) 65 gtttgagatt
aaagtaaaag gacaggacat gctctcagac gccgctctca tggtgttgca 60 66 60 DNA
Artificial Sequence Synthetic Oligonucleotide for 3ABC(3ABC-24) 66
cggcgagagt accacaacgt ggcaccctta gcgcacgcac tgtagtgctt tgtgaaagca
60 67 60 DNA Artificial Sequence Synthetic Oligonucleotide for
3ABC(3ABC-25) 67 acatcacgaa acactttcgt gacgtagcga gaatgaagaa
gggaaccccc gtcgtcggtg 60 68 60 DNA Artificial Sequence Synthetic
Oligonucleotide for 3ABC(3ABC-26) 68 cccttggggg cagcagccac
actagttgtt acgactgcag ccctctgagt ataagagacc 60 69 60 DNA Artificial
Sequence Synthetic Oligonucleotide for 3ABC(3ABC-27) 69 gggagactca
tattctctgg tgtagccctc acttacaagg acatcgtcgt gtgtatggat 60 70 60 DNA
Artificial Sequence Synthetic Oligonucleotide for 3ABC(3ABC-28) 70
tgtagcagca cacataccta cctctgtggt acggacccga gaaacggatg tcccgtaggt
60 71 60 DNA Artificial Sequence Synthetic Oligonucleotide for
3ABC(3ABC-29) 71 ctttgcctac agggcatcca ccaaggcagg ctactgcgga
ggagccgtcc tggcaaagga 60 72 60 DNA Artificial Sequence Synthetic
Oligonucleotide for 3ABC(3ABC-30) 72 cctcggcagg accgtttcct
gccccggctt tgcaagtagc aaccgtgggt gaggcgtcca 60 73 60 DNA Artificial
Sequence Synthetic Oligonucleotide for 3ABC(3ABC-31) 73 ttggcaccca
ctccgcaggt ggaaacggca taggatactg ttcgtgtgtt tcccgatcaa 60 74 60 DNA
Artificial Sequence Synthetic Oligonucleotide for 3ABC(3ABC-32) 74
aagcacacaa agggctagtt acgaggactt ctacttccgt gtgtagctgg gacttggtgt
60 75 49 DNA Artificial Sequence Synthetic Oligonucleotide for
3ABC(3ABC-33) 75 tgcaagcttt tactcgtggt gtggttcagg gtcgatgtgt
gccttcatc 49 76 35 DNA Artificial Sequence Synthetic
Oligonucleotide for 3ABC(3ABC-34) 76 ctttaaaagt gaaagcaaag
aacttgatag tcact 35 77 35 DNA Artificial Sequence Synthetic
Oligonucleotide for 3ABC(3ABC-35) 77 agtgactatc aagttctttg
ctttcacttt taaag 35 78 20 DNA Artificial Sequence Synthetic
Oligonucleotide for 3ABC(3ABC-36) 78 ccgtcgtgtt cggtagggaa 20 79 20
DNA Artificial Sequence Synthetic Oligonucleotide for 3ABC(3ABC-37)
79 aaagtaaaag gacaggacat 20 80 42 DNA Artificial Sequence Synthetic
Oligonucleotide for 3D(3d-1A) 80 gctatcggat ccgggttgat cgttgatacc
agagatgtgg aa 42 81 60 DNA Artificial Sequence Synthetic
Oligonucleotide for 3D(3d-2) 81 tgggtgcaag cttggttttg cgcattacat
ggacgcgctc ttccacatct ctggtatcaa 60 82 60 DNA Artificial Sequence
Synthetic Oligonucleotide for 3D(3d-3) 82 caaaaccaag cttgcaccca
ccgtcgcgca cggtgtgttc aatcctgagt tcgggcctgc 60 83 60 DNA Artificial
Sequence Synthetic Oligonucleotide for 3D(3d-4) 83 aacaccttcg
ttcagacgtg ggtccttgtt agacaaggcg gcaggcccga actcaggatt 60 84 60 DNA
Artificial Sequence Synthetic Oligonucleotide for 3D(3d-5) 84
cacgtctgaa cgaaggtgtt gtcctcgatg aagtcatttt ctccaagcat aaaggagaca
60 85 60 DNA Artificial Sequence Synthetic Oligonucleotide for
3D(3d-6) 85 cagcggcgga acagcgcttt gtcctcctca gacatctttg tgtctccttt
atgcttggag 60 86 60 DNA Artificial Sequence Synthetic
Oligonucleotide for 3D(3d-7) 86 aaagcgctgt tccgccgctg cgctgctgac
tacgcgtcac gcctgcacag tgtgctgggt 60 87 60 DNA Artificial Sequence
Synthetic Oligonucleotide for 3D(3d-8) 87 ccttgattgc ctcgtaaatg
ctcagtgggg catttgccgt acccagcaca ctgtgcaggc 60 88 60 DNA Artificial
Sequence Synthetic Oligonucleotide for 3D(3d-9) 88 catttacgag
gcaatcaagg gcgttgacgg actcgacgcc atggagccag acaccgcacc 60 89 60 DNA
Artificial Sequence Synthetic Oligonucleotide for 3D(3d-10) 89
tgcaccgcgg cgtttcccct ggagggccca gggaaggcca ggtgcggtgt ctggctccat
60 90 60 DNA Artificial Sequence Synthetic Oligonucleotide for
3D(3d-11) 90 aggggaaacg ccgcggtgca cttatcgatt tcgagaacgg cacggtcgga
cccgaggttg 60 91 60 DNA Artificial Sequence Synthetic
Oligonucleotide for 3D(3d-12) 91 aacttgtatt ctcttttctc catgagcttc
aaggcagcct caacctcggg tccgaccgtg 60 92 60 DNA Artificial Sequence
Synthetic Oligonucleotide for 3D(3d-13) 92 gagaaaagag aatacaagtt
tgtttgccag accttcctga aggacgaaat tcgcccgatg 60 93 60 DNA Artificial
Sequence Synthetic Oligonucleotide for 3D(3d-14) 93 aaacgtcgac
aatgcgagtc ttgccggcac gtactttctc catcgggcga atttcgtcct 60 94 60 DNA
Artificial Sequence Synthetic Oligonucleotide for 3D(3d-15) 94
gactcgcatt gtcgacgttt tgcctgttga acacattctt tacaccagga tgatgattgg
60 95 60 DNA Artificial Sequence Synthetic Oligonucleotide for
3D(3d-16) 95 ctgcggcccg ttgtttgagt gcatttgtgc acaaaatctg ccaatcatca
tcctggtgta 60 96 60 DNA Artificial Sequence Synthetic
Oligonucleotide for 3D(3d-17) 96 actcaaacaa cgggccgcag attggctcag
cggtcggttg caaccctgat gttgattggc 60 97 60 DNA Artificial Sequence
Synthetic Oligonucleotide for 3D(3d-18) 97 cacacgtttc tgtattgggc
gaagtgtgtg ccgaatctct gccaatcaac atcagggttg 60 98 60 DNA Artificial
Sequence Synthetic Oligonucleotide for 3D(3d-19) 98 gcccaataca
gaaacgtgtg ggacgtggac tattcggcct ttgatgcaaa ccactgcagc 60 99 60 DNA
Artificial Sequence Synthetic Oligonucleotide for 3D(3d-20) 99
ccgtgcggaa cacctcttca aacatgatgt tcatggcatc gctgcagtgg tttgcatcaa
60 100 60 DNA Artificial Sequence Synthetic Oligonucleotide for
3D(3d-21) 100 tgaagaggtg ttccgcacgg agttcggctt ccacccgaat
gctgagtgga ttctgaagac 60 101 60 DNA Artificial Sequence Synthetic
Oligonucleotide for 3D(3d-22) 101 gatgcgcttg ttctcatagg cgtgttccgt
gttcacgaga gtcttcagaa tccactcagc 60 102 60 DNA Artificial Sequence
Synthetic Oligonucleotide for 3D(3d-23) 102 cctatgagaa caagcgcatc
actgttgaag gcgggatgcc atctggctgt tccgcaacaa 60 103 60 DNA
Artificial Sequence Synthetic Oligonucleotide for 3D(3d-24) 103
tagagcacgt agatgttatt caaaattgtg ttgatgatgc ttgttgcgga acagccagat
60 104 60 DNA Artificial Sequence Synthetic Oligonucleotide for
3D(3d-25) 104 aataacatct acgtgctcta cgccttgcgt agacactatg
agggggttga gctggacacc 60 105 60 DNA Artificial Sequence Synthetic
Oligonucleotide for 3D(3d-26) 105 ttgccaccac gatgtcgtct ccataggaga
tcatggtgta ggtgtccagc tcaaccccct 60 106 60 DNA Artificial Sequence
Synthetic Oligonucleotide for 3D(3d-27) 106 agacgacatc gtggtggcaa
gcgattatga tctggacttt gaggccctca agcctcactt 60 107 60 DNA
Artificial Sequence Synthetic Oligonucleotide for 3D(3d-28) 107
gcttttgtca gctggagtaa tggtttggcc aagagatttg aagtgaggct tgagggcctc
60 108 60 DNA Artificial Sequence Synthetic Oligonucleotide for
3D(3d-29) 108 ttactccagc tgacaaaagc gacaaaggtt ttgttcttgg
tcactccatt actgacgtca 60 109 60 DNA Artificial Sequence Synthetic
Oligonucleotide for 3D(3d-30) 109 ccagtgccat aatccatgtg gaagtgtctt
ttgaggaaag tgacgtcagt aatggagtga 60 110 60 DNA Artificial Sequence
Synthetic Oligonucleotide for 3D(3d-31) 110 cacatggatt atggcactgg
gttttacaaa cctgtgatgg cctcgaagac cctcgaggct 60 111 60 DNA
Artificial Sequence Synthetic Oligonucleotide for 3D(3d-32) 111
acttctcctg gatggtccca cggcgtgcaa aggagaggat agcctcgagg gtcttcgagg
60 112 60 DNA Artificial Sequence Synthetic Oligonucleotide for
3D(3d-33) 112 tgggaccatc caggagaagt tgatttccgt ggcaggactc
gccgtccact ccggaccaga 60 113 60 DNA Artificial Sequence Synthetic
Oligonucleotide for 3D(3d-34) 113 aaagaggccc tggaagggct caaagagacg
ccggtactcg tctggtccgg agtggacggc 60 114 60 DNA Artificial Sequence
Synthetic Oligonucleotide for 3D(3d-35) 114 agcccttcca gggcctcttt
gagattccaa gctacagatc actttacctg cgttgggtga 60 115 55 DNA
Artificial Sequence Synthetic Oligonucleotide for 3D(3d-36A) 115
gcaatcgaat tcttatgcgt cgccgcacac ggcgttcacc caacgcaggt aaagt 55 116
20 DNA Artificial Sequence Synthetic
Oligonucleotide for 3D(pGEX5) 116 ctggcaagcc acgtttggtg 20 117 20
DNA Artificial Sequence Synthetic Oligonucleotide for 3D(pGEX3) 117
ggagctgcat gtgtcagagg 20 118 639 DNA Foot-and-mouth disease virus
CDS (1)..(639) Synthetic Nucleotide and Amino acid Sequence of VP-1
Protein 118 acc acc tct gcg ggt gag tct gcg gac ccg gtg act gcc acc
gtt gag 48 Thr Thr Ser Ala Gly Glu Ser Ala Asp Pro Val Thr Ala Thr
Val Glu 1 5 10 15 aac tac ggt ggt gag acc caa gtt cag cgt cgc cag
cac acg gac agc 96 Asn Tyr Gly Gly Glu Thr Gln Val Gln Arg Arg Gln
His Thr Asp Ser 20 25 30 gcg ttc atc ttg gac cgt ttc gtg aaa gtt
aag cca aag gaa caa gtt 144 Ala Phe Ile Leu Asp Arg Phe Val Lys Val
Lys Pro Lys Glu Gln Val 35 40 45 aat gtg ttg gac ctg atg cag atc
cct gcc cac acc ttg gta ggt gcg 192 Asn Val Leu Asp Leu Met Gln Ile
Pro Ala His Thr Leu Val Gly Ala 50 55 60 ctc ctg cgt acg gcc acc
tac tac ttc tct gac ctg gag ctg gcc gtt 240 Leu Leu Arg Thr Ala Thr
Tyr Tyr Phe Ser Asp Leu Glu Leu Ala Val 65 70 75 80 aag cac gag ggc
gat ctc acc tgg gtt cca aac ggc gcc cct gag acc 288 Lys His Glu Gly
Asp Leu Thr Trp Val Pro Asn Gly Ala Pro Glu Thr 85 90 95 gca ctg
gac aac act acc aac cca acc gct tac cac aag gaa ccg ctc 336 Ala Leu
Asp Asn Thr Thr Asn Pro Thr Ala Tyr His Lys Glu Pro Leu 100 105 110
acc cgt ctg gcg ctg cct tac acg gct cca cac cgt gtt tta gcg acc 384
Thr Arg Leu Ala Leu Pro Tyr Thr Ala Pro His Arg Val Leu Ala Thr 115
120 125 gtt tac aac ggt agc agc aag tac ggt gac acc agc act aac aac
gtg 432 Val Tyr Asn Gly Ser Ser Lys Tyr Gly Asp Thr Ser Thr Asn Asn
Val 130 135 140 cgt ggt gac ctg caa gtg tta gct cag aag gca gaa cgt
act ctg cct 480 Arg Gly Asp Leu Gln Val Leu Ala Gln Lys Ala Glu Arg
Thr Leu Pro 145 150 155 160 acc tcc ttc aac ttc ggt gcc atc aag gca
act cgt gtt act gaa ctg 528 Thr Ser Phe Asn Phe Gly Ala Ile Lys Ala
Thr Arg Val Thr Glu Leu 165 170 175 ctc tac cgt atg aag cgt gcc gag
acc tac tgt ccg cgt ccg ctg ctc 576 Leu Tyr Arg Met Lys Arg Ala Glu
Thr Tyr Cys Pro Arg Pro Leu Leu 180 185 190 gcc att caa ccg agc gac
gct cgt cac aag cag cgt att gtg gca ccg 624 Ala Ile Gln Pro Ser Asp
Ala Arg His Lys Gln Arg Ile Val Ala Pro 195 200 205 gca aaa cag ctg
ctg 639 Ala Lys Gln Leu Leu 210 119 954 DNA Foot-and-mouth disease
virus CDS (1)..(954) Synthetic Nucleotide and Amino acid Sequence
of 2C Protein 119 ctc aaa gca cgt gac atc aac gac ata ttt gcc gtt
ctt aag aac ggt 48 Leu Lys Ala Arg Asp Ile Asn Asp Ile Phe Ala Val
Leu Lys Asn Gly 1 5 10 15 gag tgg ctg gtc aaa ctg atc ctg gcc atc
cgc gac tgg att aag gca 96 Glu Trp Leu Val Lys Leu Ile Leu Ala Ile
Arg Asp Trp Ile Lys Ala 20 25 30 tgg atc gcc tca gaa gag aag ttt
gtc acc atg aca gac ctg gtg cct 144 Trp Ile Ala Ser Glu Glu Lys Phe
Val Thr Met Thr Asp Leu Val Pro 35 40 45 ggc atc ctt gaa agt caa
cgg gat ctc aat gac ccc ggc aaa tac aag 192 Gly Ile Leu Glu Ser Gln
Arg Asp Leu Asn Asp Pro Gly Lys Tyr Lys 50 55 60 gag gcc aag gaa
tgg ctg gac aac gcg cgt caa gcg tgt ttg aag agc 240 Glu Ala Lys Glu
Trp Leu Asp Asn Ala Arg Gln Ala Cys Leu Lys Ser 65 70 75 80 ggg aac
gtg cac att gcc aat ctg tgt aaa gtg gtc gct ccg gcg ccc 288 Gly Asn
Val His Ile Ala Asn Leu Cys Lys Val Val Ala Pro Ala Pro 85 90 95
agc aag tcg aga ccc gaa cca gtg gtc gtg tgc ctt cgc ggc aaa tcc 336
Ser Lys Ser Arg Pro Glu Pro Val Val Val Cys Leu Arg Gly Lys Ser 100
105 110 ggc aca agg aaa agc atc ctc gcg aac gtg ctc gcg cag gca att
tcc 384 Gly Thr Arg Lys Ser Ile Leu Ala Asn Val Leu Ala Gln Ala Ile
Ser 115 120 125 aca cac ttc act ggt agg acc gac tcg gtc tgg tac tgc
ccg ccc gac 432 Thr His Phe Thr Gly Arg Thr Asp Ser Val Trp Tyr Cys
Pro Pro Asp 130 135 140 cct gac cac ttt gac ggt tac aat cag cag acc
gtc gtc gtg atg gac 480 Pro Asp His Phe Asp Gly Tyr Asn Gln Gln Thr
Val Val Val Met Asp 145 150 155 160 gac ttg ggc caa aac cca gac ggc
aaa gac ttc aag tac ttt gcc caa 528 Asp Leu Gly Gln Asn Pro Asp Gly
Lys Asp Phe Lys Tyr Phe Ala Gln 165 170 175 atg gtc tcc acc acg ggg
ttc atc ccg cct atg gcc tcg ctc gag gat 576 Met Val Ser Thr Thr Gly
Phe Ile Pro Pro Met Ala Ser Leu Glu Asp 180 185 190 aag ggt aaa ccc
ttc aac agc aag gtc ata ata gct aca acc aac ctg 624 Lys Gly Lys Pro
Phe Asn Ser Lys Val Ile Ile Ala Thr Thr Asn Leu 195 200 205 tac tcg
gga ttc acc cca aag acc atg gtg tgc ccc gat gcg ctt aac 672 Tyr Ser
Gly Phe Thr Pro Lys Thr Met Val Cys Pro Asp Ala Leu Asn 210 215 220
cgg agg ttt cac ttt gac atc gac gtg agc gcc aaa gac ggg tac aag 720
Arg Arg Phe His Phe Asp Ile Asp Val Ser Ala Lys Asp Gly Tyr Lys 225
230 235 240 atc aac aac aaa ctg gac ata gtc aaa gca ctt gaa gac acc
cac gct 768 Ile Asn Asn Lys Leu Asp Ile Val Lys Ala Leu Glu Asp Thr
His Ala 245 250 255 aac ccg gtg gcg atg ttc caa tac gac tgc gct ctt
ctc aac gga atg 816 Asn Pro Val Ala Met Phe Gln Tyr Asp Cys Ala Leu
Leu Asn Gly Met 260 265 270 gcc gtt gaa atg aag aga atg cag caa gac
atg ttc aag cct caa cca 864 Ala Val Glu Met Lys Arg Met Gln Gln Asp
Met Phe Lys Pro Gln Pro 275 280 285 ccc ttc cag aac atc tac cag ctc
gtt cag gag gtg att gag cgg gtg 912 Pro Phe Gln Asn Ile Tyr Gln Leu
Val Gln Glu Val Ile Glu Arg Val 290 295 300 gaa cta cac gaa aag gtg
tcg agc cac ccg ata ttt aaa cag 954 Glu Leu His Glu Lys Val Ser Ser
His Pro Ile Phe Lys Gln 305 310 315 120 1281 DNA Foot-and-mouth
disease virus CDS (1)..(1281) Synthetic Nucleotide and Amino acid
Sequence of 3ABC Protein 120 att tca atc cct tcc cag aag tcc gtg
ttg tac ttc ctc att gag aag 48 Ile Ser Ile Pro Ser Gln Lys Ser Val
Leu Tyr Phe Leu Ile Glu Lys 1 5 10 15 ggt cag cac gaa gca gcg atc
gag ttc ttc gag ggg atg gtc cac gat 96 Gly Gln His Glu Ala Ala Ile
Glu Phe Phe Glu Gly Met Val His Asp 20 25 30 tcc atc aaa gag gaa
ctc cga ccc ctc att cag cag acc tcg ttc gta 144 Ser Ile Lys Glu Glu
Leu Arg Pro Leu Ile Gln Gln Thr Ser Phe Val 35 40 45 aaa cgc gcc
ttc aag cgc ctg aaa gag aac ttt gaa gtt gta gcc ctg 192 Lys Arg Ala
Phe Lys Arg Leu Lys Glu Asn Phe Glu Val Val Ala Leu 50 55 60 tgt
ttg acc ctc ttg gca aac ata gtg att atg ctc cgc caa gcg cgc 240 Cys
Leu Thr Leu Leu Ala Asn Ile Val Ile Met Leu Arg Gln Ala Arg 65 70
75 80 aag agg tac caa tcg gtg gat gac cca ctg gac ggc gac gta gct
ctt 288 Lys Arg Tyr Gln Ser Val Asp Asp Pro Leu Asp Gly Asp Val Ala
Leu 85 90 95 ggc gac gcg gaa aag aac cct ctg gag acg agt gcc gct
agc cgt gtc 336 Gly Asp Ala Glu Lys Asn Pro Leu Glu Thr Ser Ala Ala
Ser Arg Val 100 105 110 ggt ttc aga gag aga tcc ccc acc gag caa ggg
acg cgc gaa gac gcg 384 Gly Phe Arg Glu Arg Ser Pro Thr Glu Gln Gly
Thr Arg Glu Asp Ala 115 120 125 aac gct gag ccc gtc gtg ttc ggt agg
gaa caa ccg cga gct gaa gga 432 Asn Ala Glu Pro Val Val Phe Gly Arg
Glu Gln Pro Arg Ala Glu Gly 130 135 140 ccc tac gct ggg cca ctc gag
cgt cag aaa cct ctt aaa gtg aaa gcc 480 Pro Tyr Ala Gly Pro Leu Glu
Arg Gln Lys Pro Leu Lys Val Lys Ala 145 150 155 160 gag ctg cca caa
cag gag gga cca tac gcc ggc cca atg gag aga cag 528 Glu Leu Pro Gln
Gln Glu Gly Pro Tyr Ala Gly Pro Met Glu Arg Gln 165 170 175 aaa ccg
cta aag gtg aaa gca aaa gcc ccc gtc gtg aag gaa gga cct 576 Lys Pro
Leu Lys Val Lys Ala Lys Ala Pro Val Val Lys Glu Gly Pro 180 185 190
tac gag gga ccg gtg aag aaa cct gtc gct tta aaa gtg aaa gca aag 624
Tyr Glu Gly Pro Val Lys Lys Pro Val Ala Leu Lys Val Lys Ala Lys 195
200 205 aac ttg ata gtc act gag agt ggt gcg cca ccg acc gac ttg caa
aag 672 Asn Leu Ile Val Thr Glu Ser Gly Ala Pro Pro Thr Asp Leu Gln
Lys 210 215 220 atg gtc atg ggc aac act aag cca gtc gag ctc atc ctc
gac ggc aag 720 Met Val Met Gly Asn Thr Lys Pro Val Glu Leu Ile Leu
Asp Gly Lys 225 230 235 240 acg gta gcc att tgc tgt gct acc gga gtg
ttc ggc act gcc tac ctc 768 Thr Val Ala Ile Cys Cys Ala Thr Gly Val
Phe Gly Thr Ala Tyr Leu 245 250 255 gtg cct cgt cat ctc ttc gcg gaa
aag tac gac aag atc atg ttg gac 816 Val Pro Arg His Leu Phe Ala Glu
Lys Tyr Asp Lys Ile Met Leu Asp 260 265 270 ggc aga gcc ttg aca gac
agt gac tac aga gtg ttt gag ttt gag att 864 Gly Arg Ala Leu Thr Asp
Ser Asp Tyr Arg Val Phe Glu Phe Glu Ile 275 280 285 aaa gta aaa gga
cag gac atg ctc tca gac gcc gct ctc atg gtg ttg 912 Lys Val Lys Gly
Gln Asp Met Leu Ser Asp Ala Ala Leu Met Val Leu 290 295 300 cac cgt
ggg aat cgc gtg cgt gac atc acg aaa cac ttt cgt gac gta 960 His Arg
Gly Asn Arg Val Arg Asp Ile Thr Lys His Phe Arg Asp Val 305 310 315
320 gcg aga atg aag aag gga acc ccc gtc gtc ggt gtg atc aac aat gct
1008 Ala Arg Met Lys Lys Gly Thr Pro Val Val Gly Val Ile Asn Asn
Ala 325 330 335 gac gtc ggg aga ctc ata ttc tct ggt gta gcc ctc act
tac aag gac 1056 Asp Val Gly Arg Leu Ile Phe Ser Gly Val Ala Leu
Thr Tyr Lys Asp 340 345 350 atc gtc gtg tgt atg gat gga gac acc atg
cct ggg ctc ttt gcc tac 1104 Ile Val Val Cys Met Asp Gly Asp Thr
Met Pro Gly Leu Phe Ala Tyr 355 360 365 agg gca tcc acc aag gca ggc
tac tgc gga gga gcc gtc ctg gca aag 1152 Arg Ala Ser Thr Lys Ala
Gly Tyr Cys Gly Gly Ala Val Leu Ala Lys 370 375 380 gac ggg gcc gaa
acg ttc atc gtt ggc acc cac tcc gca ggt gga aac 1200 Asp Gly Ala
Glu Thr Phe Ile Val Gly Thr His Ser Ala Gly Gly Asn 385 390 395 400
ggc ata gga tac tgt tcg tgt gtt tcc cga tca atg ctc ctg aag atg
1248 Gly Ile Gly Tyr Cys Ser Cys Val Ser Arg Ser Met Leu Leu Lys
Met 405 410 415 aag gca cac atc gac cct gaa cca cac cac gag 1281
Lys Ala His Ile Asp Pro Glu Pro His His Glu 420 425 121 1413 DNA
Foot-and-mouth disease virus CDS (1)..(1410) Synthetic Nucleotide
and Amino acid Sequence of 3D Protein 121 ggg ttg atc gtt gat acc
aga gat gtg gaa gag cgc gtc cat gta atg 48 Gly Leu Ile Val Asp Thr
Arg Asp Val Glu Glu Arg Val His Val Met 1 5 10 15 cgc aaa acc aag
ctt gca ccc acc gtc gcg cac ggt gtg ttc aat cct 96 Arg Lys Thr Lys
Leu Ala Pro Thr Val Ala His Gly Val Phe Asn Pro 20 25 30 gag ttc
ggg cct gcc gcc ttg tct aac aag gac cca cgt ctg aac gaa 144 Glu Phe
Gly Pro Ala Ala Leu Ser Asn Lys Asp Pro Arg Leu Asn Glu 35 40 45
ggt gtt gtc ctc gat gaa gtc att ttc tcc aag cat aaa gga gac aca 192
Gly Val Val Leu Asp Glu Val Ile Phe Ser Lys His Lys Gly Asp Thr 50
55 60 aag atg tct gag gag gac aaa gcg ctg ttc cgc cgc tgc gct gct
gac 240 Lys Met Ser Glu Glu Asp Lys Ala Leu Phe Arg Arg Cys Ala Ala
Asp 65 70 75 80 tac gcg tca cgc ctg cac agt gtg ctg ggt acg gca aat
gcc cca ctg 288 Tyr Ala Ser Arg Leu His Ser Val Leu Gly Thr Ala Asn
Ala Pro Leu 85 90 95 agc att tac gag gca atc aag ggc gtt gac gga
ctc gac gcc atg gag 336 Ser Ile Tyr Glu Ala Ile Lys Gly Val Asp Gly
Leu Asp Ala Met Glu 100 105 110 cca gac acc gca cct ggc ctt ccc tgg
gcc ctc cag ggg aaa cgc cgc 384 Pro Asp Thr Ala Pro Gly Leu Pro Trp
Ala Leu Gln Gly Lys Arg Arg 115 120 125 ggt gca ctt atc gat ttc gag
aac ggc acg gtc gga ccc gag gtt gag 432 Gly Ala Leu Ile Asp Phe Glu
Asn Gly Thr Val Gly Pro Glu Val Glu 130 135 140 gct gcc ttg aag ctc
atg gag aaa aga gaa tac aag ttt gtt tgc cag 480 Ala Ala Leu Lys Leu
Met Glu Lys Arg Glu Tyr Lys Phe Val Cys Gln 145 150 155 160 acc ttc
ctg aag gac gaa att cgc ccg atg gag aaa gta cgt gcc ggc 528 Thr Phe
Leu Lys Asp Glu Ile Arg Pro Met Glu Lys Val Arg Ala Gly 165 170 175
aag act cgc att gtc gac gtt ttg cct gtt gaa cac att ctt tac acc 576
Lys Thr Arg Ile Val Asp Val Leu Pro Val Glu His Ile Leu Tyr Thr 180
185 190 agg atg atg att ggc aga ttt tgt gca caa atg cac tca aac aac
ggg 624 Arg Met Met Ile Gly Arg Phe Cys Ala Gln Met His Ser Asn Asn
Gly 195 200 205 ccg cag att ggc tca gcg gtc ggt tgc aac cct gat gtt
gat tgg cag 672 Pro Gln Ile Gly Ser Ala Val Gly Cys Asn Pro Asp Val
Asp Trp Gln 210 215 220 aga ttc ggc aca cac ttc gcc caa tac aga aac
gtg tgg gac gtg gac 720 Arg Phe Gly Thr His Phe Ala Gln Tyr Arg Asn
Val Trp Asp Val Asp 225 230 235 240 tat tcg gcc ttt gat gca aac cac
tgc agc gat gcc atg aac atc atg 768 Tyr Ser Ala Phe Asp Ala Asn His
Cys Ser Asp Ala Met Asn Ile Met 245 250 255 ttt gaa gag gtg ttc cgc
acg gag ttc ggc ttc cac ccg aat gct gag 816 Phe Glu Glu Val Phe Arg
Thr Glu Phe Gly Phe His Pro Asn Ala Glu 260 265 270 tgg att ctg aag
act ctc gtg aac acg gaa cac gcc tat gag aac aag 864 Trp Ile Leu Lys
Thr Leu Val Asn Thr Glu His Ala Tyr Glu Asn Lys 275 280 285 cgc atc
act gtt gaa ggc ggg atg cca tct ggc tgt tcc gca aca agc 912 Arg Ile
Thr Val Glu Gly Gly Met Pro Ser Gly Cys Ser Ala Thr Ser 290 295 300
atc atc aac aca att ttg aat aac atc tac gtg ctc tac gcc ttg cgt 960
Ile Ile Asn Thr Ile Leu Asn Asn Ile Tyr Val Leu Tyr Ala Leu Arg 305
310 315 320 aga cac tat gag ggg gtt gag ctg gac acc tac acc atg atc
tcc tat 1008 Arg His Tyr Glu Gly Val Glu Leu Asp Thr Tyr Thr Met
Ile Ser Tyr 325 330 335 gga gac gac atc gtg gtg gca agc gat tat gat
ctg gac ttt gag gcc 1056 Gly Asp Asp Ile Val Val Ala Ser Asp Tyr
Asp Leu Asp Phe Glu Ala 340 345 350 ctc aag cct cac ttc aaa tct ctt
ggc caa acc att act cca gct gac 1104 Leu Lys Pro His Phe Lys Ser
Leu Gly Gln Thr Ile Thr Pro Ala Asp 355 360 365 aaa agc gac aaa ggt
ttt gtt ctt ggt cac tcc att act gac gtc act 1152 Lys Ser Asp Lys
Gly Phe Val Leu Gly His Ser Ile Thr Asp Val Thr 370 375 380 ttc ctc
aaa aga cac ttc cac atg gat tat ggc act ggg ttt tac aaa 1200 Phe
Leu Lys Arg His Phe His Met Asp Tyr Gly Thr Gly Phe Tyr Lys 385 390
395 400 cct gtg atg gcc tcg aag acc ctc gag gct atc ctc tcc ttt gca
cgc 1248 Pro Val Met Ala Ser Lys Thr Leu Glu Ala Ile Leu Ser Phe
Ala Arg 405 410 415 cgt ggg acc atc cag gag aag ttg att tcc gtg gca
gga ctc gcc gtc 1296 Arg Gly Thr Ile Gln Glu Lys Leu Ile Ser Val
Ala Gly Leu Ala Val 420 425 430 cac tcc gga cca gac gag tac cgg cgt
ctc ttt gag ccc ttc cag ggc 1344 His Ser Gly Pro Asp Glu Tyr Arg
Arg Leu Phe Glu Pro Phe Gln Gly 435 440 445 ctc ttt gag att cca agc
tac aga tca ctt tac ctg cgt tgg gtg aac 1392 Leu Phe Glu Ile Pro
Ser Tyr Arg Ser Leu Tyr Leu Arg Trp Val Asn 450 455 460 gcc gtg tgc
ggc gac gca taa 1413 Ala Val Cys Gly Asp Ala 465 470
* * * * *