U.S. patent application number 14/980673 was filed with the patent office on 2016-04-28 for porcine pseudorabies virus, vaccine composition and preparation method and use thereof.
This patent application is currently assigned to PULIKE BIOLOGICAL ENGINEERING, INC.. The applicant listed for this patent is PULIKE BIOLOGICAL ENGINEERING, INC.. Invention is credited to CHAOYONG BAI, JINZHONG SUN, FEIFEI TAN, KEGONG TIAN, RUI WU, XUKE ZHANG.
Application Number | 20160114029 14/980673 |
Document ID | / |
Family ID | 51365682 |
Filed Date | 2016-04-28 |
United States Patent
Application |
20160114029 |
Kind Code |
A1 |
ZHANG; XUKE ; et
al. |
April 28, 2016 |
PORCINE PSEUDORABIES VIRUS, VACCINE COMPOSITION AND PREPARATION
METHOD AND USE THEREOF
Abstract
Provided in the embodiments of the present invention is a
vaccine composition including an immune amount of attenuated live
vaccine, inactivated vaccine, subunit vaccine, synthetic vaccine,
or genetically engineered vaccine of the porcine pseudorabies virus
strain. The vaccine composition can effectively induce antibody
generation, and prevent infections of virulent strains of the
porcine pseudorabies virus, and provides effective protection for
pigs.
Inventors: |
ZHANG; XUKE; (LUOYANG,
CN) ; SUN; JINZHONG; (LUOYANG, CN) ; WU;
RUI; (LUOYANG, CN) ; TAN; FEIFEI; (LUOYANG,
CN) ; BAI; CHAOYONG; (LUOYANG, CN) ; TIAN;
KEGONG; (LUOYANG, CN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
PULIKE BIOLOGICAL ENGINEERING, INC. |
LUOYANG |
|
CN |
|
|
Assignee: |
PULIKE BIOLOGICAL ENGINEERING,
INC.
LUOYANG
CN
|
Family ID: |
51365682 |
Appl. No.: |
14/980673 |
Filed: |
December 28, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
14779132 |
Sep 22, 2015 |
|
|
|
PCT/CN2014/076691 |
Apr 30, 2014 |
|
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14980673 |
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Current U.S.
Class: |
424/186.1 ;
435/235.1; 536/23.72 |
Current CPC
Class: |
C12N 2710/16721
20130101; A61P 31/22 20180101; A61K 2039/5252 20130101; C07K 14/005
20130101; C12N 2710/16771 20130101; A61K 39/00 20130101; A61K
2039/552 20130101; C07H 21/04 20130101; A61K 39/12 20130101; C12N
7/00 20130101; C12N 7/04 20130101; A61K 39/245 20130101; C12N
2710/16722 20130101; C12N 2710/16734 20130101; C12N 2710/16761
20130101 |
International
Class: |
A61K 39/245 20060101
A61K039/245; C12N 7/00 20060101 C12N007/00; C07K 14/005 20060101
C07K014/005 |
Foreign Application Data
Date |
Code |
Application Number |
May 31, 2013 |
CN |
201310216881.6 |
Sep 18, 2013 |
CN |
201310428605.6 |
Claims
1. A nucleotide sequence substantially encoding the protein as
shown in SEQ ID NO. 1 of the sequence listing.
2. A porcine pseudorabies virus strain comprising gD glycoprotein
encoded by the nucleotide sequence as shown in SEQ ID NO.4 of the
sequence listing.
3. The pseudorabies virus strain as described in claim 2, wherein
said pseudorabies virus strain comprises gB glycoprotein encoded by
the nucleotide sequence shown in SEQ ID NO.5 of the sequence
listing.
4. The pseudorabies virus strain as described in claim 3, wherein
said pseudorabies virus strain is the HN1201 strain or culture
thereof, of which the accession number is CCTCC NO. V 201311; said
HN1201 strain was deposited in the China Center for Type Culture
Collection (CCTCC) on May, 20, 2013, of which the address is Wuhan
University, Wuhan City, Hubei Province.
5. A vaccine composition, comprising an immune amount of attenuated
live vaccine, inactivated vaccine, subunit vaccine, synthetic
peptide vaccine, or genetically engineered vaccine of the porcine
pseudorabies virus strain as described in claim 2.
6. The vaccine composition as described in claim 5, wherein said
pseudorabies virus strain comprises gB glycoprotein encoded by the
nucleotide sequence shown in SEQ ID NO.5 of the sequence
listing.
7. The vaccine composition as described in claim 6, wherein said
pseudorabies virus strain is the HN1201 strain or culture thereof,
of which the accession number is CCTCC NO. V 201311; said HN1201
strain was deposited in the China Center for Type Culture
Collection (CCTCC) on May, 20, 2013, of which the address is Wuhan
University, Wuhan City, Hubei Province.
8. The vaccine composition as described in claim 7, wherein said
vaccine composition comprising an immune amount of attenuated live
vaccine, inactivated vaccine, subunit vaccine, synthetic peptide
vaccine, or genetically engineered vaccine of said PRV HN1201
strain or culture thereof.
9. The vaccine composition as described in claim 8, wherein said
vaccine composition comprises an inactivated vaccine of the PRV
HN1201 strain or culture thereof, of which the concentration is not
less than 10.sup.6.0TCID.sub.50/ml.
10. The vaccine composition as described in claim 8, wherein said
vaccine composition comprises 25-100m/dose gD protein antigen of
the PRV HN1201 strain or culture thereof.
11. The vaccine composition as described in claim 5, wherein said
vaccine composition further comprises medium, adjuvants and
excipient.
12. A method for preparing said vaccine composition as described in
claim 8 comprising the steps: (1) cloning said PRV recombinant gD
gene; (2) expressing said PRV recombinant gD protein; and (3)
mixing said PRV gD protein antigen with adjuvants based on certain
ratio and emulsifying the resulting mixture.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 14/779,132, filed Sep. 22, 2015 which claims
priority to International Application No. PCT/CN2014/076691, filed
Apr. 30, 2014, which claims priority to Chinese Application No.
201310216881.6, filed May 31, 2013 and Chinese Application No.
201310428605.6, filed Sep. 18, 2013, the entire contents of which
are hereby incorporated by reference.
FIELD OF THE INVENTION
[0002] This invention relates to a vaccine composition, belonging
to the field of animal virology.
BACKGROUND
[0003] Pseudorabies, also called Aujeszky's disease, is an acute
infectious disease caused by Suid herpesvirus 1 (SuHV1) belonging
to the Alphaherpesvirinae subfamily for many kinds of livestock
such as swine, cattle and sheep, as well as poultry and wild
animals, with the main symptoms of fever, intense itching (except
swine) and encephalomyelitis. Pseudorabies in swine is found
nationwide in China causing severe damages, and is one of the major
diseases limiting the large-scale production of pig farms.
Infection can result in abortion, stillborn or mummified fetuses in
pregnant sows, and neurological signs, paralysis and a high death
rate in piglets. Pseudorabies virus (PRV) with strong pantropic
properties, neurotropic properties and latent infectivity, may
establish long-term latent infection in the peripheral nervous
system, and then the latently infected host starts to get sick when
the latent virus is activated into the infectious virus.
[0004] It has been indicated by many researches that a
corresponding protection can be provided for the vaccinated animals
by a subunit vaccine, which is a vaccine prepared by cloning the
protective antigen genes of pathogen into prokaryote and eukaryote
expression systems with methods of genetic engineering so as to
highly express those genes. It has been found so far that either of
glycoprotein B, C and D (gB, gC and gD) in the PRV glycoproteins
can make the body generate neutralizing antibodies, which have the
ability to neutralize PRV, no matter in vivo or in vitro, or no
matter with the presence or absence of complements. The article,
Progress in Subunit Vaccine against Pseudorabies Virus Development
(Chenghuai Yang, Gaoming Lou, Nanhui Chen, Jiangxi Journal of
Animal Husbandry & Veterinary Medicine 2004 Issue 3) has
disclosed that either of gB, gC and gD among 11 PRV glycoproteins
which have been found so far, can induce the body to generate
neutralizing antibodies. In the absence of complements, monoclonal
antibodies directed against gB, gC and gD can neutralize PRV. The
swine and mice injected with monoclonal antibodies directed against
gB, gC and gD can resist attacks by virulent PRV strains. Therefore
gB, gC and gD are the most preferred proteins for developing PRV
subunit vaccine. The glycoprotein, gD, is an important neutralizing
antigen as well as the main target for protective antibodies, and
it can induce better protective response. As disclosed by U.S. Pat.
No. 6,858,385 and U.S. Pat. No. 6,521,231, vaccine for preventing
pseudorabies can be prepared by use of gD of porcine pseudorabies
virus.
[0005] The porcine PRV has only one serotype, thus usually the
cross-protection immunity between strains of porcine PRV is
considered to be very strong. However, piglets may still suffer
from typical porcine pseudorabies after their injection with
commercial vaccines, with symptoms such as long-term high fever,
depression, loss of appetite, respiratory and/or neurological
signs. The significant manifestations include that infection among
swine at any ages, horizontal transmission among swine herds, short
incubation period (1.about.2 days), morbidity rates between
10%.about.100%, mortality rate in pigs between 10%.about.100%
(mortality rate in piglets can reach up to 100%), high fever in
pigs after being infected (40.degree. C..about.42.degree. C.,
lasting for more than 3 days), dyspnea, diarrhea, wheezing,
coughing, sneezing, hind limb paralysis, dog sitting, suddenly
falling down, convulsions, lying on their sides, opisthotonus,
making strokes with their arms, and finally dying of exhaustion,
and the infection also can cause reproductive disorder symptoms
such as declined semen quality of boar, as well as abortion of
pregnant sow (the abortion rate can reach up to 35%), premature
birth, stillbirth, weakened piglets (weakened piglets die by 14
days of age), etc. By means of prior art, vaccinated pigs cannot
completely resist attacks by the wild virus, and still have
symptoms like high fever, depression, partially or completely loss
of appetite, with a infection rate of more than 30% and a mortality
rate between 10% and 20%. There are no vaccines in the prior art
capable of solving the pseudorabies caused by variant strains of
porcine pseudorabies virus.
SUMMARY OF INVENTION
[0006] In order to solve the deficiency of the prior art, the
present invention aims to provide a porcine pseudorabies virus
strain for preparing vaccines, which have been proved by animal
testing to provide a good immune function for porcine
pseudorabies.
[0007] The present invention provides a nucleotide sequence
substantially encoding the protein as shown in SEQ ID NO. 1 of the
sequence listing.
[0008] The present invention provides a nucleotide sequence
substantially encoding the protein as shown in SEQ ID NO.2 of the
sequence listing.
[0009] The present invention provides a nucleotide sequence
substantially encoding the protein as shown in SEQ ID NO. 3 of the
sequence listing.
[0010] The "nucleotide sequence" in the present invention refers to
a deoxyribonucleic acid (DNA) sequence, which can be transcribed
into a corresponding RNA sequence.
[0011] To solve the deficiency of the prior art, the main aim of
present invention is to provide a porcine pseudorabies virus strain
comprising gD glycoprotein encoded by the nucleotide sequence as
shown in SEQ ID NO.4 of the sequence listing.
[0012] The term "gD glycoprotein" in the present invention refers
to a structural protein required for infection of PRV, which is one
of the major glycoproteins in the surface of envelope of mature
virus particles, also called gp50 protein.
[0013] The term "homology" in the present invention refers to the
level of similarity between two amino acid sequences or two
nucleotide sequences. The homology between amino acid sequences or
nucleotide sequences can be calculated by any appropriate methods
well known in the art, for example, the target amino acid (or
nucleotide) sequence and the reference amino acid (or nucleotide)
sequence is aligned, and gaps can be induced if necessary to
optimize the number of the identical amino acids (or nucleotides)
between two aligned sequences, and the percentage of the identical
amino acids (or nucleotides) between two aligned sequences can be
calculated accordingly. Alignment of amino acid (or nucleotide)
sequences and calculation of homology can be achieved by software
well kwon in the art. Examples of such software include, but is not
limited to, BLAST (which can be accessed through the website of the
National Center for Biotechnology Information, NCBI,
http://blast.ncbi.nlm.nih.gov/Blast.cgi or can be found in Altschul
S. F. et al, J. Mol. Biol, 215:403-410 (1990); Stephen F. et al,
Nucleic Acids Res., 25:3389-3402(1997)), ClustalW2 (which can be
accessed through the website of the European Bioinformatics
Institute, EBI, http://www.eji.ac.uk/Toolsa/clustalw2/, or can be
found in Higgins D. G et al, Methods in Enzymology,
266:383-402(1996); Larkin M. A. et al, Bioinformatics (Oxford,
England), 23(21):2947-8(2007)), and TCoffee (which can be accessed
through the website of the Swiss Institute of Bioinformatics, SIB,
http://tcoffee.vital-it.ch/cgi-bin/Tcoffee/tcoffee_cgi/index.cgi,
or can be found in, Poirot O. et al, Nucleic Acids Res.,
31(13):3503-6(2003); Notredame C. et al, J. Mol. Boil,
302(1):205-17(2000)) etc. It is all within the knowledge scope of a
person skilled in the art that when using the software to do
sequence alignment, he can use the default parameters provided by
the software or adjust the parameters provided by the software
according to the actual condition.
[0014] Preferably, said PRV strain comprises the gB glycoprotein
encoded by the nucleotide sequence shown in SEQ ID NO.5 of the
sequence listing.
[0015] Preferably, said PRV comprises the gB glycoprotein encoded
by the amino acid sequence as shown in SEQ ID NO.2 of the sequence
listing.
[0016] Preferably, said PRV according to the invention comprises
the gC glycoprotein encoded by the amino acid sequence as shown in
SEQ ID NO.3 of the sequence listing.
[0017] Preferably, said PRV strain is HN1201 strain (pseudorabies
virus, strain HN1201), or culture thereof, of which the accession
number is CCTCC NO. V 201311; the HN1201 strain was deposited in
the China Center for Type Culture Collection (CCTCC) on May, 20,
2013, of which the address is Wuhan University, Wuhan City, Hubei
Province.
[0018] The term "culture" refers to cultures of different passages
of the virus, known to those skilled in the art, which may only
have minute variations from one passage to another. Preferably said
culture is a culture within 5-35 passages.
[0019] Another aim of the invention is to provide a vaccine
composition comprising an immune amount of attenuated live vaccine,
inactivated vaccine, subunit vaccine, synthetic peptide vaccine, or
genetically engineered vaccine of the porcine pseudorabies virus
strain.
[0020] Preferably the vaccine composition comprises an immune
amount of attenuated live vaccine, inactivated vaccine, subunit
vaccine, synthetic peptide vaccine, or genetically engineered
vaccine of the said PRV HN1201 strain or culture thereof.
[0021] Preferably the vaccine composition according to the present
invention comprises said PRV or antigen thereof as an active
component. The PRV in the vaccine composition comprises gD
glycoprotein represented by the amino acid sequence of SEQ ID NO. 1
or the amino acid sequence which shares at least 98% homology to
the sequence of SEQ ID NO. 1.
[0022] Preferably the PRV in said vaccine composition is the HN1201
strain or culture thereof.
[0023] The antigen used in the invention is the antigen ingredients
in the components of virus, which induces the immune response, and
comprises gD protein with the amino acid sequence of SEQ ID NO.
1.
[0024] Optionally, the antigen may comprise gB protein with the
amino acid sequence of SEQ ID NO. 2 or the one of which the
fragment shares at least 95% homology to the sequence of SEQ ID NO.
2.
[0025] Optionally, the antigen may comprise gC protein with the
amino acid sequence of SEQ ID NO.3 or the one of which the fragment
shares at least 95% homology to the sequence of SEQ ID NO. 3.
[0026] As used herein, the term "live vaccines" refer to vaccines
prepared by viruses which still can reproduce in the host or on the
cells and in the meanwhile their virulence has been weakened. As
used herein, the term "attenuated" refers to artificially reducing
the virulence of pathogens via mutation of gene by preparing
pathogens which are deprived of pathogenicity but maintain
immunogenicity. Generally attenuation can be achieved by UV
irradiation, chemical processing or continuous high-order
subculturing in vitro. Artificial alteration of gene attenuates the
virulence via, for example, the deletion of some specific
nucleotides in the given sequence.
[0027] As used herein, the term "inactivated vaccine", also called
non-living vaccine, refers to suspension of inactivated virus used
as an antigen for producing immunity Examples of inactivated
vaccines include whole virus vaccines and split virus vaccines. By
using known methods it is easy to produce inactivated vaccines. For
instance, one can obtain inactivated whole virus vaccines by
treatment with formaldehyde solution. Split virus vaccines can be
prepared with virus envelopes after treatment with ether.
[0028] The term "subunit vaccine" refers to a vaccine prepared via
highly effectively expression of protective antigen gene of a
pathogen by cloning it into a prokaryotic or eukaryotic expression
system. A subunit vaccine has less risk of adverse reactions than a
whole virus vaccine. For example, the expressed gD protein or gC
protein of PRV can be used for preparing subunit vaccines.
[0029] The term "synthetic peptide vaccine" refers to a small
peptide only comprising the component of immunogenic determinants,
i.e. a vaccine prepared by synthesizing a protective short peptide
according to the amino acid sequence of natural proteins, and
adding in an adjuvant after connecting them to a carrier.
[0030] Preferably, said vaccine composition in the invention
comprises an inactivated vaccine of the PRV HN1201 strain or
culture thereof, of which the content is not less than
10.sup.6.0TCID.sub.50/ml.
[0031] Preferably, said vaccine composition in the present
invention may comprise 10.sup.6.0TCID.sub.50/ml PRV per pig. The
vaccine cannot effectively trigger the generation of antibodies
when the amount of said PRV used is less than
10.sup.6.0TCID.sub.50. On the other hand the excessive amount may
not be economical.
[0032] Preferably, said vaccine composition comprises 25-100 m/dose
of gD protein antigen of the PRV HN1201 strain or culture
thereof.
[0033] In addition, said pseudorabies vaccine in the present
invention can be used conjunctly with other inactivated pathogens
or antigen to prepare combined vaccines or complex vacancies
against various diseases including pseudorabies. As used herein,
the term "combined vaccine" refers to a vaccine prepared with the
virus mixture by mixing the pseudorabies virus in the present
invention with at least one different virus. The term "complex
vaccine" refers to a vaccine prepared from viruses and bacterium.
For example, the pseudorabies virus in the present invention can be
mixed or combined with classical swine fever virus, porcine
reproductive and respiratory syndrome virus, porcine circovirus
and/or haemophilus parasuis and mycoplasma.
[0034] Preferably, said vaccine composition further comprises
medium, adjuvants and excipients.
[0035] Said vaccine composition according to the present invention
also may comprise medium, adjuvants and/or excipients.
Physiological saline or distilled water can be used as medium.
Examples of adjuvants used in the vaccine composition include
Freund's incomplete adjuvant or Freund's complete adjuvant,
aluminum hydroxide gel, vegetable oil or mineral oil etc. Examples
of excipients include, but are not limited to, aluminum phosphate,
aluminum hydroxide and potassium aluminum sulfate. In practice, all
substances for preparing vaccines, known to those skilled in the
art, can be adapted to the vaccine composition in the present
invention.
[0036] One more aim of the present invention is to provide a method
for preparing said vaccine composition comprising the steps: the
PRV HN1201 strain is amplified and cultured, inactivated, and then
added with adjuvants and mixed thoroughly.
[0037] Specifically, the method comprises the steps: (1)
inoculating the PRV vaccine strains into respective susceptible
cells, and cultivating the inoculated susceptible cells; and then
harvesting cell culture; and (2) treating the viruses obtained from
step (1) with formaldehyde solution, BPL
(.beta.-beta-Propiolactone) or BEI (binary ethylenimine).
[0038] The susceptible cells can be continuous cell lines or
primary cell lines. The susceptible cells adapted to PRV culture
include but are not limited to continuous cell lines such as ST
cell line (ATCC CRL-1746), PK-15 cell line(ATCC CCL-33), African
green monkey kidney Marc-145 cell line (ATCC CRL-12219),
Madin-Darby bovine kidney MDBK cell line (ATCC CCL-22), bovine
turbinate BT cell line (ATCC CRL-1390), Vero cell line (ATCC
CCL-81), BHK-21 cell line (ATCC CCL-10), pig kidney continuous cell
line (such as IBRS-2, refer to e.g. DECASTRO, M.P.1964. Behavior of
foot and mouth disease virus in cell culture: susceptibility of the
IB-RS-2 swine cell line. Arquivos Instituto Biologica 31:63-78),
rabbit kidney continuous cell line (RK, e.g. ATCC CCL-106) etc.,
and primary cell lines such as chicken embryo fibroblasts and
porcine kidney cells etc. The primary cells can be isolated and
prepared from tissues of animals via methods well known in the
art.
[0039] The vaccine composition according to the present invention
can be prepared into oral dosage forms or non-oral dosage forms.
Non-oral dosage forms are preferred which can be administrated via
intradermal route, intramuscular route, intraperitoneal route,
intravenous route, subcutaneous route, intranasal route or epidural
route.
[0040] Another aim of the invention is to provide a method for
preparing the vaccine composition, comprising the steps: (1)
cloning said PRV recombinant gD gene; (2) expressing said PRV
recombinant gD protein; and (3) mixing said PRV gD protein antigen
with adjuvants based on certain ratio and emulsifying the resulting
mixture.
[0041] A further aim of the invention is to provide a use of said
vaccine composition for preparing medicine for treatment and
prevention of diseases relating to PRV.
[0042] As used herein, the term "diseases relating to PRV" can
further refer to diseases caused by infection of PRV. Examples
includes but are not limited to, obvious neurological signs,
lethargy, crying, vomiting diarrhea and fever in infected piglets,
and abortion, mummified or stillborn fetuses or reproductive
disorder in infected pregnant sows.
[0043] As used herein, the term "diseases relating to PRV" can
further refer to diseases with significant manifestations including
but not limited to infection among swine at any ages, horizontal
transmission among swine herds, short incubation period (1-2 days),
morbidity rates between 10%-100%, mortality rate in pigs between
10%-100% (mortality rate in piglets can reach up to 100%), high
fever of pigs after being infected (40.degree. C..about.42.degree.
C., lasting for more than 3 days), dyspnea, diarrhea, wheezing,
coughing, sneezing, hind limb paralysis, dog sitting, suddenly
falling down, convulsions, lying on their sides, opisthotonus,
making strokes with their arms, and finally dying of exhaustion,
and reproductive disorder symptoms caused by infection such as
declined semen quality of boar, as well as abortion of pregnant sow
(the abortion rate can reach up to 35%), premature birth,
stillbirth, weakened piglets (weakened piglets die by 14 days of
age), etc. The differences between above described symptoms and
symptoms caused by infection of regular pseudorabies virus in the
prior art are: in adult pigs (whose weight is above 50 kg), high
fever of infected pigs (40.degree. C..about.42.degree. C., lasting
for more than 3 days), dyspnea, diarrhea, wheezing, coughing,
sneezing, hind limb paralysis, dog sitting, suddenly falling down,
convulsions, lying on their sides, opisthotonus, making strokes
with their arms, and finally dying of exhaustion; sudden incidence
of pseudorabies in newborn piglets and piglets below the age of 4
weeks , further resulting in massive death with a mortality of more
than 90%; main manifestations in infected piglets including
increased body temperature over 41.degree. C., completely loss of
appetite, obvious neurological signs and diarrhea; and in piglets
just before or after being weaned, mainly respiratory symptoms,
such as dyspnea, coughing and runny noses, etc.
[0044] As used herein, the term "prevention" refers to all
behaviors to inhibit the infection of pseudorabies virus or delay
the onset of the disease via administration of the vaccine
composition according to the present invention. The term
"treatment" refers to all behaviors to relieve or cure the symptoms
caused by infection of PRV via administration of the vaccine
composition according to the present invention.
BRIEF DESCRIPTION OR THE DRAWINGS
[0045] FIG. 1. Result of homology analysis between amino acid
sequences of gC in HN1201 strain and SA215 strain.
[0046] FIG. 2. Result of homology analysis between amino acid
sequences of gD in HN1201 strain and SA215 strain.
SEQUENCE LISTING
[0047] SEQ ID NO. 1 is the amino acid sequence of gD in the PRV
HN1201 strain.
[0048] SEQ ID NO. 2 is the amino acid sequence of gB in the PRV
HN1201 strain.
[0049] SEQ ID NO. 3 is the amino acid sequence of gC in the PRV
HN1201 strain.
[0050] SEQ ID NO. 4 is the nucleotide sequence of gD in the PRV
HN1201 strain.
[0051] SEQ ID NO. 5 is the nucleotide sequence of gB in the PRV
HN1201 strain.
[0052] SEQ ID NO.6 is the nucleotide sequence of gC in the PRV
HN1201 strain.
DETAILED DESCRIPTION
[0053] The description of the present invention is further provided
as follows with reference to the specific embodiments, and features
and advantages of the present invention will become more apparent
from the following description. However, these embodiments are only
exemplary, but not forming any limitation to the scope of the
present invention. It should be understood by a person skilled in
the art that modifications or alternatives to details and forms of
the technical solution of the present invention without deviation
from the spirit and scope of the present invention will be allowed,
while those modification and alternatives should all fall within
the scope of the present invention.
[0054] In the invention, the term "per pig" refers to the amount of
vaccine each pig injected.
[0055] In the invention, the term "TCID.sub.50" refers to 50%
tissue culture infective dose, a way to represent viral
infectivity.
[0056] Minimum Essential Medium (MEM) liquid medium is prepared
with MEM dry powdered medium purchased from Life Technologies,
Corp. according to the instruction.
[0057] Dulbecco's Modified Eagle's Medium (DMEM) in the present
invention is prepared with reference to the preparation method from
Appendix A of GB/T18641-2002 Diagnostic Techniques for Aujeszk's
Disease.
[0058] In the present invention, the term "PBS" is the abbreviation
for Phosphate Buffer Saline, and 0.01 mM pH 7.4 PBS as used in the
present invention is prepared as described in Molecular cloning:
Laboratory manuals, 3rd edition.
EXAMPLE 1
[0059] Collection and Isolation of Viruses
[0060] Porcine brain tissue was collected under aseptic conditions
from samples isolated from samples from Henan province suspected of
having pseudorabies infection, added in MEM liquid medium in a
ratio of 1:10(V:V), and ground to prepare a tissue suspension.
After 3 times of repeated freezing-thawing, the tissue suspension
was centrifuged at 2000r/min for 15 minutes. The supernatant was
then collected, filtered through a 0.2 .mu.m pore filter,
subcultured on PK-15 cells and incubated at 37.degree. C. for lh,
and then the medium was changed by adding MEM liquid medium
supplemented with 2% fetal bovine serum, and incubated at
37.degree. C. for 5 days. PRV was detected by PRV PCR detection kit
(Beijing Anheal Laboratories Co., Ltd), and the result was
positive; PCR kit was employed to detect the exotic virus
contamination (porcine reproductive and respiratory syndrome virus
RT-PCR detection kit kit, porcine parvovirus PCR detection kit and
classical swine fever virus RT-PCR detection kit, all purchased
from Beijing Anheal Laboratories Co., Ltd) for the isolated virus
and the PCR detection result was negative, indicating a pure viral
specie.
[0061] The isolated PRV strain was deposited in the China Type
Culture Collection on May, 20, 2013 named HN1201 strain
(Pseudorabies virus, strain HN1201) of which the accession number
is CCTCC NO. V 201311 and the preservation address is Wuhan
University, Wuhan City, Hubei Province.
EXAMPLE 2
[0062] Genetic Characteristics of the Isolated Virus
[0063] Genetic characteristics of the isolated virus in Example 1
were determined by means of gene analysis. Genomic DNA prepared
from the pseudorabies virus isolated from PK-15 cells was used as
template with primers shown in Table 1 for PCR amplification
reactions. The Primer Primer 5.0 was used for designing the primer
sequence for amplifying gB, gC and gD gene, respectively.
[0064] The genomic DNA extracted was used as template to prepare
the following PCR amplification system: 100 .mu.g template DNA, 0.5
.mu.L PrimerSTAR HS DNA Polymerase (2.5U/.mu.L), 25 .mu.L 2
.times.PrimerSTAR GC Buffer, 1 .mu.L of each upstream and
downstream primer (10 pmol/.mu.L), 4 .mu.L dNTP Mix (2.5 mM each),
adjusted to a final volume of 50 .mu.L with distilled water.
Two-step PCR reaction was carried out by an initial denaturation
for 10 sec at 98.degree. C. followed by annealing and extension at
68.degree. C. (all the time is calculated by 1 kb/min) and there
were 30 cycles in total. The PCR reactions were finalized at
4.degree. C. The analysis of the amplification products was
conducted by electrophoresis on 1% agarose gel containing ethidium
bromide. The sequences of PCR products were determined. The
sequence data was analyzed by Lasergene.
TABLE-US-00001 TABLE 1 PCR primer sequences Target Size of gene
Primer sequence (5'.fwdarw.3') PCR product gB
aagcgcatctttattgtttcccg 2957 bp ggcttctaccgcttccagacgg gC
accgtcgccatgtgcgccacta 1603 bp cgggtcggactcgctgtcgtttatt gD
ttcccatacactcacctgccagc 1250 bp tcgacgccggtactgcggag
EXAMPLE 3
[0065] Pathogenicity Test of the Virus
[0066] 3.1 Pathogenicity of the Virus in Piglets at Different Days
of Age
[0067] 6 piglets at 34.about.35 days of age which were negative for
pseudorabies antibodies were randomly divided into 2 groups, one
with 4 piglets (experimental group) and the other with 2 piglets
(control group), wherein the experimental group was inoculated with
PRV HN1201 strain by intranasal instillation (challenge dosage is
2.times.10.sup.8.0TCID.sub.50/piglet) and the control group was
inoculated with DMEM medium. Meanwhile 4 piglets at 49 days of age
were inoculated with third passage of the virulent HN1201 strain
after preservation (challenge dose was
2.times.10.sup.8.0TCID.sub.50/piglet), and the control is still
piglets at 35 days of age. After inoculation of virus, the
temperature of piglets was determined daily, and clinical signs and
death status were observed. The results are shown in Table 2.
TABLE-US-00002 TABLE 2 Pathogenicity of PRV HN1201 strain in
piglets at different days of age Num- Inoculation Group ber Days
Dose Clinical signs Death status 1 A1 35 2 .times.
10.sup.8.0TCID.sub.50/ Body Temper- Died on day piglet ature
increased, 4 after viral depression, challenge A2 loss of appetite,
Died on day onset of respi- 5 after viral ratory and/or challenge
A3 neurological Died on day signs 5 after viral challenge A4 Died
on day 6 after viral challenge 2 B1 49 2 .times.
10.sup.8.0TCID.sub.50/ Body Temper- Died on day piglet ature
increased, 7 after viral depression, challenge B2 loss of appetite,
Died on day onset of respi- 7 after viral ratory and/or challenge
B3 neurological Died on day signs 5 after viral challenge B4
Survived 3 C1 35 DMEM control Normal Survived C2 Survived
[0068] It showed in the results that inoculation with PRV HN1201
strain in piglets at different days of age could lead to onset of
pseudorabies in piglets, as well as death of over 3/4 of inoculated
piglets.
[0069] 3.2 Pathogenicity of the Virus at Different Doses in
Piglets
[0070] 8 piglets at 49 days of age which were negative for
pseudorabies antibodies were randomly divided into 2 groups, each
with 4 piglets, in addition two more piglets were used as control.
The experimental groups were inoculated with
2.times.10.sup.7.0TCID.sub.50/piglet PRV HN1201 strain or
2.times.10.sup.8.0TCID.sub.50/piglet PRV HN1201 strain by
intranasal instillation, respectively, and the control group was
inoculated with DMEM medium. After inoculation of virus, the body
temperature of piglets was determined daily, and clinical signs and
death status were observed. The results are shown in Table 3.
TABLE-US-00003 TABLE 3 Pathogenicity of different doses of PRV
HN1201 strain in piglets Group Number Inoculation Dose Clinical
signs Death status 1 A1 2 .times. Significant Died on day 5
10.sup.7.0TCID.sub.50/piglet clinical signs: after viral
temperature challenge A2 increased, Died on day 6 depression, after
viral loss of challenge A3 appetite Died on day 6 after viral
challenge A4 Died on day 6 after viral challenge 2 B1 2 .times.
Significant Died on day 2 10.sup.8.0TCID.sub.50/piglet clinical
signs: after viral temperature challenge B2 increased, Died on day
3 depression, after viral loss of challenge B3 appetite Died on day
4 after viral challenge B4 Died on day 4 after viral challenge 3 C1
DMEM control Normal Survived C2 Survived
[0071] It showed in the results that inoculation with different
doses of clinically isolated PRV HN1201 strain in piglets at 49
days of age could all lead to onset of pseudorabies in piglets, of
which 4/4 died.
EXAMPLE 4
[0072] Preparation of the Inactivated PRV Vaccine
[0073] The culture of different passages of the isolated strain was
inoculated, according to Table 4, onto PK-15 cell culture to form a
seed lot which was then inoculated into a monolayer of PK cell
culture at 1% (V/V) of the amount of the liquid virus medium, and
placed in a rotary incubator at 37.degree. C. The cell medium
containing viruses was harvested when the cytopathic effect of
cells reached to 80%; the viruses were harvested after 2 times of
freezing-thawing and the virus titer was assessed. 10% (V/V)
formaldehyde solution was added to different passages of virus
solution respectively, with a final concentration of 0.2% (V/V).
The virus solution was inactivated at 37.degree. C. for 18 hours,
being stirred for 10 min every 4 hours, and diluted with pH 7.4
phosphate-buffered saline (PBS) to the content of viruses as shown
in Table 4, mixed with 206 adjuvant (SEPPIC, France) according to
the volume ratio of 54:46, and stirred at 120 rpm for 15 min at
30.degree. C.
TABLE-US-00004 TABLE 4 Preparation of each group of pseudorabies
vaccines Ratio of vaccines (inactivated virus Passage number of
Content of virus solution:206 Group HN1201 culture before
inactivation adjuvant) A 5 10.sup.8.43TCID.sub.50/mL 54:46 B 35
.sup. 10.sup.6.0TCID.sub.50/mL 54:46
EXAMPLE 5
[0074] Immunogenicity Assay of Inactivated Vaccines
[0075] 16 piglets at 21 days of age which were negative for PRV
antibodies were randomly divided into 4 groups, each with 4
piglets, and injected with vaccines according to Table 5. Two
groups injected with inactivated vaccines were injected with
2ml/piglet of the inactivated vaccines against pseudorabies
prepared in Example 4. As a control vaccine, the live vaccine SA215
strain prepared by using the method in CN101186902 was applied
according to the method for determining immunogenicity in the
specification of the patent. The control group was inoculated with
2 mL/piglet of DMEM medium.
TABLE-US-00005 TABLE 5 Grouping of immunogenicity assay Group
Vaccines injected Dose Group A injected with Group A vaccine 2
mL/piglet inactivated vaccine prepared in Example 4 Group B
injected with Group B vaccine 2 mL/piglet inactivated vaccine
prepared in Example 4 Group injected with Live Live PRV vaccine
10.sup.6.0TCID.sub.50/piglet vaccine SA215 Control group DMEM
medium 2 mL/piglet
[0076] After immunization with vaccines, neutralizing titers of
antibodies of the inactivated vaccines groups were determined
weekly according to the method of serum neutralization test from
GB/T 18641-2002 Diagnostic Techniques for Aujeszk's Disease. The
results are shown in Table 6.
TABLE-US-00006 TABLE 6 Nneutralizing titers of antibodies at
different time in piglets after immunization with PRV inactivated
vaccines Average value of neutralizing titers of antibodies at
different time (weeks) Group 1 2 3 4 Group A injected 1:4.8 1:11.2
1:16.0 1:37.7 with inactivated vaccine Group B injected 1:4.0 1:6.3
1:13.5 1:22.4 with inactivated vaccine Group injected 1:3.7 1:4.0
1:10.0 1:16.0 with live vaccine Control group Negative Negative
Negative Negative
[0077] The result from Table 6 indicated that immunization with
inactivated PRV vaccines in piglets can produce high neutralizing
titers which increase with immunization time.
[0078] The piglets were challenged with
2.times.10.sup.8.0TCID.sub.50/piglet of PRV HN1201 strain on day 28
after immunization, clinical signs and death status were observed
as shown in Table 7. After challenge the body temperature of
piglets was determined daily as shown in Table 8.
TABLE-US-00007 TABLE 7 Viral challenge for piglets after
immunization with PRV inactivated vaccines Rate of Group clinical
signs and death status protection Group A injected with Body
temperature increased 100% (4/4) inactivated vaccine for 2-3 days,
normal appetite, basically no neurological signs, survived Group B
injected with Body temperature increased 100% (4/4) inactivated
vaccine for 2-3 days, normal appetite, basically no neurological
signs, survived Group injected with live Body temperature increased
100% (4/4) vaccine for 7-10 days, loss of appetite, significant
neurological signs, survived Control group Significant signs, two
piglets 0% (0/4) died on day 2 after challenge, and all died within
3 days after challenge.
TABLE-US-00008 TABLE 8 Body temperature change after challenge for
piglets immunized with PRV vaccines Group A Group B Group injected
with injected with injected inactivated inactivated with live Day
vaccine vaccine vaccine day 1 after challenge 39.5 39.6 39.4 day 2
after challenge 41.2 41.6 41.8 day 3 after challenge 40.5 40.3 41.2
day 4 after challenge 40.2 39.7 41.6 day 5 after challenge 39.7
39.5 41.4 day 6 after challenge 39.6 39.4 41.3 day 7 after
challenge 39.7 39.5 41.4 day 8 after challenge 39.5 39.7 41.2 day 9
after challenge 39.6 39.4 41.5 day 10 after challenge 39.2 39.5
40.6 day 11 after challenge 39.5 39.7 39.7 day 12 after challenge
39.4 39.5 39.8 day 13 after challenge 39.3 39.4 39.7 day 14 after
challenge 39.2 39.1 39.4
[0079] The results from Table 7 and 8 indicated that immunization
of piglets with inactivated PRV vaccines could provide a 100% (4/4)
protection rate for piglets, even though the infection of viruses
could not be avoided (they showed clinical signs), while all
piglets in the control group died on day 4 after challenge,
therefore the inactivated PRV vaccines can provide excellent
protection. In addition, compared with the live vaccine as the
control vaccine, it took less time for the body temperature of
piglets immunized with the inactivated vaccines to increase, and
also they kept a basically normal appetite with no clinical signs,
indicating excellent immune protection.
EXAMPLE 6
[0080] Preparation of the PRV gD Protein
[0081] 1. Amplification of the PRV gD Gene
[0082] The PK-15 cells which were in excellent health were
inoculated with the PRV HN1201 strain or culture thereof of
different passages (the PRV strain was HN1201 strain (Pseudorabies
virus, strain HN1201), of which the accession number was CCTCC NO.
V 201311; the HN1201 strain was deposited in the China Center for
Type Culture Collection (CCTCC) on May, 20, 2013, of which the
address was Wuhan University, Wuhan City, Hubei Province), the
culture of different passages was the culture within 5-35 passages.
The PRV genomic DNA was extracted by using MiniBEST Viral RNA/ DNA
Extraction Kit Ver.3.0 (TAKARA) after harvesting viruses. PCR
amplification was performed by using 1 .mu.L genomic DNA as
template and gD-specific primers:
TABLE-US-00009 gDSF: 5' ATGCTGCTCGCAGCGCTATTGGC 3' and gDSR: 5'
CTACGGACCGGGCTGCGCTTTTAG3'.
[0083] The high fidelity polymerase, Prime STAR.RTM. HS DNA
Polymerase with GC Buffer (TAKARA) was used in the PCR reaction.
The amplification conditions were: 94.degree. C. 3min; 94.degree.
C. 30 s, 68.degree. C. 90 s, 30 cycles; 72.degree. C. 5min. The PCR
product was named gD, of which the nucleotide sequence is shown in
SEQ No.4, and the amino acid sequence can be derived as shown in
SEQ No.1.
[0084] 2. Acquisition and Identification of Recombinant Bacmid
[0085] The PCR product, gD obtained from amplification with the
high fidelity polymerase was cloned into the pFastBac/HBM-TOPO
vector (Invitrogen, A11339). The cloning system was as follows: 4
.mu.L PCR product, gD, 1 .mu.L salt solution, 1 .mu.L TOPO vector;
60 .mu.L in total. The mixture was mixed thoroughly and incubated
at room temperature for 5 minutes, and then used to transform One
Shot.RTM. Machl.TM. T1R competent cells. The transformation mix was
spread onto plates containing ampicillin. A single colony was
picked to identify insert directionality of gD gene, and the
plasmid with the correct insert directionality was delivered to
Invitrogen for sequencing, in order to verify the correct sequence.
The plasmid with the correct sequence was named
pFastBac/HBM-TOPO-gD.
[0086] After the pFastBac/HBM-TOPO-gD plasmid was transformed into
DH10Bac competent cells, transposition occurred between the
pFastBac/HBM-TOPO-gD plasmid and the shuttle plasmid Bacmid in the
competent cells, and the resulting recombinant plasmid was
extracted by using PureLink.TM. HiPure Plasmid DNA Miniprep Kit
(Invitrogen), and the insertion of gD was identified with pUCM13
Forward/pUCM13 Reverse primer. The positive Bacmid was named
Bacmid-gD.
[0087] 3. Transfection for Obtaining Recombinant Baculovirus
[0088] This step was carried out based on the method provided by
the instruction of Bac-to-Bac HBM TOPO Secreted Expression System
(Invitrogen). 8.times.10.sup.5 sf9 cells were layered in each well
of a 6-well plate, transfection was performed according to the
instruction of Cellfectin.RTM. II transfection agent after
adherence of the cells: 8 .mu.L Cellfectin.RTM. II and 1 .mu.g
Bacmid-gD DNA were diluted respectively with 100 .mu.L SF-900 II
medium and mixed by vortex. The diluted DNA was combined with the
diluted Cellfectin.RTM. II (total volume .about.210 .mu.L), mixed
thoroughly and incubated for 15-30 minutes at room temperature. The
transfection mixture was then added dropwise onto the cells. The
supernatant of cell culture, marked as PO recombinant baculovirus
vBac-gD, was collected 72 h after transfection or until the
cytopathic effect was visible. The PO recombinant baculovirus
vBac-gD infected sf9 cells, and after three rounds of amplification
the resulting P3 recombinant baculovirus vBac-gD was used for
expressing the recombinant protein.
[0089] 4. Infection of High-Five Cells with the Recombinant
Baculovirus for Expression of the Recombinant Protein
[0090] The P3 recombinant baculovirus vBac-gD was inoculated in
High-five cells (Invitrogen, B85502). Suspension culture of
High-five cells was performed in a 500 mL Erlenmeyer flask and the
cells were inoculated with the virus with an MOI of 1 when the cell
density reached to 7.0.times.10.sup.5 cell/mL. The supernatant of
cell culture was collected 72 h after infection. A Tangential Flow
Filtration System (Millipore) was employed to concentrate the
volume into 1/10 of the original one. 1% (V%) of Triton X-100
(Sigma) was used to inactivate the baculovirus, and the content of
protein determined by SDS-PAGE optical density method was 200
.mu.g/mL.
EXAMPLE 7
[0091] Preparation of the PRV Subunit Vaccines
[0092] The subunit antigens prepared in Example 6 were diluted with
PBS (pH=7.4) to the contents of protein in Table 9, mixed with 206
adjuvant (SEPPIC, France) at a volume ratio of 54:46, and stirred
at 120 rpm for 15 min at 30.degree. C.
TABLE-US-00010 TABLE 9 Preparation of each group of the
pseudorabies subunit vaccines Ratio of vaccines (inactivated virus
Group Content of protein solution:206 adjuvant) A 25 .mu.g/mL 54:46
B 100 .mu.g/mL 54:46
EXAMPLE 8
[0093] Immunogenicity Assay of the Vaccines in Pigs 12 piglets at
21 days of age which were negative for PRV antibodies were randomly
divided into 3 groups, each with 4 piglets, and injected with 2
ml/piglet of the subunit vaccines against PRV prepared in Example 7
according to Table 10. The control group was inoculated with 2
mL/piglet of DMEM medium.
TABLE-US-00011 TABLE 10 Grouping of immunogenicity assay Group
Vaccines injected Dose Group A injected with Group A vaccine
prepared 2 mL/piglet subunit vaccine in Example 7 Group B injected
with Group B vaccine prepared 2 mL/piglet subunit vaccine in
Example 7 Control group DMEM medium 2 mL/piglet
[0094] The piglets were challenged with
2.times.10.sup.8.0TCID.sub.50/piglet of PRV HN1201 strain on day 28
after immunization, and clinical signs and death status were
observed as shown in Table 11. After challenge the body temperature
of piglets was determined daily as shown in Table 12.
TABLE-US-00012 TABLE 11 Viral challenge for piglets after
immunization with the PRV subunit vaccines Rate of Group Clinical
signs and death status protection Group A injected with Body
temperature increased 100% (4/4) subunit vaccine for 2-3 days,
normal appetite, basically no neurological signs, survived Group B
injected with Body temperature increased 100% (4/4) subunit vaccine
for 2-3 days, normal appetite, basically no neurological signs,
survived Control group Significant signs, two died on 0% (0/4) day
2 after challenge, and all died within 3 days after challenge.
TABLE-US-00013 TABLE 12 Body temperature change of piglets
immunized with the PRV subunit vaccines after challenge Group A
injected with Group B injected with Day subunit vaccine subunit
vaccine day 1 after challenge 39.5 39.5 day 2 after challenge 41.2
41.6 day 3 after challenge 40.3 40.2 day 4 after challenge 40.1
39.7 day 5 after challenge 39.6 39.5 day 6 after challenge 39.6
39.3 day 7 after challenge 39.6 39.5 day 8 after challenge 39.5
39.4 day 9 after challenge 39.5 39.6 day 10 after challenge 39.2
39.3 day 11 after challenge 39.3 39.4 day 12 after challenge 39.4
39.4 day 13 after challenge 39.2 39.4 day 14 after challenge 39.2
39.1
[0095] The results from Table 11 and 12 indicated that immunization
with the PRV subunit vaccines for piglets could provide a 100%
(4/4) protection rate for piglets, even though the infection of
viruses could not be avoided (they showed clinical signs), while
all the control piglets died on day 4 after challenge, therefore
the PRV subunit vaccines can provide excellent protection.
[0096] Those are only preferred embodiments of the present
invention as described above, which cannot be used to limit the
present invention. Any change, substitution or modification etc.,
which are within the spirit and principle of the invention, should
be included within the scope of protection of the present
invention.
Sequence CWU 1
1
61402PRTpseudorabies virus 1Met Leu Leu Ala Ala Leu Leu Ala Ala Leu
Val Ala Arg Thr Thr Leu 1 5 10 15 Gly Ala Asp Val Asp Ala Val Pro
Ala Pro Thr Phe Pro Pro Pro Ala 20 25 30 Tyr Pro Tyr Thr Glu Ser
Trp Gln Leu Thr Leu Thr Thr Val Pro Ser 35 40 45 Pro Phe Val Gly
Pro Ala Asp Val Tyr His Thr Arg Pro Leu Glu Asp 50 55 60 Pro Cys
Gly Val Val Ala Leu Ile Ser Asp Pro Gln Val Asp Arg Leu 65 70 75 80
Leu Asn Glu Ala Val Ala His Arg Arg Pro Thr Tyr Arg Ala His Val 85
90 95 Ala Trp Tyr Arg Ile Ala Asp Gly Cys Ala His Leu Leu Tyr Phe
Ile 100 105 110 Glu Tyr Ala Asp Cys Asp Pro Arg Gln Ile Phe Gly Arg
Cys Arg Arg 115 120 125 Arg Thr Thr Pro Met Trp Trp Thr Pro Ser Ala
Asp Tyr Met Phe Pro 130 135 140 Thr Glu Asp Glu Leu Gly Leu Leu Met
Val Ala Pro Gly Arg Phe Asn 145 150 155 160 Glu Gly Gln Tyr Arg Arg
Leu Val Ser Val Asp Gly Val Asn Ile Leu 165 170 175 Thr Asp Phe Met
Val Ala Leu Pro Glu Gly Gln Glu Cys Pro Phe Ala 180 185 190 Arg Val
Asp Gln His Arg Thr Tyr Lys Phe Gly Ala Cys Trp Ser Asp 195 200 205
Asp Ser Phe Lys Arg Gly Val Asp Val Met Arg Phe Leu Thr Pro Phe 210
215 220 Tyr Gln Gln Pro Pro His Arg Glu Val Val Asn Tyr Trp Tyr Arg
Lys 225 230 235 240 Asn Gly Arg Thr Leu Pro Arg Ala Tyr Ala Ala Ala
Thr Pro Tyr Ala 245 250 255 Ile Asp Pro Ala Arg Pro Ser Ala Gly Ser
Pro Arg Pro Arg Pro Arg 260 265 270 Pro Arg Pro Arg Pro Arg Pro Lys
Pro Glu Pro Ala Pro Ala Thr Pro 275 280 285 Ala Pro Pro Gly Arg Leu
Pro Glu Pro Ala Thr Arg Asp His Ala Ala 290 295 300 Gly Gly Arg Pro
Thr Pro Arg Pro Pro Arg Pro Glu Thr Pro His Arg 305 310 315 320 Pro
Phe Ala Pro Pro Ala Val Val Pro Ser Gly Trp Pro Gln Pro Ala 325 330
335 Glu Pro Phe Pro Pro Arg Thr Thr Ala Ala Pro Gly Val Ser Arg His
340 345 350 Arg Ser Val Ile Val Gly Thr Gly Thr Ala Met Gly Ala Leu
Leu Val 355 360 365 Gly Val Cys Val Tyr Ile Phe Phe Arg Leu Arg Gly
Ala Lys Gly Tyr 370 375 380 Arg Leu Leu Gly Gly Pro Ala Asp Ala Asp
Glu Leu Lys Ala Gln Pro 385 390 395 400 Gly Pro 2914PRTpseudorabies
virus 2Met Pro Ala Gly Gly Gly Leu Trp Arg Gly Pro Arg Gly His Arg
Pro 1 5 10 15 Gly His His Gly Gly Ala Gly Leu Gly Arg Leu Trp Pro
Ala Pro His 20 25 30 His Ala Ala Ala Ala Arg Gly Ala Val Ala Leu
Ala Leu Leu Leu Leu 35 40 45 Ala Leu Ala Ala Thr Pro Thr Cys Gly
Ala Ala Ala Val Thr Arg Ala 50 55 60 Ala Ser Ala Ser Pro Ala Pro
Gly Thr Gly Ala Thr Pro Asp Gly Phe 65 70 75 80 Ser Ala Glu Glu Ser
Leu Glu Glu Ile Asp Gly Ala Val Ser Pro Gly 85 90 95 Pro Ser Asp
Ala Pro Asp Gly Glu Tyr Gly Asp Leu Asp Ala Arg Thr 100 105 110 Ala
Val Arg Ala Ala Ala Thr Glu Arg Asp Arg Phe Tyr Val Cys Pro 115 120
125 Pro Pro Ser Gly Ser Thr Val Val Arg Leu Glu Pro Glu Gln Ala Cys
130 135 140 Pro Glu Tyr Ser Gln Gly Arg Asn Phe Thr Glu Gly Ile Ala
Val Leu 145 150 155 160 Phe Lys Glu Asn Ile Ala Pro His Lys Phe Lys
Ala His Ile Tyr Tyr 165 170 175 Lys Asn Val Ile Val Thr Thr Val Trp
Ser Gly Ser Thr Tyr Ala Ala 180 185 190 Ile Thr Asn Arg Phe Thr Asp
Arg Val Pro Val Pro Val Gln Glu Ile 195 200 205 Thr Asp Val Ile Asp
Arg Arg Gly Lys Cys Val Ser Lys Ala Glu Tyr 210 215 220 Val Arg Asn
Asn His Lys Val Thr Ala Phe Asp Arg Asp Glu Asn Pro 225 230 235 240
Val Glu Val Asp Leu Arg Pro Ser Arg Leu Asn Ala Leu Gly Thr Arg 245
250 255 Gly Trp His Thr Thr Asn Asp Thr Tyr Thr Lys Ile Gly Ala Ala
Gly 260 265 270 Phe Tyr His Thr Gly Thr Ser Val Asn Cys Ile Val Glu
Glu Val Glu 275 280 285 Ala Arg Ser Val Tyr Pro Tyr Asp Ser Phe Ala
Leu Ser Thr Gly Asp 290 295 300 Ile Val Tyr Met Ser Pro Phe Tyr Gly
Leu Arg Glu Gly Ala His Gly 305 310 315 320 Glu His Ile Gly Tyr Ala
Pro Gly Arg Phe Gln Gln Val Glu His Tyr 325 330 335 Tyr Pro Ile Asp
Leu Asp Ser Arg Leu Arg Ala Ser Glu Ser Val Thr 340 345 350 Arg Asn
Phe Leu Arg Thr Pro His Phe Thr Val Ala Trp Asp Trp Ala 355 360 365
Pro Lys Thr Arg Arg Val Cys Ser Leu Ala Lys Trp Arg Glu Ala Glu 370
375 380 Glu Met Ile Arg Asp Glu Thr Arg Asp Gly Ser Phe Arg Phe Thr
Ser 385 390 395 400 Arg Ala Leu Gly Ala Ser Phe Val Ser Asp Val Thr
Gln Leu Asp Leu 405 410 415 Gln Arg Val His Leu Gly Asp Cys Val Leu
Arg Glu Ala Ser Glu Ala 420 425 430 Ile Asp Ala Ile Tyr Arg Arg Arg
Tyr Asn Asn Thr His Val Leu Ala 435 440 445 Gly Asp Lys Pro Glu Val
Tyr Leu Ala Arg Gly Gly Phe Val Val Ala 450 455 460 Phe Arg Pro Leu
Ile Ser Asn Glu Leu Ala Gln Leu Tyr Ala Arg Glu 465 470 475 480 Leu
Glu Arg Leu Gly Leu Ala Gly Val Val Gly Pro Ala Ser Pro Ala 485 490
495 Ala Ala Arg Arg Ala Arg Arg Ser Pro Gly Pro Ala Gly Thr Pro Glu
500 505 510 Pro Pro Ala Val Asn Gly Thr Gly His Leu Arg Ile Thr Thr
Gly Ser 515 520 525 Ala Glu Phe Ala Arg Leu Gln Phe Thr Tyr Asp His
Ile Gln Ala His 530 535 540 Val Asn Asp Met Leu Ser Arg Ile Ala Ala
Ala Trp Cys Glu Leu Gln 545 550 555 560 Asn Lys Asp Arg Thr Leu Trp
Gly Glu Met Ser Arg Leu Asn Pro Ser 565 570 575 Ala Val Ala Thr Ala
Ala Leu Gly Gln Arg Val Ser Ala Arg Met Leu 580 585 590 Gly Asp Val
Met Ala Ile Ser Arg Cys Val Glu Val Arg Gly Gly Val 595 600 605 Tyr
Val Gln Asn Ser Met Arg Val Pro Gly Glu Arg Gly Thr Cys Tyr 610 615
620 Ser Arg Pro Leu Val Thr Phe Glu His Asn Gly Thr Gly Val Ile Glu
625 630 635 640 Gly Gln Leu Gly Asp Asp Asn Glu Leu Leu Ile Ser Arg
Asp Leu Ile 645 650 655 Glu Pro Cys Thr Gly Asn His Arg Arg Tyr Phe
Lys Leu Gly Gly Gly 660 665 670 Tyr Val Tyr Tyr Glu Asp Tyr Ser Tyr
Val Arg Met Val Glu Val Pro 675 680 685 Glu Thr Ile Ser Thr Arg Val
Thr Leu Asn Leu Thr Leu Leu Glu Asp 690 695 700 Arg Glu Phe Leu Pro
Leu Glu Val Tyr Thr Arg Glu Glu Leu Ala Asp 705 710 715 720 Thr Gly
Leu Leu Asp Tyr Ser Glu Ile Gln Arg Arg Asn Gln Leu His 725 730 735
Ala Leu Lys Phe Tyr Asp Ile Asp Arg Val Val Lys Val Asp His Asn 740
745 750 Val Val Leu Leu Arg Gly Ile Ala Asn Phe Phe Gln Gly Leu Gly
Asp 755 760 765 Val Gly Ala Ala Val Gly Lys Val Val Leu Gly Ala Thr
Gly Ala Val 770 775 780 Ile Ser Ala Val Gly Gly Met Val Ser Phe Leu
Ser Asn Pro Phe Gly 785 790 795 800 Ala Leu Ala Ile Gly Leu Leu Val
Leu Ala Gly Leu Val Ala Ala Phe 805 810 815 Leu Ala Tyr Arg His Ile
Ser Arg Leu Arg Arg Asn Pro Met Lys Ala 820 825 830 Leu Tyr Pro Val
Thr Thr Lys Ala Leu Lys Glu Asp Gly Val Glu Glu 835 840 845 Asp Asp
Val Asp Glu Ala Lys Leu Asp Gln Ala Arg Asp Met Ile Arg 850 855 860
Tyr Met Ser Ile Val Ser Ala Leu Glu Gln Gln Glu His Lys Ala Arg 865
870 875 880 Lys Lys Asn Ser Gly Pro Ala Leu Leu Ala Ser Arg Val Gly
Ala Met 885 890 895 Ala Thr Arg Arg Arg His Tyr Gln Arg Leu Glu Asn
Glu Asp Pro Asp 900 905 910 Ala Pro 3487PRTpseudorabies virus 3Met
Ala Ser Leu Ala Arg Ala Met Leu Ala Leu Leu Ala Leu Tyr Thr 1 5 10
15 Ala Ala Ile Ala Ala Ala Pro Ser Ser Thr Thr Ala Leu Gly Thr Thr
20 25 30 Pro Asn Gly Gly Gly Gly Gly Asn Ser Ser Ala Gly Glu Leu
Ser Pro 35 40 45 Ser Pro Pro Ser Thr Pro Glu Pro Val Ser Gly Thr
Thr Gly Ala Ala 50 55 60 Ala Ser Thr Pro Ala Ala Val Ser Thr Pro
Arg Val Pro Pro Pro Ser 65 70 75 80 Val Ser Arg Arg Lys Pro Gln Arg
Asn Gly Asn Arg Thr Arg Val His 85 90 95 Gly Asp Lys Ala Thr Ser
His Gly Arg Lys Arg Ile Val Cys Arg Glu 100 105 110 Arg Leu Phe Ser
Ala Arg Val Gly Asp Ala Val Ser Phe Gly Cys Ala 115 120 125 Val Val
Pro Arg Ala Gly Glu Thr Phe Glu Val Arg Phe Cys Arg Arg 130 135 140
Gly Arg Phe Arg Ser Pro Asp Ala Asp Pro Glu Tyr Phe Asp Glu Pro 145
150 155 160 Pro Arg Pro Glu Leu Pro Arg Glu Arg Leu Leu Phe Ser Ser
Ala Asn 165 170 175 Ala Ser Leu Ala His Ala Asp Ala Leu Ala Ser Ala
Val Val Val Glu 180 185 190 Gly Glu Arg Ala Thr Val Ala Asn Val Ser
Gly Glu Val Ser Val Arg 195 200 205 Val Ala Ala Ala Asp Ala Glu Thr
Glu Gly Val Tyr Thr Trp Arg Val 210 215 220 Leu Ser Ala Asn Gly Thr
Glu Val Arg Ser Ala Asn Val Ser Leu Val 225 230 235 240 Leu Tyr Ser
Gln Pro Glu Phe Gly Leu Ser Ala Pro Pro Val Leu Phe 245 250 255 Gly
Glu Pro Phe Arg Ala Val Cys Val Val Arg Asp Tyr Tyr Pro Arg 260 265
270 Arg Ser Val Arg Leu Arg Trp Phe Ala Asp Glu His Pro Val Asp Ala
275 280 285 Ala Phe Val Thr Asn Ser Thr Val Ala Asp Glu Leu Gly Arg
Arg Thr 290 295 300 Arg Val Ser Val Val Asn Val Thr Arg Ala Asp Val
Pro Gly Leu Ala 305 310 315 320 Ala Ala Asp Asp Ala Asp Ala Leu Ala
Pro Ser Leu Arg Cys Glu Ala 325 330 335 Val Trp Tyr Arg Asp Ser Val
Ala Ser Gln Arg Phe Ser Glu Ala Leu 340 345 350 Arg Pro His Val Tyr
His Pro Ala Ala Val Ser Val Arg Phe Val Glu 355 360 365 Gly Phe Ala
Val Cys Asp Gly Leu Cys Val Pro Pro Glu Ala Arg Leu 370 375 380 Ala
Trp Ser Asp His Ala Ala Asp Thr Val Tyr His Leu Gly Ala Cys 385 390
395 400 Ala Glu His Pro Gly Leu Leu Asn Val Arg Ser Ala Arg Pro Leu
Ser 405 410 415 Asp Leu Asp Gly Pro Val Asp Tyr Thr Cys Arg Leu Glu
Gly Met Pro 420 425 430 Ser Gln Leu Pro Ile Phe Glu Asp Thr Gln Arg
Tyr Asp Ala Ser Pro 435 440 445 Thr Ser Val Ser Trp Pro Val Val Thr
Ser Met Ile Thr Val Ile Ala 450 455 460 Gly Ile Ala Ile Leu Ala Ile
Val Leu Val Ile Met Ala Thr Cys Val 465 470 475 480 Tyr Tyr Arg Arg
Ala Gly Pro 485 41209DNApseudorabies virus 4atgctgctcg cagcgctatt
ggcggcgctg gtcgcccgga cgacgctcgg cgcggacgtg 60gacgccgtgc ccgcgccgac
cttccccccg cccgcgtacc cgtacaccga gtcgtggcag 120ctgacgctga
cgacggtccc ctcgcccttc gtcggccccg cggacgtcta ccacacgcgc
180ccgctggagg acccgtgcgg ggtggtggcg ctgatctccg acccgcaggt
ggaccggctg 240ctgaacgagg cggtggccca ccggcggccc acgtaccgcg
cccacgtggc ctggtaccgc 300atcgcggacg ggtgcgcgca cctgctgtac
tttatcgagt acgccgactg cgaccccagg 360cagatctttg ggcgctgccg
gcgccgcacc acgccgatgt ggtggacccc gtccgcggac 420tacatgttcc
ccacggagga cgagctgggg ctgctcatgg tggccccggg gcggttcaac
480gagggccagt accggcgcct ggtgtccgtc gacggcgtga acatcctcac
cgacttcatg 540gtggcgctcc ccgaggggca agagtgcccg ttcgcccgcg
tggaccagca ccgcacgtac 600aagttcggcg cgtgctggag cgacgacagc
ttcaagcggg gcgtggacgt gatgcgattc 660ctgacgccgt tctaccagca
gcccccgcac cgggaggtgg tgaactactg gtaccgcaag 720aacggccgga
cgctcccgcg ggcctacgcc gccgccacgc cgtacgccat cgaccccgcg
780cggccctcgg cgggctcgcc gaggcccagg ccccggcccc ggcccaggcc
ccggccgaag 840cccgagcccg ccccggcgac gcccgcgccc cccggccgcc
tgcccgagcc ggcgacgcgg 900gaccacgccg ccggggggcg ccccacgccg
cgacccccga ggcccgagac gccgcaccgc 960cccttcgccc cgccggccgt
cgtgcccagc gggtggccgc agcccgcgga gccgttcccg 1020ccccggacca
ccgccgcgcc gggcgtctcg cgccaccgct cggtgatcgt cggcacgggc
1080accgcgatgg gcgcgctcct ggtgggcgtg tgcgtctaca tcttcttccg
cctgaggggg 1140gcgaaggggt atcgcctcct gggcggtccc gcggacgccg
acgagctaaa agcgcagccc 1200ggtccgtag 120952745DNApseudorabies virus
5ctagggggcg tcggggtcct cgttctcgag gcgctggtag tgccggcggc gcgtggccat
60cgccccgacg cggctggcca gcagcgcggg cccgctgttc ttcttgcgcg ccttgtgctc
120ctgctgctcg agggccgaca cgatggacat gtaccggatc atgtcccggg
cctggtccag 180cttggcctcg tccacgtcgt cctcttcgac gccgtcctcc
ttgagcgcct tcgtcgtgac 240ggggtacagg gccttcatgg ggttgcggcg
caggcgcgag atgtgccggt aggccaggaa 300ggccgcgacc aggccggcca
gcaccagcag cccgatggcg agcgccccga aggggttgga 360caggaaggac
accatgccgc cgacggccga gatcacggcc cccgtggcgc ccaggaccac
420cttgccgacg gcggcgccca cgtcgccgag gccctggaag aagttggcga
tgccgcgcag 480cagcaccacg ttgtggtcca ccttgaccac gcggtcaatg
tcgtagaact tgagcgcgtg 540cagctggttg cggcgctgga tctcgctgta
gtccaggagg cccgtgtcgg cgagctcctc 600gcgcgtgtac acctcgaggg
gcaggaactc gcggtcctcg agcagcgtca ggttcagggt 660cacccgcgtg
ctgatcgtct cgggcacctc caccatgcgc acgtagctgt agtcctcgta
720gtacacgtac ccgccgccca gcttaaagta gcgccggtgg ttgccggtgc
agggctcgat 780gaggtcgcgc gagatgagga gctcgttgtc gtcgccgagc
tggccctcga tcacgcccgt 840gccgttgtgc tcgaaggtca ccagcgggcg
gctgtagcac gtgccgcgct cgccgggcac 900gcgcatggag ttctgcacgt
acacgccgcc gcgcacctcc acgcaccgcg agatggccat 960cacgtcgccg
agcatgcgcg ccgagacgcg ctggcccagc gcggccgtgg ccacggcgct
1020ggggttcagg cgcgacatct cgccccacag ggtgcggtcc ttgttctgca
gctcgcacca 1080ggcggccgcg atgcggctca gcatgtcgtt cacgtgcgcc
tggatgtggt cgtaggtgaa 1140ctgcaggcgc gcaaactcgg ccgagcccgt
ggtgatgcgc aggtgccccg tgccgttgac 1200ggccggcggc tcgggcgtcc
ccgccgggcc gggggagcgc cgggcccgac gggcggccgc 1260gggggacgcg
gggcccacga cgccggcgag gccgaggcgc tcgagctcgc gcgcgtacag
1320ctgcgccagc tcgttcgaga tcagcgggcg gaaggccacc acgaagcccc
cgcgggcgag 1380gtacacctcg ggcttgtcgc cggccagcac gtgcgtgttg
ttgtagcgcc gccggtagat 1440ggcgtcgatg gcctccgagg cctcgcggag
gacgcagtcg cccaggtgca cgcgctgcag 1500gtcgagctgc gtgacgtcgc
tgacgaagga ggcgcccagg gcccgcgacg tgaagcggaa 1560ggacccgtcg
cgcgtctcgt cgcggatcat ctcctcggcc tcgcgccact tggccaggct
1620gcacacgcgc cgcgtcttgg gggcccagtc ccaggccacc gtgaagtgcg
gcgtgcgcag 1680aaagttgcgc gtcacgctct cggaggcgcg gaggcgcgag
tccaggtcga tggggtagta 1740gtgctccacc tgctggaagc gcccgggcgc
gtagccgatg tgctccccgt gggccccctc 1800gcgcaggccg tagaaggggg
acatgtacac gatgtccccc gtggacaggg cgaaggagtc 1860gtaggggtac
acggagcgcg cctccacctc ctcgacgatg cagttgacgg aggtgcccgt
1920gtggtagaag cccgcggcgc cgatcttggt gtaggtgtcg ttggtggtgt
gccagccgcg 1980ggtgccgagc gcgttcaggc gcgaggggcg caggtccacc
tcgacggggt tctcgtcgcg 2040gtcgaaggcg gtcaccttgt ggttgttgcg
cacgtactcg gccttggaga cgcacttgcc 2100gcggcggtcg atcacgtccg
tgatctcctg cacggggacg ggcacgcggt ccgtgaagcg 2160gttcgtgatg
gccgcgtacg tgctcccgga ccacacggtc gtgacgatga cgttcttgta
2220gtagatgtgg gccttgaact tgtgcggggc gatgttctcc ttgaagagca
cggcgatccc 2280ctccgtgaag ttgcgcccct gcgagtactc ggggcaggcc
tgctcgggct ccaggcgcac 2340caccgtggag ccggacggcg gcgggcagac
gtagaagcgg tcccgctcgg tcgcggccgc 2400gcgcacggcc gtgcgcgcgt
ccaggtcgcc gtactcgccg tcgggggcgt ccgaggggcc 2460gggggagacg
gccccgtcga tctcctcgag ggactcctcc gcggagaagc cgtctggggt
2520ggcgcccgtc ccgggcgcgg gcgaggccga ggcggcccgc gtcacggccg
ccgcgccgca 2580cgtcggggtc gcggcgagcg ccagcagcag cagcgctagc
gcgacggcgc cccgcgcagc 2640tgcagcgtgg tgtggagcag gccaaagacg
tccgaggcca gcaccgccgt ggtgcccggg 2700ccgatgcccg cggggcccgc
gccaaagacc gccaccagcg ggcat 274561464DNApseudorabies virus
6atggcctcgc tcgcgcgtgc gatgctcgcg ctgctggcgc tctacacggc ggccatcgcc
60gcggcgccgt cgtccacgac ggcgctcggc acgacgccca acgggggcgg gggcggcaac
120agcagcgcgg gcgagctctc gccctcgccg ccctcgacgc ccgagcccgt
ctcggggacg 180acgggggccg cggcctccac gcccgccgcc gtctcgacgc
cccgggtccc gccgccctcg 240gtctcgcgcc ggaagcccca gcggaacggc
aacaggacgc gcgtccacgg cgacaaggcc 300acctcgcacg ggcgcaagcg
catcgtgtgc cgcgagcggc tgttctcggc gagggtgggg 360gacgcggtca
gcttcgggtg cgccgtcgtc ccgcgcgccg gggagacctt cgaggtccgc
420ttctgccgcc gcgggcgctt ccgctcgccc gacgccgacc ccgagtactt
tgacgagccc 480ccgcgcccgg agctcccgcg ggagcggctc ctcttcagct
ccgccaacgc ctccctcgcc 540cacgcggacg cgctcgcctc cgccgtcgtc
gtcgagggcg agcgcgcgac cgtcgccaac 600gtctcgggcg aggtgtccgt
gcgcgtggcc gcggcggacg ccgagaccga gggcgtctac 660acgtggcgcg
tgctgtccgc caacggcacc gaggtccgca gcgccaacgt ctcgctcgtc
720ctgtacagcc agcccgagtt cggcctgagc gcgccgcccg tcctcttcgg
cgagcccttc 780cgggcggtgt gcgtcgtccg cgactactac ccgcggcgca
gcgtgcgcct gcgctggttc 840gcggacgagc acccggtgga cgccgccttc
gtgaccaaca gcaccgtggc cgacgagctc 900gggcgccgca cgcgcgtctc
cgtggtgaac gtgacgcgcg cggacgtccc gggcctcgcg 960gccgcggacg
acgcggacgc gctcgcgccg agcctgcgct gcgaggccgt gtggtaccgc
1020gacagcgtgg cctcgcagcg cttctccgag gccctgcgcc cccacgtcta
ccacccggcg 1080gcggtctcgg tgcgcttcgt cgagggcttc gccgtctgcg
acggcctctg cgtgcccccg 1140gaggcgcgcc tcgcctggtc cgaccacgcc
gccgacaccg tctaccacct cggcgcctgc 1200gccgagcacc ccggcctgct
caacgtgcgg agcgcccgcc cgctgtcgga cctcgacggg 1260cccgtcgact
acacctgccg cctcgagggc atgccctcgc agctgcccat cttcgaggac
1320acgcagcgct acgacgcctc ccccacgtcc gtgagctggc ccgtcgtgac
cagcatgatc 1380accgtcatcg ccggcatcgc catcctagcc atcgtgctgg
tcatcatggc gacgtgcgtc 1440tactaccgcc gggcggggcc gtga 1464
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References