CN107828741B - Pseudorabies virus double-gene deletion attenuated strain and application thereof - Google Patents

Abstract

The invention provides a pseudorabies virus double-gene deletion attenuated strain and application thereof, belonging to the field of vaccines in animal medicine. Pseudorabies virus LA1206-80 strain, the preserving number is: CGMCC No. 14329. The invention also provides application of the pseudorabies virus double-gene deletion attenuated strain in preparing a pseudorabies vaccine and a recombinant vector live vaccine. The recombinant vector is obtained by inserting an exogenous protective antigen gene expression cassette into the genome of the low virulent strain. The PRV LA1206-80 strain live vaccine is 100% safe to 1 day old newborn piglets, and can be used for early immunization of the newborn piglets and blocking wild virus infection as early as possible. After being inoculated to the weaned PRV negative piglet, the vaccine can quickly generate high-efficiency protective power, and particularly, the vaccine can prevent the disease from happening and can also prevent the toxin from being discharged. By monitoring the PRV gE and gB antibodies, decontamination of PRV variants is greatly facilitated.

Description

Pseudorabies virus double-gene deletion attenuated strain and application thereof

Technical Field

The invention belongs to the field of vaccines in animal medicine, and particularly relates to a pseudorabies virus double-gene deletion attenuated strain and application thereof.

Background

Pseudorabies is an acute, virulent infectious disease caused by Pseudorabies Virus (PRV). The pseudorabies of domestic animals such as cattle and sheep and wild animals is often manifested as typical symptoms of extreme itching, as well as clinical symptoms such as fever and encephalomyelitis. The symptoms of the pseudorabies of the pigs are related to the age, and the sows often suffer abortion; the newborn piglets have nervous symptoms and high mortality rate; the fattening pigs are mainly in respiratory tract symptoms, and the adult pigs are stagnated after being infected with pseudorabies viruses and generally do not die. Because PRV gene deletion strain vaccines (such as Bartha K61 strain with gE gene deletion and the like) are invented and popularized in a large scale and matched with the establishment and application of an ELISA detection method for identifying PRV gE antibodies of immune pigs and wild virus infected pigs, pseudorabies viruses in domestic swinery are purified in many countries in the world by continuously eliminating positive pigs, PRV gE antibody negative swinery is successfully established in many swineries in China, PRV is basically purified in many swineries, and the porcine pseudorabies is well controlled.

Since 2011, many pig farms immunized with traditional porcine pseudorabies vaccines in northern China have large-scale porcine pseudorabies epidemic situations, the epidemic situations rapidly spread to most pig farms in China, the PRV gE antibody positive rate is high, and serious harm is caused to the pig industry in China. Research results of a plurality of groups prove that the epidemic situation is caused by the variant PRV wild strain, the pathogenicity of the variant strain is obviously enhanced, the main antigen gene of the variant strain is obviously different from that of the traditional vaccine strain, and in order to more effectively deal with the harm of the PRV variant strain, the novel vaccine which is developed by applying the variant strain can improve the protective force and can distinguish and diagnose the immune pig and the wild virus infected pig is the key for preventing and controlling the porcine pseudorabies in China at present. However, the safety of the existing variant live vaccines is not high enough. For example, the invention patent with the application number of 201510388390.9 discloses an LA-A strain obtained by knocking out the gE gene of an AH02LA strain of the pseudorabies virus, and a vaccine prepared by the strain has 100 percent of protection effect on a pseudorabies virus variant strain, particularly the vaccine can prevent the disease from happening and can also prevent the toxin from being discharged, but the strain is only safe for 4-week-old piglets.

Disclosure of Invention

The invention aims to provide a pseudorabies virus double-gene deletion attenuated strain which can be well adapted to ST cells, has high titer, obviously reduces toxicity and has no pathogenicity to piglets of 1 day old, thereby being a vaccine candidate attenuated strain with higher safety.

The pseudorabies virus double-gene deletion low virulent strain is a pseudorabies virus LA1206-80 strain, and the preservation number is as follows: CGMCC No. 14329.

The invention also aims to provide the application of the pseudorabies virus double-gene deletion attenuated strain in the preparation of the pseudorabies vaccine, the vaccine can only provide 100 percent of protection for immune pigs after being immunized, not only can prevent the onset of disease, but also can prevent the detoxification, and the strain has obviously reduced toxicity and no pathogenicity to 1-day-old piglets, so the strain can be used for early immunization of newborn piglets and can block the wild virus infection as early as possible, and the live vaccine has higher safety.

The invention further aims to provide a vaccine taking the pseudorabies virus double-gene deletion low virulent strain as an active ingredient. The vaccine adopting the pseudorabies virus LA1206-80 strain is safe for 1-day-old newborn piglets, and can achieve 100 percent of protection effect on the pseudorabies virus variant strain 7 days after inoculating weaned PRV negative susceptible piglets, particularly, the vaccine can prevent diseases and toxin expelling, and has unexpected technical effects on safety and immune efficacy. By monitoring the antibody, the animals immunized with the vaccine can be distinguished from infected animals, and purification of PRV variant strains is facilitated.

The invention also provides a recombinant vector live vaccine, and the active component of the recombinant vector live vaccine is obtained by inserting the exogenous protective antigen gene expression cassette into the genome of the pseudorabies virus double-gene deletion low virulent strain. The promoter can be selected from pMCMV IE, SV40, pHCMV IE and the like, the insertion sites of exogenous genes comprise non-coding regions between UL22 and UL21, between UL46 and UL27, between UL51 and UL50, between UL35 and UL36, between UL40 and UL41, between UL44 and UL26.5 and between UL4 and UL3.5 of the genome, and the inserted exogenous genes comprise E protein gene of classical swine fever virus, Cap protein gene of porcine circovirus, G protein gene of rabies virus, VP1 gene of foot-and-mouth disease virus, HA gene of porcine influenza virus, VP60 gene of rabbit fever virus and the like.

The live vaccine of the invention takes the pseudorabies virus LA1206-80 strain as an active component, immune adjuvant, vaccine excipient and the like can be added into the vaccine, and the vaccine can be easily produced by a person skilled in the art.

The applicant obtains a pseudorabies virus gene deletion attenuated strain, namely PRV LA1206-80 strain, by a great amount of creative labor. The gC and gD genes of LA1206-80 strain are completely consistent with AH02LA strain (gC gene GenBank: KR 605320; gD gene GenBank: KR605321), and are highly homologous with the currently disclosed variant strain. The pseudorabies virus LA1206-80 strain is obtained by firstly knocking out 13 th to 1298 th bases of gE gene (GenBank: KR605321) of PRV AH02LA strain, then continuously knocking out 184 th to 530 th bases of TK gene (SEQ ID No:1), and then carrying out cell passage weakening and cloning screening. The pseudorabies virus LA1206-80 strain can be well adapted to ST cells, and the growth titer is as high as 10^8.5TCID₅₀and/mL. The live vaccine of the pseudorabies virus LA1206 strain is 100 percent safe for 1-day-old newborn piglets, and can be used for early immunization of the newborn piglets and blocking the wild virus infection as early as possible. After being inoculated to a weaned PRV negative piglet, the vaccine can quickly generate high-efficiency protective power and protect 100 percent of pseudorabies virus variant strains, and particularly, the vaccine can prevent the disease from happening and can also prevent the toxin from being discharged, thereby obtaining unexpected technical effects. By monitoring PRV gE and gB antibodies, animals immunized with the vaccine can be distinguished from infected animals, and purification of PRV variant strains is facilitated. The protective antigen is used as a live vector to express the protective antigen of the pig susceptible pathogen, and the effect of preventing two or more epidemic diseases by inoculating one needle can be achieved.

Drawings

FIG. 1 shows the structure of the transfer vector pPRV (AH02LA) -GFP (gE-).

FIG. 2, structural diagram of transfer vector pPRV (AH02LA) -gE.DELTA..

FIG. 3 is a schematic diagram of the construction of the strain PRV AH02LA (Δ gE). (A) Shown is the genome of strain PRV AH02LA, U_LMeans a unique long region, U_sRefers to the unique short region, and IR and TR refer to the internal and terminal repeat regions, respectively. (B) Shown is the replacement of part of the sequence of the gE gene by the GFP expression cassette by homologous recombination using the upstream homology arm Δ gE-H1 and the downstream homology arm Δ gE-H2. (C) Shown as passing againHomologous recombination is carried out to knock out a GFP expression cassette, and a PRV AH02LA (delta gE) strain with deletion of 1286bp nucleotides at 13 th-1298 th positions of the gE gene is obtained.

FIG. 4 shows the structure of the transfer vector pPRV (AH02LA) -GFP (TK-).

FIG. 5, structural diagram of transfer vector pPRV (AH02LA) -TK. DELTA..

FIG. 6 shows a schematic diagram of the construction of the pseudorabies virus LA1206 strain. (A) Shown is the genome of strain PRV AH02LA (. DELTA.gE), U_LMeans a unique long region, U_sRefers to the unique short region, and IR and TR refer to the internal and terminal repeats, respectively. (B) Shown is the partial sequence of the GFP expression cassette replacing the TK gene by homologous recombination via the upstream homology arm Δ TK-H1 and the downstream homology arm Δ TK-H2. (C) The GFP expression cassette is knocked out again through homologous recombination to obtain LA1206 strain which lacks 1286bp nucleotides from 13 th to 1298 th sites of the gE gene and 347bp nucleotides from 184 th to 530 th sites of the TK gene.

FIG. 7, LA1206-80 strains and parental viruses in vitro growth characteristics comparison. A: titer of virus in infected cell supernatant after infection of ST cells; b: viral titer of cell-associated virus after infection of ST cells.

Detailed Description

The present invention is further illustrated by the following specific examples, but the scope and spirit of the present invention are not limited to the examples.

Example I construction and identification of Pseudorabies Virus gE Gene-deleted Strain

1. Obtaining of transfer vectors

In order to knock out part of gE gene of the pseudorabies virus PRV AH02LA strain, a gene fragment consisting of the upstream homology arm delta gE-H1 of the gE gene, a green fluorescent protein expression cassette and the downstream homology arm delta gE-H2 of the gE gene was artificially synthesized, and then the fragment was cloned into a pUC57 vector (Nanjing King Shirui Biotech Co., Ltd.), thereby obtaining a GFP transfer vector pPRV (AH02LA) -GFP (gE-), the structure of which is shown in FIG. 1. The sequence of the upstream homology arm delta gE-H1 of the gE gene is shown as SEQ ID No: 2, the sequence of the downstream homology arm delta gE-H2 of the gE gene is shown as SEQ ID No: 3, the sequence of the GFP expression cassette is the same as that of the invention patent with the application number of 201510388390.9.

Then, a gene fragment consisting of the upstream homology arm Δ gE-H1 of the gE gene and the downstream homology arm Δ gE-H2 of the gE gene was artificially synthesized, and this fragment was cloned into a pUC57 vector (tokyo kasry biotechnology limited), to obtain a transfer vector pPRV (AH02LA) -gE Δ, the structure of which is shown in fig. 2. The sequence of the upstream homology arm delta gE-H1 of the gE gene is shown as SEQ ID No: 2, the sequence of the downstream homology arm delta gE-H2 of the gE gene is shown as SEQ ID No: 3, respectively.

2. Acquisition of recombinant GFP pseudorabies Virus AH02LA Strain

Primary Chick Embryo Fibroblasts (CEF) are prepared by using SPF chick embryos of 9-10 days old, and after the cells grow into a monolayer, transfection is performed. When transfection is carried out according to a conventional calcium phosphate transfection method, 0.5-1 mu g of GFP transfer vector pPRV (AH02LA) -GFP (gE-) DNA and 0.2-1 mu g of DNA of pseudorabies virus AH02LA strain are co-transfected into primary CEF for homologous recombination (figure 3), culture solution is sucked out after 24 hours, 10% of fetal calf serum and 0.5% of methylcellulose DMEM medium are added for covering, culture is continued for 24-48 hours, recombinant virus plaques emitting green fluorescence under the excitation of 488nm ultraviolet light are picked up and inoculated into fresh CEF, and the operation is repeated for more than 3 cycles to obtain purified recombinant virus, namely PRV AH02LA (delta gE) -GFP strain. A gene fragment (delta gE-H2) consisting of a gE gene upstream homology arm (delta gE-H1), a green fluorescent protein expression cassette (GFP) and a gE gene downstream homology arm is subjected to PCR amplification by using DNA of a PRV AH02LA (delta gE) -GFP strain as a template and a pair of primers (an upstream primer PRV gE site ckeck F sequence is 5'-TCTGGAGGGGCCCTCGCCGA-3'; and a downstream primer PRV gE site ckeck R sequence is 5'-AGAGAGAGGACGGAGGCGTGTCATC-3'), and an amplification product is subjected to electrophoresis and sequence determination to verify whether the sequence is correctly inserted. The PCR reaction system (Table 1) and the reaction procedure were as follows.

TABLE 125. mu.l PCR reaction System

LA Taq DNA polymerase and 2 XGC Buffer II in the PCR reaction system were purchased from Takara.

And (3) uniformly mixing the reaction system, and carrying out PCR amplification, wherein the reaction procedure is as follows: pre-denaturation at 94 ℃ for 4 min; denaturation at 94 ℃ for 40s, annealing at 57 ℃ for 40s, extension at 72 ℃ for 4min for 30s, 30 cycles; extension at 72 ℃ for 10 min. And (3) carrying out 1% agarose gel electrophoresis on the PCR product, detecting whether a target band of about 4585bp exists or not, cutting the target band, recovering by adopting a DNA gel recovery kit, and sending the recovered product to Huada Gene company for sequencing.

The identification result shows that after the recombinant virus PRV AH02LA (delta gE) -GFP strain is subjected to PCR amplification by adopting the primers, a target product of about 4585bp is obtained, and the sequence determination result is correct, wherein the recombinant virus is obtained by replacing 1286bp nucleotides from 13 th site to 1298 th site of the gE gene of the pseudorabies virus AH02LA strain with a GFP expression cassette (figure 3), and is completely consistent with the expectation.

3. Obtaining and identifying pseudorabies virus gE gene deletion strain

The GFP expression cassette was knocked out by performing a second homologous recombination by transfecting primary CEF with 1-3. mu.g of DNA of the recombinant virus PRV AH02LA (Δ gE) -GFP strain and 700-2000 ng of DNA of pPRV (AH02LA) -gE Δ according to a conventional calcium phosphate transfection method (FIG. 3). Absorbing the culture solution 24 hours after cotransfection, adding DMEM medium containing 10% fetal calf serum and 0.5% methyl cellulose for covering, continuously culturing for 24-48 hours, selecting virus plaques which do not emit green fluorescence under the excitation of ultraviolet light with the wavelength of 488nm, inoculating fresh CEF, repeating the steps for 3-5 cycles, and obtaining the purified virus, namely PRV AH02LA (delta gE) strain, and taking the purified virus as seed virus. Extracting DNA of PRV AH02LA (delta gE) strain as a template, applying primers PRV gE site ckeck F and PRV gE site ckeck R for PCR amplification, detecting whether a target band of about 2937bp exists and cutting the target band, recovering by using a DNA gel recovery kit, and performing sequence determination.

The result shows that 2900-3000 bp specific fragments are obtained by PCR amplification by using PRV AH02LA (delta gE) strain DNA as a template, and the sequencing result is consistent with the expectation, namely 1286bp nucleotides from 13 th to 1298 th of the gE gene are successfully knocked out.

Example II construction and identification of double-gene deletion strain of pseudorabies virus gE and TK

1. Obtaining of transfer vectors

In order to knock out part of TK gene of the pseudorabies virus PRV AH02LA (delta gE) strain, Nanjing Kinshire Biotechnology GmbH is sent to artificially synthesize a gene fragment consisting of an upstream homology arm delta TK-H1 of the TK gene, a green fluorescent protein expression cassette (GFP) and a downstream homology arm delta TK-H2 of the TK gene, and then the fragment is cloned into a pUC57 vector to obtain a GFP transfer vector pPRV (AH02LA) -GFP (TK-), the structure of which is shown in FIG. 4. The sequence of an upstream homology arm delta TK-H1 of the TK gene is shown as SEQ ID No: 4, the sequence of the down-stream homologous arm delta TK-H2 of the TK gene is shown as SEQ ID No: 5, the sequence of the GFP expression cassette is the same as that of the invention patent application No. 201510388390.9.

Sent to Nanjing Kingsry Biotechnology Ltd to artificially synthesize a gene fragment consisting of an upstream homology arm delta TK-H1 of a TK gene and a downstream homology arm delta TK-H2 of the TK gene, and then the fragment is cloned into a pUC57 vector to obtain a transfer vector pPRV (AH02LA) -TK delta, and the structure of the transfer vector is shown in figure 5.

2. Acquisition of recombinant pseudorabies Virus GFP AH02LA (Δ gE) Strain

Primary Chick Embryo Fibroblasts (CEF) are prepared by using SPF chick embryos of 9-10 days old, and after the cells grow into a monolayer, transfection is performed. When transfection is carried out according to a conventional calcium phosphate transfection method, 0.5-1 mu g of GFP transfer vector pPRV (LA-A) -GFP (TK-) DNA and 0.2-1 mu g of DNA of pseudorabies virus PRV AH02LA (delta gE) strain are co-transfected into primary CEF, homologous recombination is carried out (figure 6), the culture solution is aspirated after 24 hours, 10% of fetal calf serum and 0.5% of methyl cellulose are added into DMEM culture medium for covering, culture is continued for 24-48 hours, recombinant virus plaques emitting green fluorescence under the excitation of 488nm ultraviolet light are picked and inoculated into fresh CEF, and the steps are repeated for more than 3 cycles to obtain purified recombinant virus, namely PRV AH02LA (gE-) -GFP (TK-) strain. The DNA of PRV AH02LA (gE-) -GFP (TK-) strain is used as a template, a pair of primers (the sequence of an upstream primer PRV delta TK ckeck F is 5'-CGCACCCCGAGGTTGACTTCAA-3', the sequence of a downstream primer PRV delta TK ckeck R is 5'-TTGTACGCGCCGAAGAGGGTGT-3') are used for PCR amplification of a gene segment consisting of a TK gene upstream homology arm (delta TK-H1), a green fluorescent protein expression cassette (GFP) and a TK gene downstream homology arm (delta TK-H2), and the amplified product is subjected to electrophoresis and sequence determination to verify whether the sequence is correctly inserted. The PCR reaction system (Table 2) and the reaction procedure were as follows.

TABLE 225 ul PCR reaction System

LA Taq DNA polymerase and 2 XGC Buffer II in the PCR reaction system were purchased from Takara.

And (3) uniformly mixing the reaction system, and carrying out PCR amplification, wherein the reaction procedure is as follows: pre-denaturation at 94 ℃ for 4 min; denaturation at 94 ℃ for 40s, annealing at 58 ℃ for 40s, extension at 72 ℃ for 2min for 30s, 30 cycles; extension at 72 ℃ for 10 min. And (3) carrying out 1% agarose gel electrophoresis on the PCR product, detecting whether a target band of about 2200bp exists or not, cutting the target band, recovering by adopting a DNA gel recovery kit, and sending the recovered product to Huada Gene company for sequencing.

The identification result shows that after the primer is adopted to carry out PCR amplification on the recombinant virus PRV AH02LA (gE-) -GFP (TK-), the target product of about 2200bp is obtained, and the sequence determination result is correct, wherein the recombinant virus is obtained by replacing 347bp nucleotides from 184 th site to 530 th site of the TK gene of the pseudorabies virus PRV AH02LA (delta gE) strain with a GFP expression box (figure 6), and is completely consistent with the expectation.

3. Obtaining and identifying pseudorabies virus gE and TK double-gene deletion strain

The GFP expression cassette was knocked out by performing a second homologous recombination by transfecting primary CEF with 1-3. mu.g of DNA of recombinant virus PRV AH02LA (gE-) -GFP (TK-) strain and 700-2000 ng of DNA of pPRV (AH02LA) -TKdelta) according to a conventional calcium phosphate transfection method (FIG. 6). Absorbing the culture solution 24 hours after cotransfection, adding DMEM culture medium containing 10% fetal calf serum and 0.5% methyl cellulose for covering, continuously culturing for 24-48 hours, selecting virus plaques which do not emit green fluorescence under the excitation of ultraviolet light with the wavelength of 488nm to inoculate fresh CEF, repeating the steps for 3-5 cycles, and obtaining the purified virus which is named as PRV LA1206 strain and is used as seed virus. Extracting DNA of PRV LA1206 strain as a template, applying primers PRV delta TK ckeck F and PRV delta TK ckeck R for PCR amplification, detecting whether a 347bp target band exists or not and cutting off the target band, recovering by adopting a DNA gel recovery kit, and performing sequence determination.

The result shows that PCR amplification by taking PRV LA1206 strain DNA as a template obtains a specific fragment of 300-400 bp, and the sequencing result is consistent with the expectation, namely 347bp nucleotides from 184 th to 530 th sites of TK gene of PRV AH02LA (delta gE) strain are successfully knocked out.

4. Passage of pseudorabies virus LA1206 strain seed virus

In order to further improve the safety of the pseudorabies virus LA1206 strain, the pseudorabies virus LA1206 strain seed virus is continuously passaged on CEF, one batch of seeds is preserved in every 5 generations and is transmitted to 120 generations, wherein single virus plaque clone is picked from 75 generations to 80 generations through continuous 5 generations to obtain F80 generations. The generation 1 of the pseudorabies virus LA1206 strain seed virus is marked as the generation F1 of the pseudorabies virus LA1206 strain seed virus, and the rest are analogized in turn. The seeds preserved in each generation are stored at-70 ℃ for later use.

5. Determination of gC and gD Gene sequences of Pseudorabies Virus LA1206 Strain

The DNA of the F2 generation and F80 generation of the pseudorabies virus LA1206 seed virus is taken, the gC and gD gene segments are amplified by PCR and sequence determination is carried out. Firstly according to PRV ZJ01 strain in GenBank

(GenBank accession: KM061380.1) genome sequence, using Vector NTI software, 2 pairs of PCR primers (see Table 3) were designed, the amplified fragments of which covered the main protective antigen gC and gD gene sequences of PRV. All designed primers were synthesized by Nanjing Kingsrei Biotech Ltd.

TABLE 3 PCR primers for amplification of PRV gC and gD genes

The gC and gD gene sequences are amplified respectively by using DNA of F2 generation and F80 generation of pseudorabies virus LA1206 strain seed virus as templates. The primers in the PCR reaction system shown in Table 1 were replaced with the primers shown in Table 3, and the others were not changed. The PCR reaction conditions are as follows: 1min at 94 ℃; 30s at 94 ℃, 30s of annealing (the annealing temperature is determined according to the Tm value of the primer), 72 ℃ of extension (the extension time is determined according to the size of the amplified fragment), and 30 cycles; extension at 72 ℃ for 5 min. After the completion of PCR, the gel block containing the PCR target band of the same size as expected was cut out for DNA recovery by 1% agarose gel electrophoresis, and the recovered 2 glycoprotein gene fragments were sent to Huada Gene Co for sequencing.

Sequencing results show that the gC and gD gene sequences of the F2 generation and the F80 generation of the pseudorabies virus LA1206 strain seed virus are identical with those of the AH02LA strain, have the typical characteristics of a PRV variant strain, and have no variation in passage. gC and gD are main protective antigens of the pseudorabies virus, and genes of the protective antigens are not changed by passage, which indicates that the immunogenicity of the pseudorabies virus LA1206 strain seed virus is completely maintained.

The seed virus F80 generation of the pseudorabies virus LA1206 strain is named as the pseudorabies virus LA1206-80 strain, namely PRV LA1206-80 strain.

EXAMPLE III characterization of growth characteristics of PRV LA1206-80 Strain

Inoculating virulent strains PRV AH02LA, PRV LA-A and PRV LA1206-80 respectively to full monolayer ST cells at MOI of 0.004, and placing on 5% CO₂After incubation for 1h at 37 ℃ in a constant temperature incubator, the supernatant was aspirated, washed 3 times with PBS, and then cultured in a cell maintenance solution (DMEM medium (Gibco) containing 3% Fetal Bovine Serum (FBS)), and the contents of free virus in the supernatant of infected cells and cell-bound virus in infected cells were measured at 0h, 6h, 12h, 24h, 36h, 48h, and 72h after inoculation, respectively.

The free virus content of the infected cell supernatant was manipulated as follows: collecting 1ml of infected cell supernatant at a specified time point, centrifuging for 5min at 376 Xg, taking 100 mu l of supernatant, adding the supernatant into 900 mu l of DMEM medium, mixing uniformly, sucking 100 mu l to 900 mu l of DMEM medium from the supernatant, and repeating the steps to obtain 10-10 of infected cell supernatant⁹And (4) diluting by multiple times. Virus dilutions were seeded in 96-well cell plates of pre-confluent monolayers of ST cells, 8 replicates per dilutionIncubate at 37 ℃ for 2h, discard the virus solution, and add 100. mu.l of cell maintenance medium (DMEM medium (Gibco) containing 3% Fetal Bovine Serum (FBS)) per well. Observing cytopathic condition after 12h for 3 days, and calculating virus TCID according to Reed-Muench method₅₀Counting the TCID of the virus liquid at all time points₅₀And drawing a growth curve.

The detection of the cell-bound virus content of infected cells was performed as follows: sucking cell supernatant, washing infected cells with PBS for 2 times, scraping cells, resuspending with 2ml DMEM, repeatedly freezing and thawing at-70 deg.C and 37 deg.C for 3 times, centrifuging at 500 Xg for 5min, and collecting 100 μ l supernatant as 10-10⁹And (4) diluting by multiple times. ST cells were inoculated and virus TCID was determined in the same manner as described above for free virus content detection₅₀Counting the TCID of the virus liquid at all time points₅₀And drawing a growth curve.

As can be seen from the in vitro growth curve (FIG. 7) of the virus, the TK and gE double-gene-deleted strain attenuated strain PRV LA1206-80, the gE gene-deleted virus PRV LA-A and the parental virus PRV AH02LA have similar growth kinetics, and reach the peak value 24-48 h after inoculation, although PRV LA1206-80 is slightly reduced compared with LA-A and AH02LA, the titer still reaches 10^8.0TCID₅₀More than mL, can meet the requirements of the production of the live vaccine.

EXAMPLE four safety of PRV LA1206-80 Strain

1. Test materials

1.1 viruses

Inoculating PRV LA1206-80 strain to ST cell, culturing in DMEM culture medium containing fetal calf serum 3 vol% for 40 hr, collecting virus culture, freeze thawing, centrifuging to obtain supernatant as PRV LA1206-80 strain virus liquid.

Pseudorabies virus LA1206 strain seed virus F2 generation virus liquid (the preparation method is the same as PRV LA1206-80 strain virus liquid) and PRV LA1206-80 strain virus liquid, the virus content is 10 respectively^8.0TCID₅₀mL and 10^8.1TCID₅₀Per mL; PRV AH02LA virus liquid with virus content of 10^9.0TCID₅₀Per mL; PRV LA-A strain virus liquid with virus content of 10^8.3TCID₅₀and/mL. VirusStoring at-70 deg.C.

1.2 diagnostic kits

PRV gE antibody diagnostic kit, CIVTESTSIUS ADVgB HIPRA company products. PRV gB antibody diagnostic kit, CIVTESTSIUS ADVgB HIPRA company products.

2. Test animal

The test piglets are healthy piglets aged 1 day in the same litter, and all the PRV antibodies are negative. The diets, mental status and clinical status of the pigs were verified before vaccination, and tests were excluded from unhealthy pigs. The identification is carried out through the ear tag number and the cage number. Is fed together with lactating sows in an isolated environment. The special person is responsible for raising, and the cleaning and sanitation are carried out for 2 times every day.

The test mice are 17-22 g Kunming clean-grade mice purchased from Shanghai Jiesi laboratory animals Co. The special people are raised in a clean environment, and are clean and sanitary every day.

3. Grouping

The piglets for the test of 1 day of age in the same litter are randomly divided into 3 groups (A-1, A-2 and A-3), and 4 piglets in each group are subjected to the safety test of nasal drip inoculation of PRV LA1206 strain F2 generation, PRV LA1206-80 strain and PRV LA-A strain. Another test piglet of 1 day old in one litter was randomly divided into 3 groups (B-1, B-2, B-3), each group had 4 heads, and safety tests were performed for intramuscular injection of PRV LA1206 strain F2, PRV LA1206-80 strain and PRV LA-A strain.

The test mice were randomly divided into 20 groups of 4 mice each, and safety tests of PRV AH02LA strain, PRV LA-A strain, PRV LA1206 strain seed virus F2 generation and PRV LA1206-80 strain were performed, and the specific grouping method is shown in Table 4.

TABLE 4 grouping of test mice

4. Test method

4.1 method for inoculating piglets aged 1 day in the same litter and clinical observation

Respectively diluting PRV LA1206 strain seed virus F2 generation, PRV LA1206-80 strain and PRV LA-A strain to 10 by DMEM medium^7.0TCID₅₀and/mL, a group of piglets with 1 day old (4 piglets) are inoculated in a nasal drip inoculation mode and an intramuscular injection inoculation mode respectively, and the inoculation amount is 1 mL/head, and the specific table is shown in a table 5. The body temperature of the test piglets was measured daily from the day of inoculation to 14 days after inoculation, and the diet and mental condition as well as clinical symptoms were observed.

4.2 mouse inoculation method and Observation

Respectively diluting PRV AH02LA strain and PRV LA1206 strain with F2 generation, PRV LA1206-80 strain and PRV LA-A strain in DMEM medium to 10^6.0TCID₅₀0.2mL, then diluted with DMEM medium to appropriate concentrations, and inoculated subcutaneously, the amount of each virus was as shown in Table 4, and the inoculation volume of the virus solution was 0.2 mL/virus. The test mice were observed for mortality from the day of inoculation to 14 days after inoculation. Half of the lethal dose to mice was calculated according to Reed and Muench methods.

5. Test results

Test results for 5.11 day-old piglets

The safety test results of each strain on piglets are shown in table 5. The PRV LA-A strain without the gE gene still has stronger pathogenicity to PRV antibody negative piglets of 1 day old, the pathogenicity is obviously reduced after TK and gE gene double deletion (namely PRV LA1206 strain seed toxicity F2 generation), but the body temperature of the test pigs is increased no matter nose dropping inoculation or intramuscular injection inoculation, and 1 test pig dies (table 4) in 4 test pigs, which shows that the toxicity still exists, and after continuous passage to F80 generation, the body temperature of the test pigs is not increased no matter nose dropping inoculation or intramuscular injection inoculation, and no die death caused by diseases shows that the TK and gE double gene deletion strain is further weakened through continuous passage, and the PRV LA1206-80 strain is proved to have good safety.

Safety test grouping and results for table 51 day-old piglets

5.2 mouse results

LD of PRV AH02LA strain for mice₅₀Is 10^2.57TCID₅₀LD of PRV LA-A Strain on mice₅₀Is 10^4.96TCID₅₀PRV LA1206 strain seed virus F2 generation and PRV LA1206-80 strain 10 generation^6.0TCID₅₀Vaccinated mice did not cause death.

The deposit information for PRV LA1206-80 strain is as follows:

the preservation unit: china general microbiological culture Collection center.

Unit address: the institute of microbiology, Zhongke institute No. 3, Xilu No.1, Beijing, Chaoyang, North.

According to the biological materials (strains): PRV LA 1206-80.

And (3) classification and naming: porcine herpesvirus type 1.

The preservation number is as follows: CGMCC No. 14329.

The preservation date is as follows: 7 and 24 days in 2017.

EXAMPLE V immunopotency of PRV LA1206-80 Strain

1. Test materials

1.1 Virus and Pseudorabies live vaccines

PRV LA1206-80 strain virus liquid with virus content of 10^8.1TCID₅₀/mL。

Pseudorabies live vaccine (Bartha K61 strain) purchased from Biotech, Inc., Nanjing Tianbang, batch 150826.

PRV AH02LA strain for use in challenge test, LD against PRV-negative pigs of 45 days old₅₀Is 10^6.5TCID₅₀。

1.2 diagnostic kits

PRV gE antibody diagnostic kit and PRV gB antibody diagnostic kit, from the company IDEXX, Edwardsient Henschel, Beijing.

2. Test animal

24 healthy piglets of 28-35 days old are negative in PRV antigen and PRV gE and gB antibodies, and negative in Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), classical Swine Fever Virus (SFV), porcine circovirus type 2 (PCV2) and type O foot and mouth disease virus (FMDV (O)) antigen and antibodies. The diets, mental status and clinical status of the pigs were verified before vaccination, and tests were excluded from unhealthy pigs. And identifying through the ear tag number, the cage number and the ring number. Raising the seeds in cages in an isolation environment. The feed is complete compound feed (commercial product), drinking water is tap water, and the feed is fed once in the morning and afternoon every day. The special person is responsible for raising, and the cleaning and sanitation are carried out for 2 times every day.

3. Grouping

The test animals were introduced once and randomly divided into groups A-F, each group having 4 heads, as shown in Table 6.

4. Test methods and indices of study

(1) Test method

Immunization: diluting PRV LA1206-80 strain virus solution and pseudorabies live vaccine (Bartha K61 strain) with sterile DMEM culture solution to appropriate concentration, intramuscular injecting, and inoculating 1000TCID to group A, group B and group C respectively₅₀、10000TCID₅₀And 100000TCID₅₀PRV LA1206-80 strain virus liquid, group D inoculated 100000TCID₅₀The pseudorabies live vaccine (Bartha K61 strain) of (3), E, F groups were each intramuscularly infused with 2mL of DMEM solution (Table 6).

TABLE 6 groups of animal immunity challenge test

Group of	Immunization dose	Inoculation of the Strain
			Group A	1000TCID50	PRV LA1206-80 Strain
Group B	10000TCID50	PRV LA1206-80 Strain
			Group C	100000TCID50	PRV LA1206-80 Strain
Group D	100000TCID50	Pseudorabies live vaccine (Bartha K61 strain)
			Group E	Blank control	/
Group F	Control of offensive toxin	/

Counteracting toxic substances: all pigs except the blank control group were challenged 1 week after inoculation with PRV AH02LA strain at 2LD₅₀2mL for each nasal drop, and 14 days after the drug is attacked.

Serum gB and gE antibody detection: all pig serum samples were taken at three time points before immunization, before challenge and at the end of challenge (i.e. 14 days after challenge), and the PRV gE and gB antibodies were detected using the kit.

Observation of clinical symptoms: all pigs were observed daily for mental, dietary status and various respiratory, nervous and systemic clinical manifestations.

Body temperature measurement: all pigs were body temperature measured once a day (rectal temperature) from the start before inoculation to the end of the trial (14 days after challenge).

And (3) toxin expelling condition detection: before and after the challenge, and after the test (14 days after the challenge), a nasal swab sample of the pig is collected every day, the sample is stored at the temperature of-70 ℃, and BHK-21 cells are inoculated to observe lesions after dilution in a multiple ratio during detection so as to determine whether the virus is discharged.

(2) Indexes of study

Morbidity, mortality, hyperthermia, detoxification, and serum antibody positive conversion.

5. Test results

PRV gB and gE antibodies of all test pigs before inoculation were negative, PRV gB and gE antibodies of blank control pigs (group E) to the end of the test were negative, PRV gB antibodies of all test pigs of group a, group B, group C and group D were positive 1 week after inoculation (i.e. before challenge), and PRV gE antibodies of all immunized pigs and challenge control live pigs were positive after challenge (table 7).

TABLE 7 statistical table of positive conversion of pig serum antibody in immune challenge test

After the detoxification, the pigs in the blank control group have no morbidity and mortality, no body temperature rise and no toxin expelling; 4 pigs in the toxicity attacking control group all have symptoms of food reduction, mental depression, sneezing, rhinorrhea, dyspnea and the like from the 2 nd day after toxicity attacking, the body temperature is all raised to be more than 40.5 ℃, toxin is removed from the nasal swab sample, and 3 pigs die continuously from the 4 th to 6 th day after toxicity attacking; indicating that the experimental control is true (table 8). All the test pigs in the A group, the B group and the C group have no clinical symptoms, no body temperature rise and no toxin expelling detection, and the PRV LA1206-80 strain can completely protect the PRV variant strain; group D test pigs did not die, had clinical symptoms in 1 pig and all 4 pigs had increased body temperature, and all were detoxified from nasal swab samples, indicating that PRV Bartha K61 strain produced only partial protection (table 8).

TABLE 8 statistical Table of clinical conditions of swine in immune challenge test

In conclusion, PRV LA1206-80 immunized pigs all gave complete protection of PRV wild strain AH02LA, with a minimum of 1000TCID₅₀The inoculation amount of (1) can also resist morbidity and detoxification after immunization, and 100000TCID₅₀The PRV Bartha K61 strain can only produce partial protection and cannot prevent body temperature reaction and expel toxin, and the LA1206-80 strain serving as a vaccine strain has a significantly better protection effect on a variant strain than the Bartha K61 strain. The live vaccine inoculation pigs prepared from PRV LA1206-80 strains only detect PRV gB antibodies, but not detect PRV gE antibodies, and the DIVA vaccine can identify vaccine immunized animals and wild virus infected animals, is very favorable for purification of PRV variant strains in a pig farm and has wide prospect when being popularized and applied. The LA1206-80 strain has obviously reduced toxicity and no pathogenicity to piglets of 1 day, so the LA1206-80 strain is a vaccine candidate low virulent strain with higher safety, can be used for early immunization of newborn piglets and can block wild virus infection as early as possible.

SEQUENCE LISTING

<110> agricultural science and academy of Jiangsu province

<120> pseudorabies virus double-gene deletion attenuated strain and application thereof

<130> 20171103

<160> 5

<170> PatentIn version 3.3

<210> 1

<211> 963

<212> DNA

<213> PRV AH02LA Strain

<400> 1

atgcgcatcc tccggatcta cctcgacggc gcctacggca ccggcaagag caccacggcc 60

cgggtgatgg cgctcggcgg ggcgctgtac gtgcccgagc cgatggcgta ctggcgcact 120

ctgttcgaca cggacacggt ggccggtatt tacgatgcgc agacccggaa gcagaacggc 180

agcctgagcg aggaggacgc ggccctcgtc acggcgcagc accaggccgc cttcgcgacg 240

ccgtacctgc tgctgcacac gcgcctggtc ccgctcttcg ggcccgcggt cgagggcccg 300

cccgagatga cggtcgtctt tgaccgccac ccggtggccg cgacggtgtg cttcccgctg 360

gcgcgcttca tcgtcgggga catcagcgcg gcggccttcg tgggcctggc ggccacgctg 420

cccggggagc cccccggcgg caacctggtg gtggcctcgc tggacccgga cgagcacctg 480

cggcgcctgc gcgcccgcgc gcgcgccggg gagcacgtgg acgcgcgcct gctcacggcc 540

ctgcgcaacg tctacgccat gctggtcaac acgtcgcgct acctgagctc ggggcgccgc 600

tggcgcgacg actgggggcg cgcgccgcgc ttcgaccaga ccgtgcgcga ctgcctcgcg 660

ctcaacgagc tctgccgccc gcgcgacgac cccgagctcc aggacaccct cttcggcgcg 720

tacaaggcgc ccgagctctg cgaccggcgc gggcggccgc tcgaggtgca cgcgtgggcg 780

atggacgcgc tcgtggccaa gctgctgccg ctgcgcgtct ccaccgtcga cctggggccc 840

tcgccgcgcg tctgcgccgc ggccgtggcg gcgcaggcgc gcggcatgga ggtgacggag 900

tccgcgtacg gcgaccacat ccggcagtgc gtgtgcgcct tcacgtcgga gatgggggtg 960

tga 963

<210> 2

<211> 1023

<212> DNA

<213> artificial

<220>

<223> upstream homology arm of gE Gene Δ gE-H1

<400> 2

tgtttctgga ggggccctcg ccgagcaact acagcgggcg cgtggagctg ctgcgcctcg 60

accccaagcg cgcctgctac acgcgcgagt acgccgccga gtacgacctc tgcccccgcg 120

tgcaccacga agccttccgc ggctgcctgc gcaagcgcga gccgctcgcc cggcgcgcgt 180

ccgccgcggt ggaggcgcgc cggctgctgt tcgtctcgcg cccggcctcg ggggacgcgg 240

ggtcgtacgt gctgcgggtc cgcgtgaacg ggaccacgga cctctttgtg ctgacggccc 300

tggtgccgcc gagggggcgc cccgtcccca cgtcgccgcc cgcggacgag tgccggcccg 360

tcgtcggatc gtggcacgac agcctgcgcg tcgtggaccc cgccgaggac gccgtgttca 420

ccacccagcc cccgcccgag cccgagccgc cgacgacccc cgcgcccccc cgggggaccg 480

gcgccacccc cgagccccga tcggacgagg aggaggaggt gacgcggaga cgacgacgcc 540

gactgacccc cgcgcccggg accctggacg cgaacggcac gatggtgctg aacgccagcg 600

tcgtgtcgcg cgtcctgctc gccgccgcca acgccacggc gggcgcccgg agccccggga 660

agatagccat ggtgctgggg cccacgatcg tcgtcctcct gatcttcctg ggcgggatcg 720

cctgcgtggc ccggcgctgc gcgcggaatc gcatctaccg gccgcgaccc gggcgcggat 780

cggcggtcca tgcggcgccc ccgcggcgcc cgcccccaac cccggtcgcc ggggcgcccg 840

tcccccagcc caagatgacg ttggccgagc tgcgccagaa gctcgccacc atcgcagaag 900

aacaataaaa aggtggtgtt tgcataattt tgtgggtggc gttttatctc cgtccgcgcc 960

gttttaaacc tgggcacccc cgcgagtctc gcacacaccg gggttgagac catgcggccc 1020

ttt 1023

<210> 3

<211> 1113

<212> DNA

<213> artificial

<220>

<223> downstream homology arm of gE Gene Δ gE-H2

<400> 3

gctggcgctg ggctccttcg tgatgacgtg cgtcgtcggg ggggccatct ggctctgcgt 60

gctgtgctcc cggcgccggg cggcctcgcg gccgttccgg gtgccgacgc gggcgcggac 120

gcacatgctc tctccggtgt acaccagcct gcccacgcac gaggactact acgacggcga 180

cgacgacgac gacgaggagg cgggcgtcat ccgccggcgg cccgcctccc ccagcggaga 240

cagcggctac gaggggccgt acgcgagcct ggaccccgag gacgagttca gcagcgacga 300

ggacgacggg ctgtacgtgc gccccgagga ggcgccccgc tccggcttcg acgtctggtt 360

ccgcgatccg gagaaaccgg aagtgacgaa tggacccaac tatggcgtga ccgccaaccg 420

cctgttgcat gtcccgcccc gctaaaatac cgggagaacc ggtccgcccg cattccgaca 480

tgcccggcgc cgcctccgtc gacatggaca cgtttgaccc cagcgccccc gtcccgacga 540

gcgtctcgaa cccggccgcc gacgtcctgc tggcccccaa gggaccccgc tccccgctgc 600

gcccccagga cgactcggac tgctactaca gcgagagcga caacgagacg cccagcgagt 660

tcctgcgccg cgtgggacgc cggcaggcgg cgcgtcggag acgccgccgc tgcctgatgg 720

gcgtcgcgat cagcgccgcc gcgctggtca tctgctcgct gtccgcgcta ctcgggggca 780

tcgtcgccag gcacgtgtag cgagcgagcg agcgaacggg agcgggggcc cgcccccatc 840

cgccgcgccc aggagagggg ggagagagcg gggggttggg cgcgccacgt ggtgtgggca 900

cggactcgga cttgtcacaa taaatgggcc ccggcgtgtc cgggcgcaca cagcagcctt 960

cctctcctcc gcgtctctgt tccgcccgtc tctcgccgga ctcttcttct ccaccgcctc 1020

caccgtcgca gttgtcgcga gcgcgttcgc accatggggg tgacggccat caccgtggtc 1080

acgctgatgg acggggccgg gcgcatcccc gcc 1113

<210> 4

<211> 1204

<212> DNA

<213> artificial

<220>

<223> TK Gene upstream homology arm DeltaTK-H1

<400> 4

cgtcgtaggc gagcgccacg tcgttcttgg cgatctgcac ctcgcggccc tcgtccgcgg 60

ccgcgaccgc ctcgggcgcc tcctcggcgg cgcgcgccgc gtccgcctcc tcggcggcgc 120

gcgccgtctc ctcgagcacc acgagcgcct cggccacgcg ctccacctgc cgctccagcg 180

gccgcagctg ctcgtccacc tcggcctcga ggcgcgcgcc cgcggccatg gcgttgtcca 240

gcgccgcggc ggccgcgcgg cggcgcgcgt tcgcgtgcgc cagcgcgagg cgcgcgtcga 300

ggccctcgcc gaagcccggg cgggcccaga agcccacggg gaacgggggc gcgatgaagt 360

ggcgcgcgct gcccgggatc gcagcggcct cgaaggcgaa ccacgcgcgg tccatggcgc 420

ggggggacat gggccgcgcg gcggcgcgcg ccgccttatc atccccgctc cccgccgccg 480

cccggcccgg ccccgcgcgc gccgcgatcg cgatcaccgc cgcggcccgg cgacgtactc 540

ggcgaggccg cgcacggtcg cggccatcgc gctcgcgttg ccgcgcgtct gggtgcaggg 600

caggcgcgtc acgtcgagca cgcgcatgct ccgctgggcc acaaacacca gcaggggcac 660

gagcgtgatc tcctcgccgc ccgggggcac ggcggcggcg aggaggcgcg ccgagtcgcg 720

cagctggcac agcccctcgt gccgctgccc gcgcttgctg ggcgtgttga ggttccgggg 780

gaagcggcac gtcttgagct cgatgaggaa gcacaggtgc gggcccgccc ccagccgcac 840

cacgcacacg cagtcggggc ggcgcacccc gaggttgact tcaaaggcca gggtcaagga 900

cgccttctta agcgtctctc ggggaagccc gaagagactc tcgccgtacg cggacgggtc 960

gcgtcgcagg cgttcgtaga agcggttgtg gcagcggatc cccgcccgga agcgcgccgg 1020

gatgcgcatc ctccggatct acctcgacgg cgcctacggc accggcaaga gcaccacggc 1080

ccgggtgatg gcgctcggcg gggcgctgta cgtgcccgag ccgatggcgt actggcgcac 1140

tctgttcgac acggacacgg tggccggtat ttacgatgcg cagacccgga agcagaacgg 1200

cagc 1204

<210> 5

<211> 1216

<212> DNA

<213> artificial

<220>

<223> TK Gene downstream homology arm Delta TK-H2

<400> 5

gctcacggcc ctgcgcaacg tctacgccat gctggtcaac acgtcgcgct acctgagctc 60

ggggcgccgc tggcgcgacg actgggggcg cgcgccgcgc ttcgaccaga ccgtgcgcga 120

ctgcctcgcg ctcaacgagc tctgccgccc gcgcgacgac cccgagctcc aggacaccct 180

cttcggcgcg tacaaggcgc ccgagctctg cgaccggcgc gggcggccgc tcgaggtgca 240

cgcgtgggcg atggacgcgc tcgtggccaa gctgctgccg ctgcgcgtct ccaccgtcga 300

cctggggccc tcgccgcgcg tctgcgccgc ggccgtggcg gcgcaggcgc gcggcatgga 360

ggtgacggag tccgcgtacg gcgaccacat ccggcagtgc gtgtgcgcct tcacgtcgga 420

gatgggggtg tgaccctcgc ccctcccacc cgcgccgcgg ccggatggag accgcgacgg 480

aggcaacgac gacggcgtgg gagggggctc ggggcgcgta taaagccatg tgtatgtcat 540

cccaataaag tttgccgtgc ccgtcaccat gcccgcgtcg tccgtgcgcc tcccgctgcg 600

cctcctgacc ctcgcgggcc tcctggccct cgcgggggcc gccgccctcg cccgcggcgc 660

gccgcagggt gggccgccct cgccgcaggg gggtcccgcg cccaccgcgg cgcccgcgcg 720

cgggcccacc ctgttcgtcc tggacggcga cggctccgcg tggttcgtct tccagctcgg 780

cgggctgggg gcgctcaacg acacgcgcat ccgcgggcac ctgctcggcc ggtacctcgt 840

ctcgtaccag gtggtgcccc cgcccgtctc cgcgtggtac tttgtgcagc gcccgcgcga 900

gcgcccgcgc ctctcggggc cgccctcggg cgcggagctc gtggccttcg acgcgcccgg 960

cgtccggcgc acgtacacca cggcggcggt gtggcccgcg gaggtggccg tcctcgcgga 1020

cgcggaggcg cgctgccccg cggccgtctt caacgtgacg ctgggcgagg ccttcctcgg 1080

cctgcgcgtc gcgctgcgct ccttcctgcc gctggaggtc atcatctccg ccgagcggat 1140

gcgcatgatc gcgcccccgg cgctcggctc ggacctggag ccgccgggcc cgcccgcggg 1200

ccgcttccac gtgtac 1216

Claims (6)

1. The pseudorabies virus double-gene deletion low virulent strain is a pseudorabies virus LA1206-80 strain, and the preservation number is as follows: CGMCC No. 14329.

2. The use of the double-gene deletion attenuated strain of pseudorabies virus of claim 1 in the preparation of a vaccine for pseudorabies.

3. A vaccine comprising the double-gene deletion attenuated strain of pseudorabies virus of claim 1 as an active ingredient.

4. The vaccine of claim 3, wherein the virus fluid of the pseudorabies virus double-gene deletion low virulent strain is prepared by the following method: inoculating the pseudorabies virus LA1206-80 strain to ST cells, culturing for 36-48 hours by adopting a DMEM medium containing fetal bovine serum, harvesting virus culture, freezing and thawing, and centrifuging to obtain supernatant serving as pseudorabies virus LA1206-80 strain virus liquid.

5. The vaccine of claim 4, wherein said vaccine is a live vaccine.

6. A recombinant vector live vaccine, characterized in that the active component is obtained by inserting a foreign protective antigen gene expression cassette into the genome of the double-gene deletion attenuated strain of the pseudorabies virus as claimed in claim 1.

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