U.S. patent application number 15/054404 was filed with the patent office on 2016-09-29 for gene-deleted variant strain of porcine pseudorabies virus, vaccine composition, method of making the same and use thereof.
The applicant listed for this patent is Pulike Biological Engineering, Inc.. Invention is credited to Jinzhong SUN, Feifei TAN, Kegong TIAN, Xuke ZHANG.
Application Number | 20160279232 15/054404 |
Document ID | / |
Family ID | 52185894 |
Filed Date | 2016-09-29 |
United States Patent
Application |
20160279232 |
Kind Code |
A1 |
TIAN; Kegong ; et
al. |
September 29, 2016 |
Gene-Deleted Variant Strain Of Porcine Pseudorabies Virus, Vaccine
Composition, Method Of Making The Same And Use Thereof
Abstract
The present invention provides an attenuated strain of porcine
pseudorabies virus (PRV), in which said attenuated strain of PRV is
a variant strain of PRV with inactivation of gI/gE/11K/28K
proteins. In addition, the present invention also provides a
vaccine composition comprising the attenuated strain of PRV as an
antigen, a preparation method and use thereof. Proved by
immunogenicity and pathogenicity testing of the live vaccine, said
live PRV vaccine can provide a good protection for pigs with no
clinical signs observed, indicating excellent immune
protection.
Inventors: |
TIAN; Kegong; (Luoyang City,
CN) ; ZHANG; Xuke; (Luoyang City, CN) ; SUN;
Jinzhong; (Luoyang City, CN) ; TAN; Feifei;
(Luoyang City, CN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Pulike Biological Engineering, Inc. |
Luoyang City |
|
CN |
|
|
Family ID: |
52185894 |
Appl. No.: |
15/054404 |
Filed: |
February 26, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14901981 |
Dec 29, 2015 |
|
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PCT/CN2015/070221 |
Jan 6, 2015 |
|
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15054404 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 7/00 20130101; C12N
2710/16734 20130101; A61K 39/245 20130101; C12N 2710/16011
20130101; A61K 2039/545 20130101; A61K 39/12 20130101; A61P 31/22
20180101; C12N 2710/16762 20130101; A61K 2039/5252 20130101; C12N
2770/20071 20130101; C12N 2710/16634 20130101; C12N 7/04 20130101;
A61K 2039/5254 20130101; A61K 45/06 20130101; A61K 2039/543
20130101; C12N 2710/16721 20130101; A61K 2039/552 20130101; C12N
2710/16771 20130101 |
International
Class: |
A61K 39/245 20060101
A61K039/245; C12N 7/00 20060101 C12N007/00 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 22, 2014 |
CN |
201410418379.8 |
Claims
1. An attenuated strain of porcine pseudorabies virus comprising a
first attenuated porcine pseudorabies virus strain, wherein the
first attenuated strain of porcine pseudorabies comprises a porcine
pseudorabies virus strain with inactivation of gI/gE/11K/28K
protein.
2. The attenuated strain of porcine pseudorabies virus of claim 1,
wherein the first attenuated porcine pseudorabies virus strain is a
variant strain of pseudorabies virus.
3. The attenuated strain of porcine pseudorabies virus of claim 1,
wherein a whole ORF is deleted from a gI/gE/11K/28K gene of the
first attenuated porcine pseudorabies virus strain.
4. The attenuated strain of porcine pseudorabies virus of claim 2,
wherein the variant strain of pseudorabies virus comprises a virus
strain in which gE protein has 95% or greater homology with SEQ ID
NO. 05.
5. The attenuated strain of porcine pseudorabies virus of claim 4,
wherein said variant strain of pseudorabies virus is obtained
through isolation, and when infection with said variant strain
occurs in pigs previously immunized with a second attenuated gene
deleted porcine pseudorabies virus strain, wherein the second
attenuated gene deleted porcine pseudorabies virus strain is
prepared according to the prior art, the pigs display clinical
signs of infection selected from the group consisting of high
fever, depression, and partial or complete loss of appetite.
6. The attenuated strain of porcine pseudorabies virus of claim 5,
wherein when exposed to said variant strain of pseudorabies virus,
pigs previously immunized with the second attenuated porcine
pseudorabies virus strain develop a pseudorabies infection, and
wherein the second attenuated porcine pseudorabies vaccine is a
prior art strain with deletion of one or more of the group
consisting of gE, TK and gI genes.
7. The attenuated strain of claim 6, wherein the pseudorabies
infection comprises clinical signs of infection selected from the
group consisting of depression and loss of appetite among piglets
at the age of 9-10 days.
8. The attenuated strain of porcine pseudorabies virus of claim 1,
wherein the first porcine pseudorabies virus strain is selected
from the group consisting of an HN1201 strain, HN1202 strain,
JS-2012 strain, PRV HeN1 strain, NVDC-PRV-BJ strain, NVDCPRV-HEB
strain or NVDC-PRV-SD strain, PRV TJ strain, and PRV-ZJ01
strain.
9. The attenuated strain of porcine pseudorabies virus of claim 1,
wherein the first attenuated strain of porcine pseudorabies virus
strain comprises a gene variation resulting in inactivation of TK
protein.
10. The attenuated strain of porcine pseudorabies virus of claim 9,
wherein the gene variation comprises a nucleotide sequence located
at a site corresponding to a TK gene of the first attenuated
porcine pseudorabies virus strain, and wherein the nucleotide
sequence encodes for SEQ ID NO. 4.
11. A vaccine composition, comprising an immunizing amount of an
antigen of an attenuated strain of porcine pseudorabies and a
carrier, wherein the attenuated strain of porcine pseudorabies
comprises a genetic variation resulting in inactivation of
gI/gE/11K/28K protein.
12. The vaccine composition of claim 11, wherein the immunizing
amount comprises at least 10.sup.6.0TCID.sub.50/ml of the
attenuated strain of porcine pseudorabies virus.
13. The vaccine composition of claim 11, wherein the antigen
comprises a live attenuated strain of porcine pseudorabies virus,
and wherein the vaccine composition further comprises a
cryoprotectant.
14. The vaccine composition of claim 11, further comprising an
inactivated pathogen or antigen.
15. The vaccine composition of claim 14, wherein the inactivated
pathogen or antigen is selected from the group consisting classical
swine fever virus, antigen of porcine reproductive and respiratory
syndrome virus, antigen of porcine circovirus, antigen of
haemophilus parasuis, and antigen of mycoplasma.
16. A method of treating and preventing pseudorabies infection,
comprising immunizing a pig with an antigen comprising a porcine
pseudorabies virus strain with inactivation of gI/gE/11K/28K
protein.
17. The method of claim 16, wherein the porcine pseudorabies virus
strain comprises a virus strain in which gE protein has 95% or
greater homology with SEQ ID NO. 05.
Description
[0001] This application is a continuation of U.S. Patent
Application No. 14/901981, filed Dec. 29, 2015, which is a National
Stage of PCT/CN2015/070221, filed Jan. 6, 2015, which claims the
benefit of priority to CN 201410418379.8, filed Aug. 22, 2014.
FIELD OF THE INVENTION
[0002] This invention relates to a gene-deleted variant strain of
porcine pseudorabies virus, a vaccine composition prepared
therefrom, a method of making the same and a use thereof, 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] According to recent researches, there are reports of new
features of pseudorabies, of which 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%. According to literatures in the prior
art, for example, Jin-mei Peng, et al., Identification and
antigenic variation of new epidemiology of pseudorabies virus from
swine. Chinese Journal of Preventive Veterinary Medicine, 2013, 35
(1):1-4; Tong Wu et al., Identification and Characterization of a
pseudorabies virus isolated from a dead piglet born to vaccinated
sow. Chinese Journal of Animal infectious diseases, 2013, 21
(3):1-7; Yu et al., Pathogenic Pseudorabies Virus, China, 2012.
Emerging infectious Diseases. 2014, 20 (1):102-104; An et al.,
Pseudorabies virus variant in Bartha-K61-vaccinated pigs, China,
2012. Emerging infectious Diseases. 2013. 19 (11): 1749-1755, there
is no vaccines capable of solving the pseudorabies caused by
variant strains of porcine pseudorabies virus in the prior art.
[0005] The Chinese patent application CN103756977A has disclosed a
gE and gI deleted variant strain of porcine pseudorabies virus,
PRV-ZJ011G strain (of which the accession number is CGMCC No. 7957)
and a vaccine prepared therefrom, of which the content of virus
prior to being inactivated is 10.sup.6.0TCID.sub.50. Immunization
with said vaccine could provide a 100% protection rate for 5
healthy piglets at 45 days of age. Said patent application,
however, cannot successfully provide an attenuated live vaccine
against new PRV strains, due to both humoral and cellular immunity
resulted from proliferation of attenuated live PRV vaccine in
vivo.
[0006] The Chinese patent application CN103981153A has disclosed
the construction of a gene deleted variant strain of the
pseudorabies virus labeled with two fluorescent markers, wherein a
variant strain of pseudorabies virus with deletion of gE, gI, US9
and TK gene is inserted with GRP gene and RFP gene as labels at the
site of deleted genes through homologous recombination. Whereas in
this patent application said a gene deleted variant strain is used
as a vector of antigen, we don't know if it has immunological
competence itself, or how strong the virulence of the variant
strain is, since there are more than 70 gene fragments for
pseudorabies virus.
[0007] It has been disclosed by Chun-Hua Wang et al. (Chun-Hua
Wang, Jin Yuan, Hua-Yang Qin, et al, A novel gE-deleted
pseudorabies virus (PRV) provides rapid and complete protection
from lethal challenge with the PRV variant emerging in
Bartha-K61-vaccinated swine population in China, Vaccine 32
(2014)3379-3385) that 6-week-old piglets injected with
PRVTJ-gE.sup.- live vaccines got a complete protection from
challenge and didn't exhibit fever. Whereas as is known to those
skilled in the art, the virulence of the PRV live vaccines depends
on the age of piglets. Also according to the general experience in
the art, the vaccines may not be safe for 7-day-old piglets even
though they are safe for 6-week-old piglets. The live vaccines may
spread among pigs, therefore the vaccine which is only safe for
6-week-old piglets cannot be ensured to be used clinically and a
live vaccine which is safe for 7-day-old piglets, is required in
the prior art to prevent effectively infection with the new variant
strains of PRV.
SUMMARY OF INVENTION
[0008] In order to solve the deficiency of the prior art, the
present invention provides an attenuated live strain of porcine
pseudorabies virus for prevention and treatment of pseudorabies
caused by mutated pseudorabies virus.
[0009] The main aim of present invention is to provide an
attenuated strain of porcine pseudorabies virus, wherein said
attenuated strain of porcine pseudorabies virus is a pseudorabies
virus strain with inactivation of gI/gE/11K/28K proteins,
preferably said PRV strain is a variant strain of PRV.
[0010] The inactivation of gI/gE/11K/28K proteins can be achieved
by using well known methods in the art, including deletion of
nucleotide sequence expressing the functional fragments of those
above proteins from the gene, deletion of the whole ORF from the
gene, or deletion or addition of one or more nucleotides whereby
the gene cannot express functional proteins normally or the
proteins expressed don't have their original function or have an
extremely weak function.
[0011] As an embodiment of the present invention, the present
invention provides an attenuated genetically engineered strain of
porcine pseudorabies virus with deletion of gI/gE/11K/28K
genes.
[0012] As an embodiment of the present invention, the whole ORF of
gI/gE/11K/28K genes was deleted from the genome of said attenuated
genetically engineered strain of pseudorabies virus.
[0013] As an embodiment of the present invention, said variant
strain of pseudorabies virus is a virus strain of which gE protein
has the sequence of SEQ ID NO. 5 or shares at least 95% homology to
the sequence of SEQ ID NO. 5; preferably, said variant strain of
pseudorabies virus is obtained through isolation, and when
infection with said variant strain recurs in pigs previously
immunized with attenuated gene-deleted strain of pseudorabies virus
according to the prior art, the pigs still display clinical signs
of infection with said variant strain, selected from high fever,
depression and partial or complete loss of appetite; more
preferably, said variant strain of pseudorabies virus is a variant
strain of pseudorabies virus and when infection with said variant
strain recurs in pigs previously immunized with attenuated strain
of PRV with deletion of one or more of gE, TK and gI genes,
according to the prior art, the pigs are still infected with
pseudorabies, which optionally causes clinical signs of infection,
selected from depression and loss of appetite among piglets at the
age of 9-10 days.
[0014] Most preferably, said variant strain of pseudorabies virus,
includes, but are not limited to, PRV HN1201 strain (pseudorabies
virus, strain HN1201)(deposited in the China Center for Type
Culture Collection on May, 20, 2013, of which the accession number
is CCTCC NO. V 201311 and the address is Wuhan University, Wuhan,
China); JS-2012 strain (Wu Tong, Qingzhan Zhang, Hao Zheng et al.
Isolation and identification of PRV from piglets infected after
immunization [J]. Chinese Journal of Animal Infectious Diseases.
2013, 21 (3): 1-7); PRV HeN1 strain (deposited in the China General
Microbiological Culture Collection Center on May, 20, 2013, of
which the accession number is CGMCC NO. 6656 and has been disclosed
in the patent application CN102994458A); NVDC-PRV-BJ strain,
NVDC-PRV-HEB strain and NVDC-PRV-SD strain (Xiuling Yu, Zhi Zhou,
Dongmei Hu,et al. Pathogenic Pseudorabies Virus, China, 2012
Emerging Infectious Diseases, www.cdc.gov/eid ol. 20, No. 1,
January 2014); PRV HN1202 strain (pseudorabies virus, strain
HN1202) (deposited in the China Center for Type Culture Collection
on Aug. 26, 2013, of which the accession number is CCTCC NO. V
201335 and the address is Wuhan University, Wuhan, China); PRV TJ
strain (Chun-Hua Wang Jin Yuan, Hua-Yang Qin, et al, A novel
gE-deleted pseudorabies virus (PRV) provides rapid and complete
protection from lethal challenge with the PRV variant emerging in
Bartha-K61-vaccinated swine population in China. Vaccine. 32 (2014)
3379-3385); a variant strain of pseudorabies virus PRV-ZJ01 (with
the accession number, CGMCC No. 8170, and disclosed in
CN103627678A).
[0015] As an embodiment of the present invention, said PRV strain
is HN1201 strain, HN1202 strain, JS-2012 strain, PRV HeN1 strain,
NVDC-PRV-BJ strain, NVDC-PRV-HEB strain or NVDC-PRV-SD strain, PRV
TJ strain or PRV-ZJ01 strain.
[0016] As an embodiment of the present invention, said attenuated
strain of porcine pseudorabies virus is an attenuated strain of
porcine pseudorabies virus with further inactivation of TK protein;
preferably the nucleotide sequence at the location of TK in the
genome of said attenuated strain of porcine pseudorabies virus
encodes and expresses the amino acid sequence shown in SEQ.NO. 4 of
the sequence listing.
[0017] As a preferred embodiment of the present invention, the
nucleotide sequence at the location of TK in the genome of said
attenuated strain of porcine pseudorabies virus is the nucleotide
sequence shown in SEQ.NO. 3 of the sequence listing.
[0018] As a preferred embodiment of the present invention, the
present invention provides an attenuated genetically engineered
strain of porcine pseudorabies virus with deletion of
gI/gE/11K/28K/TK genes.
[0019] As used herein, the term "variant strain of pseudorabies
virus", also called highly pathogenic PRV strain, refers to
diseases with significant manifestations including 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 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. Preferably, said variant strain of pseudorabies virus
obtained through isolation, and when infection with said variant
strain recurs in pigs previously immunized with attenuated
gene-deleted strain of pseudorabies virus according to the prior
art, the pigs still display clinical signs of infection with said
variant strain, selected from selected from high fever, depression
and partial or complete loss of appetite. Preferably, said variant
strain of pseudorabies virus is a virus strain of which gE protein
has the sequence of SEQ ID NO. 5 or shares at least 95% homology to
the sequence of SEQ ID NO. 5. More preferably, said variant strain
of pseudorabies virus is a variant strain of pseudorabies virus
wherein, when infection with said variant strain recurs in pigs
previously immunized with attenuated strain of porcine pseudorabies
virus with deletion of one or more of gE, TK and gI genes,
according to the prior art, the pigs are still infected with
pseudorabies, which optionally causes clinical signs of infection
selected from depression and loss of appetite among piglets at the
age of 9-10 days. 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. Biol, 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.
[0020] The term "gI protein" is encoded by US7, and comprises 366
amino acids, with ORF located between 122298-123398.
[0021] The term "gE protein" is encoded by USB, and comprises 577
amino acids, with ORF located between 123502-125235.
[0022] The term "11K" is encoded by US9, and comprises 98 amino
acids, with ORF located between 125293-125589.
[0023] The term "28K" is encoded by US2, and comprises 256 amino
acids, with ORF located between 125811-126581.
[0024] The term "TK", also called thymidine kinase, is encoded by
UL23, and comprises 320 amino acids, with ORF located between
59512-60474.
[0025] The term "gI/gE/11K/28K" and "gI/gE/11K/28K/TK" in the
present invention refers to "gI, gE, 11K and 28K" and "gI, gE, 11K,
28K and TK", respectively, wherein "I" in the present invention
refers to "and", for example, "inactivation of gI/gE/11K/28K
proteins" refers to inactivation of gI, gE, 11K and 28K
proteins.
[0026] Unless otherwise stated, the term "PRV-gI-gE-11K-28K-TK-" in
the present invention refers to deletion of gI, gE, 11K, 28K and TK
genes.
[0027] As a preferred embodiment of the present invention, said
attenuated genetic strain of porcine pseudorabies virus with
deletion of gI/gE/11K/28K genes is attenuated genetically
engineered virus strain of PRV HN1201 strain with deletion of
gI/gE/11K/28K genes.
[0028] As a preferred embodiment of the present invention, said
attenuated strain of porcine pseudorabies virus includes HN1201
strain, HN1202 strain, JS-2012 strain, PRV HeN1 strain, NVDC-PRV-BJ
strain, NVDC-PRV-HEB strain or NVDC-PRV-SD strain, with deletion of
gI/gE/11K/28K.
[0029] As a preferred embodiment of the present invention, said
attenuated strain of porcine pseudorabies virus is PRV HN1201
strain (pseudorabies virus, strain HN1201) with deletion of
gI/gE/11K/28K genes using genetic engineering, wherein said PRV
HN1201 strain is deposited in the China Center for Type Culture
Collection on May, 20, 2013, of which the accession number is CCTCC
NO. V 201311 and the address is Wuhan University, Wuhan, China.
[0030] As used herein, the term "attenuated" in the present
invention refers to: compared with unmodified parent strain, the
virulence of the gene-deleted pseudorabies virus strain is reduced,
of which manifestations include reduction of numbers of dead pigs,
numbers of pigs with fever, and duration of fever. If the
statistically significant difference of one or more parameters for
determination of severity of diseases for virus strains decreases,
its virulence is attenuated.
[0031] Another aspect of the invention relates to a vaccine
composition, wherein said vaccine composition comprises an immune
amount of antigen of said attenuated strain of porcine pseudorabies
virus and carrier; preferably the content of antigen of the
attenuated strain of porcine pseudorabies virus is not less than
10.sup.6.0TCID.sub.50/ml.
[0032] As a preferred embodiment of the present invention, the
antigen of said attenuated strain of porcine pseudorabies virus is
live attenuated strain of porcine pseudorabies virus; said vaccine
composition further comprises a cryoprotectant.
[0033] As an embodiment of the present invention, said vaccine
composition comprises an immune amount of attenuated live vaccine
of said variant strain of pseudorabies virus with deletion of
gI/gE/11K/28K genes and carrier.
[0034] As a preferred embodiment of the present invention, said
vaccine composition comprises an immune amount of attenuated live
vaccine of said variant strain of pseudorabies virus with deletion
of gI/gE/11K/28K/TK genes and carrier.
[0035] As a preferred embodiment of the present invention, said
vaccine composition comprises an immune amount of attenuated live
vaccine of a variant strain of pseudorabies virus with deletion of
gI/gE/11K/28K genes, such as HN1201 strain, HN1202 strain, JS-2012
strain, PRV HeN1 strain, NVDC-PRV-BJ strain, NVDC-PRV-HEB strain or
NVDC-PRV-SD strain, PRV TJ strain or PRV-ZJ01 strain and
carrier.
[0036] As a preferred embodiment of the present invention, said
vaccine composition comprises an immune amount of attenuated live
vaccine of variant strain of PRV strains with deletion of
gI/gE/11K/28K/TK genes, such as HN1201 strain, HN1202 strain,
JS-2012 strain, PRV HeN1 strain, NVDC-PRV-BJ strain, NVDC-PRV-HEB
strain or NVDC-PRV-SD strain, PRV TJ strain or PRV-ZJ01 strain and
carrier.
[0037] Preferably, the antigen of said attenuated strain of porcine
pseudorabies virus is attenuated live PRV strain; said vaccine
composition further comprises a cryoprotectant.
[0038] As an embodiment of the present invention, said vaccine
composition is attenuated live vaccine of the PRV strain with
deletion of gI/gE/11K/28K genes.
[0039] Optionally, one or more compounds with adjuvant activity may
be added to vaccines. It does not necessarily require such an
adjuvant to achieve the efficacy of the live attenuated
pseudorabies virus according to the present invention, but
especially for a combination vaccine comprising the live attenuated
pseudorabies virus according to the present invention and antigenic
materials from another pathogenic virus or microorganism (see
below), it will be worth adding an adjuvant. Adjuvants are
non-specific stimulators of the immune system. They improve immune
response of the host responding to a vaccine. Examples of adjuvants
known in the art is include complete/incomplete Freund's adjuvant,
vitamin E, non-ionic blocking copolymers, muramyl dipeptide, ISCOMs
(immune stimulating complexes, refer to, for example the European
patent EP 1099 42), saponins, mineral oil, vegetable oil, and
Carbopol.
[0040] Therefore, in a preferred form of said embodiment, the live
attenuated vaccine according to the present invention further
comprises an adjuvant.
[0041] Other examples of pharmaceutically acceptable carriers or
diluents can be used in the present invention, include stabilizers
such as SPGA, carbohydrates (e.g., sorbitol, mannitol, starch,
sucrose, glucose, dextran), proteins such as albumin or casein,
protein-containing agents such as bovine serum or skimmed milk and
buffers (e.g. phosphate buffer).
[0042] Especially when such stabilizers are added to the vaccine,
the vaccine is very suitable for freeze-drying. Therefore, in a
more preferred form of said embodiment, the live attenuated vaccine
is in a freeze-dried form.
[0043] 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.
[0044] As an embodiment of the present invention, said vaccine
further comprises inactivated pathogens or antigen, preferably,
said antigen comprises antigen of classical swine fever virus,
antigen of porcine reproductive and respiratory syndrome virus,
antigen of porcine circovirus and/or haemophilus parasuis or
antigen of mycoplasma.
[0045] As an embodiment of the present invention, the attenuated
virus strain in the present invention can be inserted by exogenous
genes, said exogenous genes encode one or more antigens selected
from a plurality of pathogens infecting pigs, said pathogens
consist of porcine reproductive respiratory syndrome (PRRS) virus,
porcine Influenza virus, porcine parvovirus, transmissible
gastroenteritis virus, rotavirus, type 1 or type 2 porcine
circovirus virus, Escherichia coli, Erysipelothrix rhusiopathiae,
Bordetella bronchiseptica, Haemophilus parasuis, Mycoplasma
hyopneumoniae and Streptococcus suis.
[0046] Preferably, said vaccine composition may further comprise
medium, adjuvants and excipients.
[0047] The vaccine composition according to the present invention
may also comprises medium, adjuvants and/or excipients.
Physiological saline or distilled water can be used as medium.
[0048] Another aspect of the present invention relates to a method
for preparing said vaccine composition, wherein said method
comprises the steps: (1) said attenuated strain of porcine
pseudorabies virus is amplified and cultured; and (2) a
cryoprotectant is added into said attenuated strain of porcine
pseudorabies virus amplified and cultured.
[0049] Another aspect of the present invention relates to a use of
said vaccine composition for preparing medicine for treatment and
prevention of pseudorabies.
[0050] As an embodiment of the present invention, said pseudorabies
is pseudorabies caused by a variant strain of pseudorabies
virus.
[0051] 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.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 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.
[0052] 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.
ADVANTAGES OF THE PRESENT INVENTION
[0053] The strain in the present invention with less virulence
could provide better immune protection, induce an earlier
generation of antibodies and the challenge result after
immunization shows that gE antibody is still negative.
BRIEF DESCRIPTION OF THE DRAWINGS
[0054] FIG. 1 is a schematic diagram showing construction of
plasmid pUCgI/gE/11K/28KA-GFP-B;
[0055] FIG. 2 is a schematic diagram showing the location for
deletion of gI/gE/11K/28K genes and location of homologous arms,
USA and USB in the genome;
[0056] FIG. 3 is a gel electrophoresis pattern for comparing PCR
fragments of PRV HN1201 strain before and after deletion of
gI/gE/11K/28K genes through PCR method;
[0057] FIG. 4 is a schematic diagram showing the location for
deletion of TK gene and location of homologous arms of TKA and TKB
in the genome;
[0058] FIG. 5 is a gel electrophoresis pattern for comparing PCR
fragments of PRV HN1201 strain before and after deletion of TK gene
through PCR method.
SEQUENCE LISTING
[0059] SEQ ID NO. 1 is the nucleotide sequence of gI/gE/11K/28K
genes in the PRV HN1201 strain.
[0060] SEQ ID NO. 2 is the nucleotide sequence of TK gene in the
PRV HN1201 strain.
[0061] SEQ ID NO. 3 is the nucleotide sequence of location for
deletion of TK gene in the PRV HN1201 strain.
[0062] SEQ ID NO. 4 is the amino acid sequence of location for
deletion of TK gene in the PRV HN1201 strain.
[0063] SEQ ID NO. 5 is the amino acid sequence of gE in the PRV
HN1201 strain.
DETAILED DESCRIPTION
[0064] 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.
[0065] In the invention, the term "per pig" refers to the amount of
vaccine each pig injected.
[0066] In the invention, the term "TCID.sub.50" refers to 50%
tissue culture infective dose, a way to represent viral
infectivity.
[0067] Minimum Essential Medium (MEM) liquid medium is prepared
with MEM dry powdered medium purchased from Life Technologies,
Corp. according to the instruction.
[0068] 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.
[0069] 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.
[0070] The PRV HN1201 strain (pseudorabies virus, strain HN1201)
used in the embodiments is deposited in the China Center for Type
Culture Collection on May, 20, 2013, of which the accession number
is CCTCC NO. V 201311 and the address is Wuhan University, Wuhan,
China.
[0071] The PRV HN1202 strain (pseudorabies virus, strain HN1202)
used in the embodiments is deposited in the China Center for Type
Culture Collection on Aug. 26, 2013, of which the accession number
is CCTCC NO. V 201335 and the address is Wuhan University, Wuhan,
China.
[0072] PRV is the abbreviation for the term Pseudorabies virus.
[0073] In the following specific embodiments, the description of
the present invention is further provided with examples of PRV
HN1201 strain, NVDC-PRV-BJ strain, NVDCPRV-HEB strain, NVDC-PRV-SD
strain and HN1202 strain.
EXAMPLE 1
Preparation of PRV HN1201 Strain With Deletion of gI/gE/11K/28K
1.1 Construction of a Transfer Vector for Recombinant PRV HN1201GFP
Virus
[0074] According to the sequence of US segment (gI/gE/11K/28K) to
be deleted, the homologous arms were designed at its two ends,
called USA and USB, respectively. USA and USB were cloned into
pUC19 vector and named pUCUSAB. Then GFP gene was cloned into
pUCUSAB, to obtain a transfer vector for recombinant virus which
was called pUCUSA-GFP-B. The homologous arms in the transfer vector
are sequences of two sides of US, therefore the recombinant virus
obtained after recombination, was US segment deleted, which
comprised gI/gE/11K/28K. FIG. 1 is a schematic diagram showing
construction of the transfer vector, and FIG. 2 shows the location
of the homologous arms, USA and USB in the genome.
1.1.1 Amplification and Cloning of the Homologous Recombinant
Arms
1.1.1.1 Design of Primers and Preparation of Templates
[0075] Two pairs of primers were designed for amplifying the
homologous arms at two sides of segment to be deleted according to
the gene sequence of HN1201 virus:
[0076] The upstream and downstream primers for the homologous arm
USA at the left side are, respectively:
TABLE-US-00001 USAF: CCGGAATTCTCGTCGTGGGCATCGTCATCAT (the underline
portion refers to the EcoR I cutting site) USAR:
CTATCTAGAataacttcgtataatgtatgctatacgaagttat CGGTACTGCGGAGGCTACGGAC
(the underline portion refers to the Xba I cutting site, lowercase
letters refer to the loxp site
[0077] The upstream and downstream primers for the homologous arm
USB at the right side are, respectively:
TABLE-US-00002 USBF: ACATGCATGCataacttcgtatagcatacattatacgaagttat
ACGGCAGGATCTCTCCGCGTCCC (the underline portion refers to the SphI
cutting site, lowercase letters refer to the loxp site) USBR:
CCCAAGCTTAGGAGGGGGCGGGGAGCGCGAGC (the underline portion refers to
the Hind III cutting site)
[0078] Vero cells were transfected with PRV HN1201, and part of
supernatant was harvested when the cytopathic effect of cells
reached to 80%, for extracting genomic DNA of virus by using
Geneaid Viral Nucleic Acid Extraction kit as the template for
amplification of the homologous arms.
1.1.1.2 Amplification and Cloning of the Homologous Arms, USA and
USB
[0079] USA and USB were amplified through PCR method by using
TAKARA PrimeSTAR, of which the system and condition is as
follows:
TABLE-US-00003 PRV HN1201 DNA 1 .mu.L PrimeSTAR 0.5 .mu.L
2*primeSTAR GC buffer 25 .mu.L dNTP(25 mM) 4 .mu.L Upstream primer
0.5 .mu.L downstream primer 0.5 .mu.L Water Used for adjusting to a
final volume of 50 .mu.L
TABLE-US-00004 98.degree. C. 2 min 98.degree. C. 10 s 68.degree. C.
1 min } 30 cycles 15 s 68.degree. C. 5 min
[0080] USA and USB fragments amplified by PCR were separated by
electrophoresis on agarose gel, and the target fragments were
recovered with TIANGEN Gel Recovery Kit. USA fragment and pUC19
vector was digested by both of EcoR I and XbaI, and the target
fragments were recovered, connected by T4 DNA ligase, and the
product was transformed into DH5.alpha.. The transformation mix was
spread onto plates containing ampicillin, and incubated at
37.degree. C. overnight. A single colony was picked to extract the
plasmid and the plasmid was identified using enzyme digestion, and
the correct plasmid after identification was named pUCUSA. pUCUSA
and USB was digested by both of SalI and HindIII, and the target
fragments were recovered, linked by T4 DNA ligase, and the product
was transformed into DH5.alpha.. The transformation mix was spread
onto plates containing ampicillin, and incubated at 37.degree. C.
overnight. A single colony was picked to extract the plasmid and
the plasmid was identified by sequencing after enzyme digestion,
and the correct plasmid after identification was named pUCUSAB.
1.1.3 Amplification of Label Gene GFP
1.1.2.1 Removal of Multiple Cloning Site (MCS) of GFP Vector
pAcGFP-C1
[0081] The pAcGFP-C1 plasmid (purchased from Clontech, Catalog No.
632470) was digested by Bgl II and Sma I, and the linearized vector
was recovered, linked by T4 DNA Ligase after filling-in with DNA
Polymerase I Large (Klenow) Fragment, and transformed into the
competent cell DH5.alpha. to obtain MCS deleted GFP plasmid, named
pAcGFP.DELTA.MCS.
1.1.2.2 Amplification of GFP Gene
[0082] The primers for amplifying GFP were designed according to
the sequence of pAcGFP-C 1 vector.
TABLE-US-00005 Upstream primer CMVU:
ACGCGTCGACTAGTTATTAATAGTAATCAATTACG (the underline portion refers
to the SalI cutting site.) Downstream primer SV40R:
ACATGCATGCCTAGAATGCAGTGAAAAAAATGC ((the underline portion refers to
the Sph I cutting site.)
[0083] GFP gene was amplified with pAcGFP.DELTA.MCS plasmid as the
template, of which the system and condition is as follows:
TABLE-US-00006 pAcGFP.DELTA.MCS 1 .mu.L primeSTAR 0.5 .mu.L
2*primeSTAR GC buffer 25 .mu.L dNTP(25 mM) 4 .mu.L Upstream primer
CMVU 0.5 .mu.L Downstream primer SV40R 0.5 .mu.L Water Used for
adjusting to a final volume of 50 .mu.L
TABLE-US-00007 94.degree. C. 2 min 94.degree. C. 30 s 60.degree. C.
30 s 30 cycles {close oversize brace} 72.degree. C. 2 min
72.degree. C. 5 min
[0084] A target band was recovered by electrophoresis on agarose
gel for further linking.
1.1.3 Linking of GFP Label Gene and pUCUSAB
[0085] GFP was digested with both of Sal and Sph I, and the target
fragments were recovered, linked to pUCUSB plasmid which had been
through the same double enzyme digestion, and the product was
transformed into the competent cell DH5.alpha.. The transformation
mix was spread onto plates containing ampicillin, and incubated at
37.degree. C. overnight. A single colony was picked to extract the
plasmid and the plasmid was identified by sequencing after enzyme
digestion, and the correct plasmid after identification was named
pUCUSA-GFP-B.
1.2 Acquisition of Recombinant Virus Containing GFP
1.2.1 Acquisition of Recombinant Virus Through Co-Transfection of
Vero Cells With the Transfer Vector and HN1201 DNA
[0086] Co-transfection of vero cells was conducted by using
lipofectin technique, wherein 3 .mu.g PRV-HN1201 viral genomic DNA
and 5 .mu.g the transfer vector pUCUSA-GFP-B was transfected, in
accordance with procedures of Lipofectamine 2000 Protocol
(Invitrogen, Catalog No. 11668030). Cells were incubated at
37.degree. C. in an incubator containing 5% CO.sub.2. The
supernatant of cell culture, i.e. P0 recombinant virus, named
rPRV-GFP-US-, was collected 36-48h after transfection, or until the
cytopathic effect was visible and infected cells exhibited
fluorescence.
1.2.2 Plaque Purification of Recombinant Viruses
[0087] When infected with the obtained PO recombinant virus
rPRV-GFP-US-, vero cells infected were covered with 2% agarose with
low melting point. After 48h when the cytopathic effect became
apparent and infected cells exhibited obvious fluorescence, a
plaque with a green fluorescence was picked and freeze-thawed 3
times in -70.degree. C., inoculated at 10-fold serial dilutions
into vero cells previously laid in six-well plates. Such plaque
with a green fluorescence was continued to be picked for
purification. After 8 rounds of plaques purification, a purified
recombinant virus rPRV-GFP-US-which was free of wild-type virus
HN1201 and with deletion of gI/gE/US9/US2 (i.e. gI/gE/11K/28K) was
obtained.
1.3 Deletion of GFP Label Gene in the gI/gE/US9/US2 (i.e.
gI/gE/11K/28K) Segment-Deleted Recombinant Virus
[0088] pBS185 plasmid expressing Cre enzyme (purchased from
addgene, Cre enzyme recognizes loxP sites at downstream of USA and
upstream of USB, wherein USA and USB are homology arms, and deletes
sequence between two loxp sites) and genomic DNA of recombinant
virus rPRV-GFP-US- was co-transfected into vero cells, with the
results showing relatively obvious cytopathic effect and more
single fluorescence 24 h after transfection. After serial dilution,
PO virus harvested was inoculated for plaque screening;
fluorescence-negative plaque was picked for the next round of
purification. After 2 rounds of screening and purification, a
fluorescence-negative virus was obtained, and named vPRV-US-. PCR
identification result after extraction and purification of viral
genomic DNA, showed deletion of gI/gE/US9/US2 (i.e. gI/gE/11K/28K)
segment, and indicated that GFP label gene had been deleted. The
result showed a successful purification of gI/gE/US9/US2 (i.e.
gI/gE/11K/28K) segment-deleted virus containing no GFP label
gene.
1.4 Confirmation of PRV HN1201 Strain With Deletion of US
Segment
[0089] The viral genome of gI/gE/US9/US2 (i.e. gI/gE/11K/28K)
segment-deleted virus and wild-type virus, was extracted and
identified by PCR, with the following primers:
TABLE-US-00008 USDCF: TACATCGTCGTGCTCGTCTTTGGC USDCR:
AGCTCGTGCGTCTCGGTGGTG
[0090] The size of PCR amplification product of the wild-type virus
was 6286 bp, the size of PCR amplification fragment of
gI/gE/US9/US2 (i.e. gI/gE/11K/28K) segment-deleted virus was 1960
bp.
[0091] PCR assay result confirmed that ORF of gI/gE/US9/US2 (i.e.
gI/gE/11K/28K) segment had been completely missing.
EXAMPLE 2
Preparation of PRV HN1201 Strain With Deletion of
gI/gE/11K/28K/TK
2.1 Construction of a Transfer Vector for Recombinant PRV HN1201GFP
Virus
[0092] According to the sequence of TK gene to be deleted, the
homologous arms at its two ends were designed, called TKA and TKB,
respectively. TKA and TKB were cloned into pUC19 vector and named
pUCTKAB. Then GFP gene was cloned into pUCTKAB, to obtain a
transfer vector for recombinant virus which was called
pUCTKA-GFP-B. The homologous arms in the transfer vector are
sequences of two sides of TK, therefore the recombinant virus
obtained after recombination, was TK gene deleted. FIG. 4 shows the
location of homologous arms, TKA and TKB in the genome.
2.1.1 Amplification and Cloning of the Homologous Recombinant
Arms
2.1.1.1 Design of Primers and Preparation of Template
[0093] Two pairs of primers were designed for amplifying the
homologous arms at two sides of TK gene according to the gene
sequence of HN1201 virus:
[0094] The upstream and downstream primers for the homologous arm
TKA at the left side are, respectively:
TABLE-US-00009 TKAF: CCGGAATTCGTAGTGCCGGTTGCCCACGTACA (the
underline portion refers to the EcoR I cutting site) TKAR:
CTAGTCTAGAataacttcgtatagtacacattatacgaagttat CGCTCAGGCTGCCGTTCTGC
(the underline portion refers to the Xba I cutting site, lowercase
letters refer to the loxp site)
[0095] The upstream and downstream primers for the homologous arm
TKB at the right side are, respectively:
TABLE-US-00010 TKBF: ACATGCATGCataacttcgtataatgtgtactatacgaagttat
AACGACGACGGCGTGGGAGG (the underline portion refers to the SphI
cutting site, lowercase letters refer to the loxp site) TKBR:
CCCAAGCTTAGGGCGACGGCGAAGAAGAGC (the underline portion refers to the
Hind III cutting site)
[0096] Vero cells were transfected with PRV HN1201, and part of
supernatant was harvested when the cytopathic effect of cells
reached to 80%, for extracting genomic DNA of virus by using
Geneaid Viral Nucleic Acid Extraction kit as the template for
amplification of the homologous arms.
2.1.1.2 Amplification and Cloning of the Homologous Arms, TKA and
TKB
[0097] TKA and TKB were amplified through PCR method by using
TAKARA PrimeSTAR, of which the system and condition is as
follows:
TABLE-US-00011 PRV HN1201 DNA 1 .mu.L PrimeSTAR 0.5 .mu.L
2*primeSTAR GC buffer 25 .mu.L dNTP(25 mM) 4 .mu.L Upstream primer
0.5 .mu.L downstream primer 0.5 .mu.L Water Used for adjusting to a
final volume of 50 .mu.L
TABLE-US-00012 98.degree. C. 2 min 98.degree. C. 10 s 68.degree. C.
1 min } 30 cycles 15 s 68.degree. C. 5 min
[0098] TKA and TKB fragments amplified by PCR were separated by
electrophoresis on agarose gel, and the target fragments were
recovered with TIANGEN Gel Recovery Kit. TKA fragment and pUC19
vector was digested with both of EcoR I and XbaI, and the target
fragments were recovered, linked by T4 DNA ligase, and the product
transformed into DH5.alpha.. The transformation mix was spread onto
plates containing ampicillin, and incubated at 37.degree. C.
overnight. A single colony was picked to extract the plasmid and
the plasmid was identified after enzyme digestion, and the correct
plasmid after identification was named pUCTKA. pUCTKA and TKB was
digested with both of SalI and HindIII, and the target fragments
were recovered, linked by T4 DNA ligase, and the product
transformed into DH5.alpha.. The transformation mix was spread onto
plates containing ampicillin, and incubated at 37.degree. C.
overnight. A single colony was picked to extract the plasmid and
the plasmid was identified by sequencing after enzyme digestion,
and the correct plasmid after identification was named pUCTKAB.
2.1.2 Amplification of Label Gene GFP
2.1.2.1 Removal of Multiple Cloning Site (MCS) of GFP Vector
pAcGFP-C1
[0099] The pAcGFP-C1 plasmid (purchased from Clontech, Catalog No.
632470) was digested with Bgl II and Sma I, and the linearized
vector was recovered, linked by T4 DNA Ligase after filling-in with
DNA Polymerase I Large (Klenow) Fragment, and the product was
transformed into the competent cell DH5.alpha. to obtain MCS
deleted GFP plasmid, named pAcGFP.DELTA.MCS.
2.1.2.2 Amplification of GFP Gene
[0100] The primers for amplifying GFP were designed according to
the sequence of pAcGFP-C1 vector.
TABLE-US-00013 Upstream primer CMVU:
ACGCGTCGACTAGTTATTAATAGTAATCAATTACG (the underline portion refers
to the SalI cutting site.) Downstream primer SV40R:
ACATGCATGCCTAGAATGCAGTGAAAAAAATGC ((the underline portion refers to
the Sph I cutting site.)
[0101] GFP gene was amplified with the template of pAcGFP.DELTA.MCS
plasmid, of which the system and condition is as follows:
TABLE-US-00014 pAcGFP.DELTA.MCS 1 .mu.L PrimeSTAR 0.5 .mu.L
2*primeSTAR GC buffer 25 .mu.L dNTP(25 mM) 4 .mu.L Upstream primer
CMVU 0.5 .mu.L Downstream primer SV40R 0.5 .mu.L Water Used for
adjusting to a final volume of 50 .mu.L
TABLE-US-00015 94.degree. C. 2 min 94.degree. C. 30 s 60.degree. C.
30 s 72.degree. C. 2 min 30 cycles {close oversize brace}
72.degree. C. 5 min
[0102] A target band was recovered by electrophoresis on agarose
gel for further linking.
2.1.3 Linking of GFP Label Gene and pUCTKAB
[0103] GFP was digested with both of Sal and Sph I, and the target
fragments were recovered, linked to pUCTKAB plasmid which had been
through the same double enzyme digestion, and the linked product
was transformed into the competent cell DH5.alpha.. The
transformation mix was spread onto plates containing ampicillin,
and incubated at 37.degree. C. overnight. A single colony was
picked to extract the plasmid and the plasmid was identified by
sequencing after enzyme digestion, and the correct plasmid after
identification was named pUCTKA-GFP-B.
2.2 Acquisition of Recombinant Virus Containing GFP
2.2.1 Acquisition of Recombinant Virus Through Co-Transfection of
Vero Cells With the Transfer Vector and vPRV-gI-gE-11K-28K-DNA
[0104] Co-transfection of vero cells was conducted by using
lipofectin technique, wherein 3 .mu.g vPRV-gI-gE-11K-28K- viral
genomic DNA and 5 .mu.g the transfer vector pUCTKA-GFP-B was
transfected, in accordance with procedures of Lipofectamine 2000
Protocol (Invitrogen, Catalog No. 11668030). Cells were incubated
at 37 .degree. C. in an incubator containing 5% CO.sub.2. The
supernatant of cell culture, i.e. PO recombinant virus, named
rPRV-GFP-gI-gE-11K-28K-TK-, was collected 36-48 h after
transfection, or until the cytopathic effect was visible and
infected cells exhibited fluorescence.
2.2.2 Plaque Purification of Recombinant Virus
rPRV-GFP-gI-gE-11K-28K-TK-
[0105] When infected with the obtained PO recombinant virus
rPRV-GFP-gI-gE-11K-28K-TK-, vero cells infected were covered with
2% agarose with low melting point. After 48 h when the cytopathic
effect became apparent and infected cells exhibited obvious
fluorescence, a plaque with a green fluorescence was picked and
freeze-thawed 3 times in -70 .degree. C., inoculated at 10-fold
serial dilutions into vero cells previously laid in six-well
plates. Such plaque with a green fluorescence was continued to be
picked for purification. After 11 rounds of plaques purification, a
purified recombinant virus rPRV-GFP-gI-gE-11K-28K-TK- which was
free of PRV-gI-gE-11K-28K-TK- and with deletion of five genes was
obtained.
2.3 Deletion of GFP Label Gene in gI/gE/11K/28K/TK Deleted
Recombinant Virus
[0106] The pBS185 plasmid expressing Cre enzyme (purchased from
addgene, Cre enzyme recognizes mutated loxP sites at downstream of
TKA and upstream of TKB, wherein TKA and TKB are homology arms, and
deletes sequence between two loxp sites) and genomic DNA of
recombinant virus rPRV-GFP-gI-gE-11K-28K-TK- was co-transfected
into vero cells, with the results showing relatively obvious
cytopathic effect and more single fluorescence 24 h after
transfection. After serial dilution, P0 virus harvested was
inoculated for plaque screening; fluorescence-negative plaque was
picked for the next round of purification. After 2 rounds of
screening and purification, a fluorescence-negative virus was
obtained, and named PRV-gI-gE-11K-28K-TK-. PCR identification
result after extraction and purification of viral genomic DNA,
showed deletion of TK gene, and also indicated that GFP label gene
had been deleted. The result showed a successful purification of
gI-gE-11K-28K-TK- deleted virus containing no GFP label gene.
2.4 Confirmation of PRV HN1201 Strain With Deletion of
gI/gE/11K/28K/TK
[0107] The primers used for identifying deletion of gI/gE/11K/28K
were the same as above.
[0108] The viral genome of gI/gE/11K/28K/TK-deleted virus and
wild-type virus, was extracted and identified by PCR, with the
following primers: [0109] TKDCF: cctacggcaccggcaagagca [0110]
TKDCR: cgcccagcgtcacgttgaagac
[0111] The size of PCR amplification product of the wild-type virus
was 1566 bp, the size of PCR amplification fragment of TK deleted
virus was 742 bp (refer to FIG. 5).
EXAMPLE 3
Preparation of PRV HN1201 Strain With Deletion of gI/gE
[0112] PRV HN1201 strain with deletion of gI/gE was prepared by
reference to the method in Example 1 of CN103756977A.
EXAMPLE 4
Pathogenicity Test of Gene-Deleted PRV Strain
[0113] 25 7-day-old piglets which were negative for pseudorabies
antibodies and pseudorabies antigen were randomly divided into 5
groups (A, B, C, D and blank control group), each with 5 piglets.
Grouping conditions and challenge conditions are shown in Table
1.
TABLE-US-00016 TABLE 1 Grouping of animals in the pathogenicity
test Group Strain used for inoculation Dose A PRV HN1201 strain
with inoculated with 1 ml deletion of gI/gE/11K/28K (10.sup.7.0
TCID.sub.50/ml)/piglet by prepared in Example 1 intranasal
instillation B PRV HN1201 strain with inoculated with 1 ml deletion
of (10.sup.7.0 TCID.sub.50/ml)/piglet by gI/gE/11K/28K/TK
intranasal instillation prepared in Example 2 C PRV HN1201 strain
with inoculated with 1 ml deletion of gI/gE prepared (10.sup.7.0
TCID.sub.50/ml)/piglet by in Example 3 intranasal instillation D
PRV HN1201 strain inoculated with 1 ml (10.sup.7.0
TCID.sub.50/ml)/piglet by intranasal instillation Blank DMEM medium
inoculated with 1 ml/piglet control by intranasal instillation
[0114] 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-00017 TABLE 2 Pathogenicity of different genes-deleted PRV
HN1201 strains in 7-day-old piglets Group Number Clinical signs
Death status A A1 Normal body temperature, no clinical Survived
signs A2 Body temperature increased for 1 day, Survived no clinical
signs A3 Normal body temperature, no clinical Survived signs A4
Normal body temperature, no clinical Survived signs A5 Body
temperature increased for 1 day, Survived no clinical signs B B1
Normal body temperature, no clinical Survived signs B2 Normal body
temperature, no clinical Survived signs B3 Normal body temperature,
no clinical Survived signs B4 Normal body temperature, no clinical
Survived signs B5 Normal body temperature, no clinical Survived
signs C C1 Body temperature increased for 1 day, Survived slightly
depression, loss of appetite C2 Body temperature increased for 1
day, Survived slightly depression, loss of appetite C3 Body
temperature increased for 1 day, Survived slightly depression, loss
of appetite C4 Body temperature increased for 1 day, Survived
slightly depression, loss of appetite C5 Body temperature increased
for 1 day, Survived slightly depression, loss of appetite D D1 Body
temperature increased for 3 days, Died on day depression,
completely loss of 3 after appetite, neurological signs such as
challenge staying lying, dyspnea, trembling, convulsions, turning
around, and making strokes with their arms D2 Body temperature
increased for 4 days, Died on day depression, completely loss of 4
after appetite, staying lying, dyspnea, challenge trembling and
convulsions. D3 Body temperature increased for 4 days, Died on day
depression, completely loss of 4 after appetite, neurological signs
such as challenge staying lying, dyspnea, trembling, convulsions,
turning around, and making strokes with their arms D4 Body
temperature increased for 4 days, Died on day depression,
completely loss of 4 after appetite, neurological signs such as
challenge staying lying, dyspnea, trembling, convulsions, turning
around, and making strokes with their arms D5 Body temperature
increased for 4 days, Died on day depression, completely loss of 4
after appetite, neurological signs such as challenge staying lying,
dyspnea, trembling, convulsions, and making strokes with their arms
Blank K1 Normal Survived control K2 Normal Survived K3 Normal
Survived K4 Normal Survived K5 Normal Survived
[0115] It showed in the results that inoculation with PRV HN1201
strain in 7-day-old piglets could lead to 100% death (5/5) of
inoculated piglets, while the virulence of PRV HN1201 strain with
deletion of gI/gE/11K/28K was significantly decreased, which could
only make the temperature of 2 piglets increased, without any
clinical signs. Inoculation with PRV HN1201 strain with deletion of
gI/gE in 7-day-old piglets could still lead to common clinical
signs such as increased body temperature and depression etc.,
indicating remaining virulence; while PRV HN1201 strain with
deletion of gI/gE/11K/28K/TK gene had completely lost its
virulence.
EXAMPLE 5
Preparation of the Live Gene-Deleted PRV Vaccines
5.1 Proliferation of Vaccine Virus
[0116] The virus seed of PRV HN1201 strain with deletion of
gI/gE/11K/28K prepared in Example 1, PRV HN1201 strain with
deletion of gI/gE/11K/28K/TK prepared in Example 2 and PRV HN1201
strain with deletion of gI/gE prepared in Example 3 was diluted at
5.times.10.sup.4 fold, and then inoculated into a monolayer of ST
cell. After 1 h adhesion, 1000 ml of DMEM medium containing 2%
fetal calf serum was added into ST cell, which was then placed at
37.degree. C. in a roller bottle with a rotation speed of 6 rph.
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 the medium and the
virus titer was assessed. The virus solution was preserved at low
temperature.
5.2 Preparation of a Protective Agent
[0117] 40 g of sucrose and 8 g of gelatin was added into every 100
ml of deionized water, and the solution was autoclaved (under
121.degree. C. for 30 min) after fully melted.
5.3 Preparation of Vaccine Virus Suspension
[0118] The virus solution prepared and preserved in Example 5.1 was
mixed with the protective agent prepared and preserved in Example
5.2 at a volume ratio of 1:1, distributed into sterilized bottles,
each of which containing 2.6 ml and the mixed virus solution was
freeze-dried. The vaccine was tested and determined to be free of
contamination of bacterium and exogenous viruses and the content of
virus was consistent with that before freeze-drying. The batch
number of PRV HN1201 strain with deletion of gI/gE/11K/28K prepared
in Example 1, PRV HN1201 strain with deletion of gI/gE/11K/28K/TK
prepared in Example 2 and PRV HN1201 strain with deletion of gI/gE
prepared in Example 3 were 20140501, 20140502 and 20140503,
respectively.
EXAMPLE 6
Immunogenicity Assay of the Live Gene-Deleted PRV Vaccines
[0119] 12 9-day-old piglets which were negative for PRV antibodies
and PRV antigens were randomly divided into 5 groups, each with 5
piglets, and the piglets were injected with the vaccines prepared
in Example 5 according to Table 3. The vaccine control group was
inoculated with the live PRV vaccine, Bartha K-61strain purchased
from HIPRA, Spain, Batch No. 42RH, at the dosage from the protocol.
The blank control group was inoculated with 1 mL/piglet of DMEM
medium. The piglets were challenged with
1.quadrature.10.sup.7.0TCID.sub.50/piglet of PRV HN1201 strain on
day 28 after immunization. After challenge, the body temperature of
piglets was determined daily, and in the meanwhile clinical signs
and death status were observed (The results are shown in Table 3),
the blood of piglets in all the experimental groups and control
groups was collected respectively before challenge.
TABLE-US-00018 TABLE 3 Grouping of animals in the pathogenicity
test Group Vaccines injected Dose Group I Batch No. inoculated with
1 ml 20140501 10.sup.6.0 TCID.sub.50/piglet by intramuscular
injection Group II Batch No. inoculated with 1 ml 20140502
10.sup.6.0 TCID.sub.50/piglet by intramuscular injection Group III
Batch No. inoculated with 1 ml 20140503 10.sup.6.0
TCID.sub.50/piglet by intramuscular injection Vaccine control Live
PRV inoculated with 2 ml group 2 vaccine 10.sup.6.0
TCID.sub.50/piglet by intramuscular injection Blank control DMEM
inoculated with 1 mL/piglet group medium by intramuscular
injection
[0120] The piglets were challenged with
1.quadrature.10.sup.7.0TCID.sub.50/piglet (1 ml/piglet) of PRV
HN1201 strain on day 28 after immunization. After challenge, the
body temperature of piglets was determined daily, and in the
meanwhile clinical signs and death status were observed (The
results are shown in Table 5).
TABLE-US-00019 TABLE 5 clinical status and challenge status for
piglets challenged after immunization with live PRV vaccines
clinical signs and death Group status Rate of protection Group I
Normal body temperature, 100% (5/5) normal appetite, no abnormal
clinical signs, survived Group II Normal body temperature, 100%
(5/5) normal appetite, no abnormal clinical signs, survived Group
III After immunization, body 100% (5/5) temperature increased,
slightly depression and loss of appetite. After challenge, normal
body temperature, normal appetite, no abnormal clinical signs,
survived Vaccine Body temperature of three 80% (4/5) control
piglets increased for 7-10 group days, depression, loss of
appetite, one died. Blank Body temperature of three 0% (0/5)
control piglets increased for 7-10 group days, depression in all
piglets, partially or completely loss of appetite, significant
clinical signs, two piglets died on day 4 after challenge, and all
died within 5 days after challenge.
[0121] The result from Table 5 indicated that immunizing piglets
with the gene-deleted PRV vaccines prepared in example 5 can
blocked virus infection (i.e. displaying clinical signs), and
provide 100% (5/5) protection rate for piglets, while all the
piglets in the blank control group died by day 5 after challenge,
therefore the PRV vaccines in three experimental groups can provide
excellent protection, showing excellent immune protection and
safety; meanwhile it indicated that either deletion of
gI/gE/11K/28K or deletion of gI/gE/11K/28K/TK for PRV strain would
not affect the immunogenicity. For the vaccine group with only
deletion of gI/gE, the clinical signs such as increased body
temperature could not be avoided, while the vaccine still possessed
good immunogenicity. Whereas the commercial vaccines in the prior
art cannot provide a full protection to pigs.
EXAMPLE 7
Construction of Gene-Deleted Variant Strains of NVDC-PRV-BJ Strain,
NVDCPRV-HEB Strain and NVDC-PRV-SD Strain, HN1202 PRV Variant
Strain
[0122] gI/gE/11K/28K genes and gI/gE/11K/28K/TK genes were deleted
from the parent strains, NVDC-PRV-BJ strain, NVDC-PRV-HEB strain
and NVDC-PRV-SD strain (Xiuling Yu, Zhi Zhou, Dongmei Hu,et al.
Pathogenic Pseudorabies Virus, China, 2012 Emerging Infectious
Diseases, www.cdc.gov/eid ol. 20, No. 1, January 2014) (the
applicant promises to open it to public for 20 year from the patent
application date according to provisions of Guidelines for Patent
Examination), HN1202 strain (deposited in the China Center for Type
Culture Collection on Aug. 26, 2013, of which the accession number
is CCTCC NO. V 201335 and the address is Wuhan University, Wuhan,
China), according to methods in Example 1 and 2. The names of the
attenuated strains obtained were NVDC-PRV-BJ with deletion of
gI/gE/11K/28K/TK, NVDCPRV-HEB with deletion of gI/gE/11K/28K/TK,
NVDC-PRV-SD with deletion of gI/gE/11K/28K/TK, and PRVHN1202 with
deletion of gI/gE/11K/28K/TK. The deletion of genes was verified
through comparison of PCR results with that of parent strains
respectively.
EXAMPLE 8
Preparation of Vaccine Compositions of the Attenuated Variant
Strains of NVDC-PRV-BJ Strain, NVDC-PRV-HEB Strain and NVDC-PRV-SD
Strain, HN1202 PRV Strain
[0123] Each attenuated vaccine strains prepared in Example 7 was
proliferated according to the method from Example 5.1, mixed with
the protective agent (prepared by adding 40 g of sucrose and 8 g of
gelatin into every 100 ml of deionized water, and autoclaved (under
121.degree. C. for 30 min) after fully melted) at a volume ratio of
1:1 and the mixed vaccine compositions were freeze-dried. The batch
numbers of NVDC-PRV-BJ strain with deletion of gI/gE/11K/28K/TK,
NVDCPRV-HEB strain with deletion of gI/gE/11K/28K/TK, NVDC-PRV-SD
strain with deletion of gI/gE/11K/28K/TK and PRV HN1201 strain with
deletion of gI/gE/11K/28K/TK were Q01, Q02, Q03 and Q04,
respectively.
EXAMPLE 9
Pathogenicity Test of the Virus Strains Prepared in Example 7
[0124] Pathogenicity test was conducted according to the method in
Example 4, in which the piglets were randomly divided into 5
groups, each with 5 piglets, inoculated with 1 ml
(10.sup.7.0TCID.sub.50/ml) of NVDC-PRV-BJ strain with deletion of
gI/gE/11K/28K/TK, NVDC-PRV-HEB strain with deletion of
gI/gE/11K/28K/TK, NVDC-PRV-SD strain with deletion of
gI/gE/11K/28K/TK, and PRV HN1202 strain with deletion of
gI/gE/11K/28K/TK by intranasal instillation, respectively. The
results showed that all the piglets were alive in each group, with
normal body temperature and no clinical signs. It proved that the
virulence of mutated PRV strain was reduced through deletion of
gI/gE/11K/28K/TK genes.
EXAMPLE 10
Immunogenicity Assay of the Vaccines Prepared in Example 8
[0125] Immunogenicity assay of the vaccines prepared in Example 8
was conducted according to the method and dose in Example 6, in the
meanwhile the piglets in the vaccine control group were inoculated
with the live PRV vaccine, HB-98 strain Batch No. 1308011-1
(purchased from China Animal Husbandry Industry Co., Ltd. Chengdu
Medical Equipments Factory). The piglets were challenged with
1.quadrature.10.sup.7.0TCID.sub.50/piglet of PRV HN1201 strain on
day 28 after immunization. After challenge, the body temperature of
piglets was determined daily, and in the meanwhile clinical signs
and death status were observed (the results are shown in Table
6).
TABLE-US-00020 TABLE 6 clinical status and challenge status for
piglets challenged after immunization with live PRV vaccines
clinical signs and death Rate of Group Vaccines status protection
Group IV Q01 Normal body 100% (5/5) temperature, normal appetite,
no abnormal clinical signs, survived Group V Q02 Normal body 100%
(5/5) temperature, normal appetite, no abnormal clinical signs,
survived Group VI Q03 Normal body 100% (5/5) temperature, normal
appetite, no abnormal clinical signs, survived Group VII Q04 Normal
body 100% (5/5) temperature, normal appetite, no abnormal clinical
signs, survived Vaccine the live PRV Body temperature of five 80%
(4/5) control vaccine, HB- piglets increased for 7-10 group 98
strain days, loss of appetite, one Batch No. piglet died and four
1308011-1 survived. Blank DMEM Body temperature of all 0% (0/5)
control medium piglets increased, group depression in all piglets,
partially or completely loss of appetite, significant clinical
signs, two piglets died on day 4 after challenge, and all died
within 5 days after challenge.
[0126] The result from Table 6 indicated that immunizing piglets
with the PRV vaccines prepared in Example 8 can block virus
infection (i.e. displaying clinical signs), and provide 100% (5/5)
protection rate for piglets, while the vaccine control group can
only provide 80% (4/5) protection rate for piglets, and all the
piglets in the blank control group died by day 5 after challenge,
therefore the PRV vaccines of the present invention can provide
excellent protection. In addition, the piglets exhibited
substantially no clinical signs, indicating excellent immune
protection of the PRV vaccines relative to live vaccines in the
prior art.
EXAMPLE 11
Monitoring of gB Antibodies After Immunization With Different
Strain Vaccines
[0127] 15 piglets at the age of around 13 days which were negative
for PRV antigens and PRV antibodies were randomly divided into 5
groups, each with 5 piglets. Groups 1-3 were injected with the
vaccine prepared in Example 5,which is PRV HN1201 strain with
deletion of gI/gE/11K/28K/TK, with Batch No. 20140502, the live PRV
vaccine Bartha K-61 strain, with Batch No. 66KR, purchased from
HIPRA, Spain, and the live PRV vaccine, K-61, with Batch No.
195-B59B purchased from Boehringer Ingelheim (US) respectively. All
the dose for immunization is 1 ml/piglet (for commercial vaccine, 1
piglet dosage/piglet, according to protocols; the PRV HN1201 with
deletion of gI/gE/11K/28K/TK vaccine,
10.sup.6.0TCID.sub.50/piglet). The blank control group was
inoculated with 1 mL/piglet of DMEM medium. The blood of piglets
was collected on day 8, 10, 12, 14 and 21 after immunization, and
gB antibody was determined according to the protocol of gB ELISA
antibody detection kit (purchased from Biochek, Batch No. F S5763,
Expiry Date: Jan. 7, 2015) after the serum was separated. The
detailed results of detection are shown in Table 7 below.
TABLE-US-00021 TABLE 7 Results of detection of gB antibodies of
piglets after immunization. Before Day 8 after Day 10 after
immunization immunization immunization Group No. of piglet OD405nm
S/P OD405nm S/P OD405nm S/P PRV HN1201 1# 0.184 0.025 0.398 0.469
0.439 0.555 strain with 2# 0.170 -0.004 0.369 0.409 0.453 0.584
deletion of 3# 0.172 0.000 0.263 0.189 0.360 0.390 gI/gE/11K/28K/
4# 0.181 0.019 0.320 0.307 0.494 0.669 TK vaccine 5# 0.182 0.021
0.339 0.347 0.400 0.474 with Batch No. 20140502 Bartha K-61 6#
0.177 0.010 0.223 0.106 0.243 0.147 7# 0.176 0.008 0.256 0.174
0.286 0.237 8# 0.167 -0.010 0.224 0.108 0.246 0.154 9# 0.186 0.029
0.221 0.102 0.219 0.098 10# 0.175 0.006 0.242 0.145 0.277 0.218
K-61 11# 0.162 -0.019 0.195 0.059 0.185 0.035 12# 0.16 -0.023 0.174
0.009 0.192 0.052 13# 0.167 -0.007 0.182 0.028 0.218 0.113 14# 0.16
-0.023 0.199 0.068 0.201 0.073 15# 0.17 0.000 0.219 0.115 0.225
0.129 Day 12 after Day 14 after Day 21 after immunization
immunization immunization Group No. of piglet OD405nm S/P OD405nm
S/P OD405nm S/P PRV HN1201 1# 0.471 0.621 0.678 1.051 1.069 1.863
strain with 2# 0.510 0.702 0.631 0.953 0.984 1.686 deletion of 3#
0.453 0.584 0.496 0.673 0.619 0.928 gI/gE/11K/28K/ 4# 0.596 0.881
0.687 1.070 0.844 1.396 TK vaccine, 5# 0.602 0.893 0.547 0.779
0.690 1.076 Batch No. 20140502 Bartha K-61 6# 0.275 0.214 0.290
0.245 0.570 0.827 7# 0.302 0.270 0.317 0.301 0.418 0.511 8# 0.283
0.231 0.309 0.285 0.315 0.297 9# 0.211 0.081 0.223 0.106 0.316
0.299 10# 0.272 0.208 0.299 0.264 0.486 0.652 K-61 11# 0.239 0.162
0.274 0.244 0.314 0.338 12# 0.205 0.082 0.211 0.096 0.277 0.251 13#
0.248 0.183 0.25 0.188 0.449 0.655 14# 0.256 0.202 0.285 0.27 0.321
0.354 15# 0.28 0.258 0.3 0.305 0.385 0.505 Note: evaluation
criteria: negative, S/P value .ltoreq.0.499; positive, S/P value
.gtoreq.0.500.
[0128] In conclusion, the antibody test results showed that, all gB
antibodies turned positive on day 12 after immunization with PRV
HN1201 strain with deletion of gI/gE/11K/28K/TK, while not all the
gB antibodies had turned positive on day 21 after immunization with
the two control vaccine. It showed that PRV HN1201 strain with
deletion of gI/gE/11K/28K/TK could provide earlier immune
protection.
EXAMPLE 12
Monitoring of gE Antibodies After Immunization With Four Genes
Deleted Strain Vaccine and Challenge
[0129] 15 piglets at the age of around 13 days which were negative
for PRV antigens and PRV antibodies were randomly divided into 3
groups, each with 5 piglets. Groups 1-3 were injected with the
vaccine prepared in Example 5,which is PRV HN1201 strain with
deletion of gI/gE/11K/28K/TK, with Batch No. 20140502, the live PRV
vaccine, Bartha K-61 strain, with Batch No. 66KR, purchased from
HIPRA, Spain, and the live PRV vaccine, K-61, with Batch No.
195-B59B purchased from Boehringer Ingelheim (US). All the dose for
immunization is 1 ml/piglet (for commercial vaccine, 1 piglet
dosage/piglet, according to protocols; the PRV HN1201 with deletion
of gI/gE/11K/28K/TK vaccine, 10.sup.6.0TCID.sub.50/piglet). The
piglets were challenged with 10.sup.7.0TCID.sub.50/piglet, 1
ml/piglet of PRV HN1201 strain on day 21 after immunization. The
blood of piglets was collected daily continuously from day 7 to day
14 after challenge, and gE antibody was determined according to the
protocol of gE ELISA antibody detection kit (purchased from IDEXX
Co., Batch No. AK650, Expiry Date: Jun. 13, 2015) after the serum
was separated. The results showed that gE antibody was still
negative (If the value of S/N is less or equal to 0.60, the sample
should be determined as PRV gE antibody positive) on Day 14 after
challenge when the piglets were immunized with the vaccine prepared
in Example 5, PRV HN1201 with deletion of gI/gE/11K/28K/TK with
Batch No. 20140502, while gE antibody became positive at different
level when the piglets were immunized with the two commercial
vaccines. The detailed results of deletion are shown in Table 8
below.
TABLE-US-00022 TABLE 8 Results of detection of gE antibody of
piglets after immunization. No Before Day 7 after Day 8 after Day 9
after Dayb10 after of challenge challenge challenge challenge
challenge Group piglet OD650nm S/N OD650nm S/N OD650nm S/N OD650nm
S/N OD650nm S/N PRV 1# 1.024 1.041 0.917 0.932 0.956 0.972 0.860
0.874 0.863 0.877 HN1201 2# 1.006 1.008 0.979 0.980 0.931 0.932
0.889 0.890 0.780 0.781 with 3# 1.070 1.072 0.990 0.991 1.007 1.009
0.970 0.971 0.929 0.930 deletion 4# 1.052 1.054 0.795 0.796 0.899
0.872 0.972 0.943 1.000 0.970 of 5# 0.969 0.970 0.912 0.913 0.915
0.916 0.922 0.923 0.683 0.684 gI/gE/11K/ 28K/TK, vaccine with Batch
No. 20140502 Bartha 6# 1.045 1.078 0.634 0.654 0.684 0.706 0.587
0.606 0.518 0.535 K-61 7# 1.063 1.097 0.788 0.813 0.758 0.782 0.664
0.685 0.612 0.632 8# 1.008 1.040 0.897 0.926 0.857 0.884 0.784
0.809 0.783 0.808 9# 1.017 1.050 0.720 0.743 0.637 0.657 0.599
0.618 0.467 0.482 10# 0.987 1.019 0.871 0.899 0.701 0.723 0.656
0.677 0.655 0.676 K-61 11# 0.905 0.934 0.946 0.976 0.698 0.720
0.643 0.664 0.618 0.638 12# 1.024 1.057 0.898 0.927 0.773 0.798
0.688 0.710 0.760 0.784 13# 1.030 1.063 0.957 0.928 0.965 0.936
0.913 0.886 0.732 0.710 14# 0.963 0.934 0.757 0.734 0.899 0.872
0.972 0.943 1.000 0.970 15# 0.944 0.916 0.747 0.725 0.591 0.573
0.543 0.527 0.531 0.515 Day 11 after Day 12 after Day 13 after Day
14 after No of challenge challenge challenge challenge Group piglet
OD650nm S/N OD650nm S/N OD650nm S/N OD650nm S/N PRV 1# 0.884 0.898
0.877 0.891 0.871 0.885 0.880 0.894 HN1201 2# 0.854 0.855 0.780
0.781 0.793 0.794 0.732 0.733 with 3# 0.907 0.908 0.905 0.906 0.904
0.905 1.067 1.069 deletion 4# 0.965 0.936 0.864 0.838 0.997 0.967
0.929 0.901 of 5# 0.623 0.624 0.718 0.719 0.784 0.785 0.718 0.719
gI/gE/11K/ 28K/ TK vaccine, with Batch No. 20140502 Bartha 6# 0.552
0.570 0.482 0.497 0.463 0.478 0.456 0.471 K-61 7# 0.664 0.685 0.533
0.550 0.499 0.515 0.478 0.493 8# 0.749 0.773 0.647 0.668 0.700
0.722 0.753 0.777 9# 0.450 0.464 0.410 0.423 0.432 0.446 0.433
0.447 10# 0.633 0.653 0.699 0.721 0.684 0.706 0.676 0.698 K-61 11#
0.568 0.586 0.472 0.487 0.472 0.487 0.449 0.463 12# 0.745 0.769
0.659 0.680 0.659 0.680 0.714 0.737 13# 0.785 0.761 0.678 0.658
0.505 0.490 0.425 0.412 14# 0.965 0.936 0.864 0.838 0.997 0.967
0.929 0.901 15# 0.578 0.561 0.528 0.512 0.457 0.443 0.398 0.386
[0130] The above results indicated that the vaccine strains in the
present invention has a better immunogenicity than commercial
vaccine in the prior art, and after immunization therewith a faster
generation of the antibody can be achieved, and the effective
amplification of virus in the body of pigs can be blocked, and gE
antibody is negative.
[0131] 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
514320DNAUnknownNucleotide sequence of gI/gE/11K/28K genes in the
PRV HN1201 strain 1atgatgatgg tggcgcgcga cgtgacccgg ctccccgcgg
ggctcctcct cgccgccctg 60accctggccg ccctgacccc gcgcgtcggg gggcgtcctc
ttcaggggcg ccggcgtcag 120cgtgcacgtc gccggcagcg ccgtcctcgt
gcccggcgac gcgcccaacc tgacgataga 180cgggacgctg ctgtttctgg
aggggccctc gccgagcaac tacagcgggc gcgtggagct 240gctgcgcctc
gaccccaagc gcgcctgcta cacgcgcgag tacgccgccg agtacgacct
300ctgcccccgc gtgcaccacg aagccttccg cggctgcctg cgcaagcgcg
agccgctcgc 360ccggcgcgcg tccgccgcgg tggaggcgcg ccggctgctg
ttcgtctcgc gcccggcctc 420gggggacgcg gggtcgtacg tgctgcgggt
ccgcgtgaac gggaccacgg acctctttgt 480gctgacggcc ctggtgccgc
cgagggggcg ccccgtcccc acgtcgccgc ccgcggacga 540gtgccggccc
gtcgtcggat cgtggcacga cagcctgcgc gtcgtggacc ccgccgagga
600cgccgtgttc accacccagc ccccgcccga gcccgagccg ccgacgaccc
ccgcgccccc 660ccgggggacc ggcgccaccc ccgagccccg atcggacgag
gaggaggagg gtgacgcgga 720gacgacgacg ccgacgctga ccccggcgcc
cgggaccctg gacgcgaacg gcacgatggt 780gctgaacgcc agcgtcgtgt
cgcgcgtcct gctcgccgcc gccaacgcca cggcgggcgc 840ccggagcccc
gggaagatag ccatggtgct ggggcccacg atcgtcgtcc tcctgatctt
900cctgggcggg atcgcctgcg tggcccggcg ctgcgcgcgg aatcgcatct
accggccgcg 960acccgggcgc ggatcggcgg tccatgcggc gcccccgcgg
cgcccgcccc caaccccgtc 1020gccggggcgc ccgtccccca gcccaagatg
acgttggccg agctgcgcca gaagctcgcc 1080accatcgcag aagaacaata
aaaaggtggt gtttgcataa ttttgtgggt ggcgttttat 1140ctccgtccgc
gccgttttaa acctgggcac ccccgcgagt ctcgcacaca ccggggttga
1200gaccatgcgg ccctttctgc tgcgcgccgc gcagctcctg gcgctgctgg
ccctggcgct 1260ctccaccgag gccccgagcc tctccgccga gacgaccccg
ggccccgtca ccgaggtccc 1320gagtccctcg gccgaggtct gggacgacct
ctccaccgag gccgacgacg atgacctcaa 1380cggcgacctc gacggcgacg
accgccgcgc gggcttcggc tcggccctcg catccctgag 1440ggaggcgccc
ccggcccatc tggtgaacgt gtccgagggc gccaacttca ccctcgacgc
1500gcgcggcgac ggcgccgtgc tggccgggat ctggacgttc ctgcccgtcc
gcggctgcga 1560cgccgtgtcg gtgaccacgg tgtgcttcga gaccgcgtgc
cacccggacc tggtgctggg 1620ccgcgcctgc gtccccgagg ccccggagat
gggcatcggc gactacctgc cgcccgaggt 1680gccgcggctc cggcgcgagc
cgcccatcgt caccccggag cggtggtcgc cgcacctgag 1740cgtcctgcgg
gccacgccca acgacacggg cctctacacg ctgcacgacg cctcggggcc
1800gcgggccgtg ttctttgtgg cggtgggcga ccggccgccc gcgccggcgg
acccggtggg 1860ccccgcgcgc cacgagcccc gcttccacgc gctcggcttc
cactcgcagc tcttctcgcc 1920cggggacacg ttcgacctga tgccgcgcgt
ggtctcggac atgggcgact cgcgcgagaa 1980ctttaccgcc acgctggact
ggtactacgc gcgcgcgccc ccgcggtgcc tgctgtacta 2040cgtgtacgag
ccctgcatct accacccgcg cgcgcccgag tgcctgcgcc cggtggaccc
2100ggcgtgcagc ttcacctcgc cggcgcgcgc gcggctggtg gcgcgccgcg
cgtacgcctc 2160gtgcagcccg ctgctcgggg accggtggct gaccgcctgc
cccttcgacg ccttcggcga 2220ggaggtgcac acgaacgcca ccgcggacga
gtcggggctg tacgtgctcg tgatgaccca 2280caacggccac gtcgccacct
gggactacac gctcgtcgcc accgcggccg agtacgtcac 2340ggtcatcaag
gagctgacgg ccccggcccg ggccccgggc accccgtggg gccccggcgg
2400cggcgacgac gcgatctacg tggacggcgt cacgacgccg gcgccgcccg
cgcgcccgtg 2460gaacccgtac ggccggacga cgcccgggcg gctgtttgtg
ctggcgctgg gctccttcgt 2520gatgacgtgc gtcgtcgggg gggccatctg
gctctgcgtg ctgtgctccc ggcgccgggc 2580ggcctcgcgg ccgttccggg
tgccgacgcg ggcgcggacg cacatgctct ctccggtgta 2640caccagcctg
cccacgcacg aggactacta cgacggcgac gacgacgacg acgaggaggc
2700gggcgtcatc cgccggcggc ccgcctcccc cagcggagac agcggctacg
aggggccgta 2760cgcgagcctg gaccccgagg acgagttcag cagcgacgag
gacgacgggc tgtacgtgcg 2820ccccgaggag gcgccccgct ccggcttcga
cgtctggttc cgcgatccgg agaaaccgga 2880agtgacgaat ggacccaact
atggcgtgac cgccaaccgc ctgttgatgt cccgccccgc 2940ttaaataccg
ggagaaccgg tccgcccgca ttccgacatg cccggcgccg cctccgtcga
3000catggacacg ttcgacccca gcgcccccgt cccgacgagc gtctcgaacc
cggccgccga 3060cgtcctgctg gcccccaagg gaccccgctc cccgctgcgc
ccccaggacg actcggactg 3120ctactacagc gagagcgaca acgagacgcc
cagcgagttc ctgcgccgcg tgggacgccg 3180gcaggcggcg cgtcggagac
gccgccgctg cctgatgggc gtcgcgatca gcgccaccgc 3240gctggtcatc
tgctcgctgt ccgcgctact cgggggcatc atcgcccggc acgtgtagcg
3300agcgagcgag cgaacgggag cgggggcccg cccccatccg ccgcgcccag
gagagggggg 3360agagagcggg gggttgggcg cgccacgtgg tgtgggcacg
gactcggact tgtcacaata 3420aatgggcccc ggcgtgtccg ggcgcacaca
gcagccttcc tctcctccgc gtctctgttc 3480cgcccgtctc tcgccggact
cttcttctcc accgcctcca ccgtcgcagt tgtcgcgagc 3540gcgttcgcac
catgggggtg acggccatca ccgtggtcac gctgatggac ggggccgggc
3600gcatccccgc cttcgtgggc gaggcgcacc cggacctgtg gaaggtgctc
accgagtggt 3660gctacgcgtc gatggtgcag cagcggcgcg ccgccgacga
gaactcgccg cggcagcacg 3720tggtgctgcg ctcctcggag atctcccccg
gctcgctggc cctgctgccg cgcgccgtgc 3780gccccgtcgt gcggacgcgg
tccgacccca cggcgccgtt ctacatcacc accgagacgc 3840acgagctgac
gcggcgcccc ccggcggacg gctcgaagcc cggggagccc ctcaggatca
3900gccacccccg cggctggaca cggagtggtc gtccgtcctg aacgggatcc
agtacctgaa 3960ctcgggggcc cggggcacgg ccccgtccac ctgtggatcc
tgggcgccgc cgacctctgc 4020gaccaggtgc tcctggccgc ctcccgcagc
accgccgccg gagcctccca cgcccagacg 4080ggcgcgcgcc tgacccggcg
ccggcccggg ctgacggacg ccgacgccct ggacgtgatc 4140gtcgccggga
tccaggcgac ccgcgccatg ttcgcgcggg tccacaaccg ctcctggcgc
4200cacgccggcg agtggacgga ggccctgcac tcccagatcg tgacccgggg
cgacgtgcgc 4260cggcgccgag gcgggcgcgg caacggacgc gagcgcgccc
cgcgatgtac catctcctag 43202963DNAUnknownNucleotide sequence of TK
gene in the PRV HN1201 strain 2atgcgcatcc tccggatcta cctcgacggc
gcctacggca ccggcaagag caccacggcc 60cgggtgatgg cgctcggcgg ggcgctgtac
gtgcccgagc cgatggcgta ctggcgcact 120ctgttcgaca cggacacggt
ggccggtatt tacgatgcgc agacccggaa gcagaacggc 180agcctgagcg
aggaggacgc ggccctcgtc acggcgcagc accaggccgc cttcgcgacg
240ccgtacctgc tgctgcacac gcgcctggtc ccgctcttcg ggcccgcggt
cgagggcccg 300cccgagatga cggtcgtctt tgaccgccac ccggtggccg
cgacggtgtg cttcccgctg 360gcgcgcttca tcgtcgggga catcagcgcg
gcggccttcg tgggcctggc ggccacgctg 420cccggggagc cccccggcgg
caacctggtg gtggcctcgc tggacccgga cgagcacctg 480cggcgcctgc
gcgcccgcgc gcgcgccggg gagcacgtgg acgcgcgcct gctcacggcc
540ctgcgcaacg tctacgccat gctggtcaac acgtcgcgct acctgagctc
ggggcgccgc 600tggcgcgacg actgggggcg cgcgccgcgc ttcgaccaga
ccgtgcgcga ctgcctcgcg 660ctcaacgagc tctgccgccc gcgcgacgac
cccgagctcc aggacaccct cttcggcgcg 720tacaaggcgc ccgagctctg
cgaccggcgc gggcgcccgc tcgaggtgca cgcgtgggcg 780atggacgcgc
tcgtggccaa gctgctgccg ctgcgcgtct ccaccgtcga cctggggccc
840tcgccgcgcg tctgcgccgc ggccgtggcg gcgcaggcgc gcggcatgga
ggtgacggag 900tccgcgtacg gcgaccacat ccggcagtgc gtgtgcgcct
tcacgtcgga gatgggggtg 960tga 9633190DNAUnknownNucleotide sequence
of location for deletion of TK gene in the PRV HN1201 strain
3atgcgcatcc tccggatcta cctcgacggc gcctacggca ccggcaagag caccacggcc
60cgggtgatgg cgctcggcgg ggcgctgtac gtgcccgagc cgatggcgta ctggcgcact
120ctgttcgaca cggacacggt ggccggtatt tacgatgcgc agacccggaa
gcagaacggc 180agcctgagcg 190463PRTUnknownAmino acid sequence of
location for deletion of TK gene in the PRV HN1201 strain 4Met Arg
Ile Leu Arg Ile Tyr Leu Asp Gly Ala Tyr Gly Thr Gly Lys 1 5 10 15
Ser Thr Thr Ala Arg Val Met Ala Leu Gly Gly Ala Leu Tyr Val Pro 20
25 30 Glu Pro Met Ala Tyr Trp Arg Thr Leu Phe Asp Thr Asp Thr Val
Ala 35 40 45 Gly Ile Tyr Asp Ala Gln Thr Arg Lys Gln Asn Gly Ser
Leu Ser 50 55 60 5579PRTUnknownAmino acid sequence of gE in the PRV
HN1201 strain 5Met Arg Pro Phe Leu Leu Arg Ala Ala Gln Leu Leu Ala
Leu Leu Ala 1 5 10 15 Leu Ala Leu Ser Thr Glu Ala Pro Ser Leu Ser
Ala Glu Thr Thr Pro 20 25 30 Gly Pro Val Thr Glu Val Pro Ser Pro
Ser Ala Glu Val Trp Asp Asp 35 40 45 Leu Ser Thr Glu Ala Asp Asp
Asp Asp Leu Asn Gly Asp Leu Asp Gly 50 55 60 Asp Asp Arg Arg Ala
Gly Phe Gly Ser Ala Leu Ala Ser Leu Arg Glu 65 70 75 80 Ala Pro Pro
Ala His Leu Val Asn Val Ser Glu Gly Ala Asn Phe Thr 85 90 95 Leu
Asp Ala Arg Gly Asp Gly Ala Val Leu Ala Gly Ile Trp Thr Phe 100 105
110 Leu Pro Val Arg Gly Cys Asp Ala Val Ser Val Thr Thr Val Cys Phe
115 120 125 Glu Thr Ala Cys His Pro Asp Leu Val Leu Gly Arg Ala Cys
Val Pro 130 135 140 Glu Ala Pro Glu Met Gly Ile Gly Asp Tyr Leu Pro
Pro Glu Val Pro 145 150 155 160 Arg Leu Arg Arg Glu Pro Pro Ile Val
Thr Pro Glu Arg Trp Ser Pro 165 170 175 His Leu Ser Val Leu Arg Ala
Thr Pro Asn Asp Thr Gly Leu Tyr Thr 180 185 190 Leu His Asp Ala Ser
Gly Pro Arg Ala Val Phe Phe Val Ala Val Gly 195 200 205 Asp Arg Pro
Pro Ala Pro Ala Asp Pro Val Gly Pro Ala Arg His Glu 210 215 220 Pro
Arg Phe His Ala Leu Gly Phe His Ser Gln Leu Phe Ser Pro Gly 225 230
235 240 Asp Thr Phe Asp Leu Met Pro Arg Val Val Ser Asp Met Gly Asp
Ser 245 250 255 Arg Glu Asn Phe Thr Ala Thr Leu Asp Trp Tyr Tyr Ala
Arg Ala Pro 260 265 270 Pro Arg Cys Leu Leu Tyr Tyr Val Tyr Glu Pro
Cys Ile Tyr His Pro 275 280 285 Arg Ala Pro Glu Cys Leu Arg Pro Val
Asp Pro Ala Cys Ser Phe Thr 290 295 300 Ser Pro Ala Arg Ala Arg Leu
Val Ala Arg Arg Ala Tyr Ala Ser Cys 305 310 315 320 Ser Pro Leu Leu
Gly Asp Arg Trp Leu Thr Ala Cys Pro Phe Asp Ala 325 330 335 Phe Gly
Glu Glu Val His Thr Asn Ala Thr Ala Asp Glu Ser Gly Leu 340 345 350
Tyr Val Leu Val Met Thr His Asn Gly His Val Ala Thr Trp Asp Tyr 355
360 365 Thr Leu Val Ala Thr Ala Ala Glu Tyr Val Thr Val Ile Lys Glu
Leu 370 375 380 Thr Ala Pro Ala Arg Ala Pro Gly Thr Pro Trp Gly Pro
Gly Gly Gly 385 390 395 400 Asp Asp Ala Ile Tyr Val Asp Gly Val Thr
Thr Pro Ala Pro Pro Ala 405 410 415 Arg Pro Trp Asn Pro Tyr Gly Arg
Thr Thr Pro Gly Arg Leu Phe Val 420 425 430 Leu Ala Leu Gly Ser Phe
Val Met Thr Cys Val Val Gly Gly Ala Ile 435 440 445 Trp Leu Cys Val
Leu Cys Ser Arg Arg Arg Ala Ala Ser Arg Pro Phe 450 455 460 Arg Val
Pro Thr Arg Ala Arg Thr His Met Leu Ser Pro Val Tyr Thr 465 470 475
480 Ser Leu Pro Thr His Glu Asp Tyr Tyr Asp Gly Asp Asp Asp Asp Asp
485 490 495 Glu Glu Ala Gly Val Ile Arg Arg Arg Pro Ala Ser Pro Ser
Gly Asp 500 505 510 Ser Gly Tyr Glu Gly Pro Tyr Ala Ser Leu Asp Pro
Glu Asp Glu Phe 515 520 525 Ser Ser Asp Glu Asp Asp Gly Leu Tyr Val
Arg Pro Glu Glu Ala Pro 530 535 540 Arg Ser Gly Phe Asp Val Trp Phe
Arg Asp Pro Glu Lys Pro Glu Val 545 550 555 560 Thr Asn Gly Pro Asn
Tyr Gly Val Thr Ala Asn Arg Leu Leu Asn Ala 565 570 575 Arg Pro
Ala
* * * * *
References