CN102994458A - Porcine pseudorabies virus virulent strain, and gene deletion vaccine strain thereof and applications thereof - Google Patents

Abstract

The invention discloses a virulent strain of porcine pseudorabies virus, its gene deletion vaccine strain and application thereof. A virulent strain of porcine pseudorabies virus isolated by the present invention is named HeN1, and its microbial preservation number is CGMCC NO.6656. On the basis of the virulent strain HeN1, the gE gene is deleted to obtain the gene deletion vaccine strain rPRV- ^gE- - EGFP ⁺ , its microbial deposit number is CGMCC NO.6657. The virulent strain of the present invention can be prepared into an inactivated vaccine (single vaccine or combined vaccine), and the gene deletion vaccine strain rPRV-gE ^- -EGFP ⁺ can be prepared into a live vaccine or an inactivated vaccine (single vaccine or combined vaccine), etc. It can effectively prevent or treat porcine pseudorabies, and can also be prepared as a diagnostic reagent for diagnosing porcine pseudorabies. The gene deletion vaccine strain rPRV-gE ^- -EGFP ⁺ of the present invention has the advantages of good safety, high protection efficiency, favorable differential diagnosis and the like.

Description

Porcine pseudorabies virus virulent strain, its gene deletion vaccine strain and application thereof

technical field

The present invention relates to a virulent virus strain and a gene-deleted vaccine strain obtained on the basis thereof, in particular to a virulent strain of porcine pseudorabies virus HeN1 and a gene-deleted vaccine strain rPRV obtained by deleting the gE gene of the virulent strain HeN1 -gE ^- -EGFP ⁺ , the present invention also relates to the application of HeN1 and vaccine strain rPRV-gE ^- -EGFP ⁺ in preventing or treating porcine pseudorabies, and the present invention belongs to the field of biomedicine.

Background technique

Pseudorabies (PR, also known as Aujeszky's disease, AD) is an acute infectious disease caused by a variety of domestic animals, wild animals, companion animals and experimental animals caused by Pseudorabies Virus (PRV). Under natural conditions, the disease is most common in pigs, cattle, sheep, dogs, cats and rodents. Pseudorabies virus has a lethal course in all susceptible animals except pigs. For pigs, the main manifestations are neurological symptoms, salivation, dyspnea, reproductive impairment, growth arrest, weight loss and high mortality. The clinical manifestations of suckling piglets infected with pseudorabies are the most typical, severe and consistent. They all appear neurological symptoms, paralysis, exhaustion and death, and the mortality rate is almost as high as 100%. Under normal circumstances, adult pigs do not get sick, but are invisible, and only mild symptoms such as sneezing, coughing, and elevated body temperature are seen; pregnant sows infected with pseudorabies virus can cause abortion, stillbirth, weak offspring, and mummified fetuses; Cause orchiditis. The disease is highly contagious, and once found in pig herds, it will quickly spread to other pigs and farms through air and contact through the respiratory tract. Recent studies have shown that pseudorabies virus can also be infected through milk and reproductive tract.

In large-scale pig farms, PR is the target of routine immunization prevention and control. Serological epidemiological surveys using ELISA to identify strong and weak viruses found that a certain proportion of pig farms and pigs still had wild virus antibodies after immunization with gene deletion vaccines, and there were occasional reports of virus isolation. Due to the limited economic loss caused, it has not attracted enough attention. However, in the past year, a number of large-scale pig farms using routine vaccine immunization have appeared suspected PR epidemics, mainly manifested as weak piglets, stillbirths, neurological symptoms and death of piglets. PCR identification and virus isolation were carried out on the submitted tissues, which proved that there was indeed PRV wild virus infection in the pig farm. At present, our laboratory has detected PRV wild virus from the disease materials submitted by pig farms in Henan, Heilongjiang, Jilin, Liaoning, Inner Mongolia and other provinces. Since the PRV genome is relatively large, about 145kb, we amplified the gE gene of PRV by PCR from the samples submitted by each pig farm for sequencing. The results showed that the two genes of the popular strains in each pig farm were highly homologous More than 99%, the lowest homology with PRV Kaplan strain (Hungary, GenBank accession number JQ809328) was 97.5%.

Immunization is the most effective way to prevent and control PRV. my country introduced the PRV Bartha k61 strain from Hungary in the 1970s. This virus is recognized as an excellent vaccine strain in the world. Most domestic large-scale pig farms use this vaccine, and PR has also been well controlled. But the current situation is that the pig farms affected by PR have been immunized with the vaccine according to the routine procedures. Is there an antigenic variation in the new epidemic strain that makes the immunized pigs unable to effectively resist the virus? We used serum neutralization test and sheep immunization test to confirm that the neutralizing antibody produced by Bartha k61 immunized pigs could not effectively neutralize the newly isolated virus, and the sheep immunized with Bartha k61 could not completely resist the attack of the new virus. Therefore, there is an urgent need to develop new safe and effective PRV vaccines to deal with new circulating strains.

Contents of the invention

One of the objectives of the present invention is to provide a virulent strain of porcine pseudorabies virus.

Two of the object of the present invention is to provide a strain by genetic engineering method deletion gE gene and the attenuated vaccine strain that inserts EGFP mark by this virulent strain of porcine pseudorabies virus, the vaccine prepared with this attenuated vaccine strain has the effect to the new epidemic strain of PRV Good protective effect.

The third object of the present invention is to provide a vaccine composition for preventing or treating porcine pseudorabies.

Above-mentioned purpose of the present invention is achieved by the following technical solutions respectively:

The brain tissue of diseased pigs was collected from a pig farm suspected of PRV infection in Henan Province, my country, and the genomic DNA was extracted. PCR identification was performed using PRV gE-specific primers (Figure 1). The positive brain tissue homogenate was centrifuged, filtered, and the filtrate was frozen. Save for later use. After Vero cells were cultured at 37°C for 48 hours to form a monolayer, each bottle was inoculated with 1ml of the supernatant of the filtered disease material suspension, and after adsorption for 1 hour, the DMEM culture medium containing 2% fetal bovine serum was changed to continue culturing at 37°C. After 3 to 4 days, there were no cytopathic changes (Figure 2). When 60-70% of the cells have CPE changes, the virus is collected, and the virus strains with lesions are passed on for 2 to 3 generations, and then PCR amplification is performed with specific primers of PRV gE and virus particles are observed and identified by electron microscopy. Tests have shown that the use of PRV gE specific primers can amplify fragments of the expected size. After the fragments are sequenced, they are compared with the sequences published on GenBank. The homology is above 97%. The sequence result is shown in SEQ ID NO:2 Show. Observing the culture with an electron microscope, it can be observed that the virus particles are round, with two characteristics: hollow and solid, some with capsules and some without capsules (Figure 3). According to the above results, it is proved that the isolated strain is PRV, named as HeN1 strain, and classified as Pseudorabies virus. No. 3 Institute of Microbiology, Chinese Academy of Sciences, the strain preservation number is: CGMCC NO.6656, and the preservation date is October 12, 2012. After BALB/c mice were inoculated with the PRV HeN1 strain, typical symptoms of pseudorabies appeared: biting the injection site with their mouths, resulting in local hair loss, skin bleeding, severe hind limbs were bitten off, and the injected virus content was between 10 ^3.0 and 10 ^6.0 Mice with TCID ₅₀ died within 60 hours after inoculation. The LD ₅₀ of PRV HeN1 on BALB/c mice was calculated as 237TCID ₅₀ . The control mice injected with DMEM medium showed no abnormalities. After the SPF weaned piglets were inoculated with the PRV HeN1 strain, there was only a transient fever reaction, and the body temperature reached 41°C, which lasted for 3-5 days. In addition, there were no other clinical symptoms and autopsy changes.

The antigenic difference between the newly isolated strain (PRV HeN1 strain) and the vaccine strain Bartha k61 was confirmed by microneutralization test and immune challenge test of sheep. The results found that the level of neutralizing antibodies produced by Bartha k61-vaccinated pigs was generally lower than that of HeN1-vaccinated pigs, and the serum dilutions that could neutralize their own virus by 50% were all within 1:40, and the neutralizing ability of the newly isolated virus HeN1 Even lower, the serum dilution for 50% neutralizing virus is around 1:10. HeN1-vaccinated pigs can produce high levels of neutralizing antibodies 2 weeks after infection, the serum dilution of 50% neutralizing virus is above 1:80, and the neutralizing ability of the two viruses is high.

The transfer vector plasmid pUCUsAB-EGFP was constructed by using partial gI and Us2 genes of the PRV HeN1 strain as homology arms and the green fluorescent protein gene (EGFP) as a screening marker by gene cloning technology, and the PRVHeN1 genome and transfer vector were co-transfected into Vero cells , After four rounds of plaque purification and identification, it was confirmed that the recombinant pseudorabies virus rPRV-gE ^- -EGFP ⁺ with deletion of gE gene and insertion of EGFP marker was obtained. The growth ^{characteristics} of recombinant pseudorabies virus rPRV-gE ^- -EGFP ⁺ were studied by means of plaque test and one-step growth curve determination ^. There was no significant difference compared with the viral HeN1 strain, indicating that deletion and insertion did not affect the propagation speed of the recombinant virus in vitro. The recombinant virus rPRV-gE ^- -EGFP ⁺ was blindly passed on Vero cells for 12 generations, and the green fluorescent protein could still be stably expressed. The inserted foreign fragment and the viral gene sequence on both sides of the insertion site could be detected by PCR identification, indicating that the The obtained recombinant virus rPRV-gE ^- -EGFP ⁺ can be inherited stably.

Therefore the present invention also proposes a strain of porcine pseudorabies virus vaccine strain, it is characterized in that described vaccine strain is on the basis of described porcine pseudorabies virus virulent strain, deletes gE gene and inserts EGFP mark by genetic engineering method owned.

In a specific embodiment of the present invention, the insertion sequence and its flanking nucleotide sequence are shown in SEQ ID NO:3.

In a specific embodiment of the present invention, the porcine pseudorabies virus vaccine strain is named as rPRV-gE ^- -EGFP ⁺ , and is classified as pseudorabies virus. The center is located at the Institute of Microbiology, Chinese Academy of Sciences, No. 1, Beichen West Road, Chaoyang District, Beijing. .

By evaluating the immune efficacy of PRV HeN1 strain and rPRV-gE--EGFP ^{+ ,} it was found that the level of neutralizing antibodies ^produced by HeN1-vaccinated pigs was higher than that of Bartha k61-vaccinated pigs; and HeN1 strain have good protective effects, but Bartha k61 immunized sheep can not completely resist the challenge of the newly isolated strain HeN1 strain, so rPRV-gE ^- -EGFP ⁺ can be used as a vaccine candidate strain to deal with the emerging epidemic.

Therefore, further, the present invention also proposes a vaccine composition for treating or preventing porcine pseudorabies, characterized in that: the inactivated product or porcine pseudorabies virus gene The composition of missing vaccine strain and pharmaceutically acceptable adjuvant.

Furthermore, the present invention also proposes the use of the virulent strain of porcine pseudorabies virus in the preparation of drugs for preventing or treating porcine pseudorabies. and

Use of the virulent strain of porcine pseudorabies virus in preparing medicines for diagnosing or detecting porcine pseudorabies. And the use of the gene-deleted vaccine strain in the preparation of medicines for preventing or treating porcine pseudorabies. and

Use of the gene-deleted vaccine strain in preparing medicines for diagnosing or detecting porcine pseudorabies.

The virulent strain of the present invention can be prepared into an inactivated vaccine (single vaccine or combined vaccine), and the gene deletion vaccine strain rPRV-gE ^- -EGFP ⁺ can be prepared into a live vaccine or an inactivated vaccine (single vaccine or combined vaccine), etc. It can effectively prevent or treat porcine pseudorabies, and can also be prepared as a diagnostic reagent for diagnosing porcine pseudorabies. The gene deletion vaccine strain rPRV-gE ^- -EGFP ⁺ of the present invention has the advantages of good safety, high protection efficiency, favorable differential diagnosis and the like.

Description of drawings

Figure 1 PCR identification of brain tissue samples sent for inspection by a pig farm;

1: Negative control, 2-8: Submitted samples 9: Positive control, 10: DNA marker

Fig. 2 Cytopathy produced by brain tissue homogenate infected Vero cells;

A: normal Vero cells; B: infected Vero cells

Electron microscope observation of Fig. 3 virus particle;

Figure 4 compares the levels of neutralizing antibodies induced by two strains of viruses;

A: Pig serum injected with PRV Bartha k61 strain.

B: Pig serum injected with PRV HeN1 strain.

Figure 5 Schematic diagram of the recombination site of rPRV-gE ^- -EGFP ⁺ ;

Fig. 6 Enzyme digestion identification of pUCUsAB-EGFP.

M: DL15000; 1. BamH I digestion plasmid pUCUsAB-EGFP

Detailed ways

The present invention will be further described through experiments and examples below. It should be understood that these examples are only for the purpose of illustration, and in no way limit the protection scope of the present invention. Those of ordinary skill in the art understand that many changes, modifications, and even equivalent changes can be made within the spirit and scope defined by the claims of the present invention, but all will fall within the protection scope of the present invention

Example 1 Isolation and identification of porcine pseudorabies virus virulent strain (PRV HeN1 strain)

The brain tissue of diseased pigs was collected from a pig farm suspected of PRV infection in Henan Province, my country, and the genomic DNA was extracted. PCR identification was performed using PRV gE-specific primers (Figure 1). The positive brain tissue homogenate was centrifuged, filtered, and the filtrate was frozen. Save for later use. After Vero cells were cultured at 37°C for 48 hours to form a monolayer, each bottle was inoculated with 1ml of the supernatant of the filtered disease material suspension, and after adsorption for 1 hour, the DMEM culture medium containing 2% fetal bovine serum was changed to continue culturing at 37°C. After 3 to 4 days, there were no cytopathic changes (Figure 2). When 60-70% of the cells have CPE changes, the virus is collected, and the virus strains with lesions are passed on for 2 to 3 generations, and then PCR amplification is performed with specific primers of PRV gE and virus particles are observed and identified by electron microscopy. Tests have shown that the use of PRV gE specific primers can amplify fragments of the expected size. After the fragments are sequenced, they are compared with the sequences published on GenBank. The homology is above 97%. The sequence result is shown in SEQ ID NO:2 Show. Observing the culture with an electron microscope, it can be observed that the virus particles are round, with two characteristics: hollow and solid, some with capsules and some without capsules (Figure 3). According to the above results, it is proved that the isolated strain is PRV, named as HeN1 strain, and classified as Pseudorabies virus. No. 3 Institute of Microbiology, Chinese Academy of Sciences, the strain preservation number is: CGMCC NO.6656, and the preservation date is October 12, 2012. After BALB/c mice were inoculated with the PRV HeN1 strain, typical symptoms of pseudorabies appeared: biting the injection site with their mouths, resulting in local hair loss, skin bleeding, severe hind limbs were bitten off, and the injected virus content was between 10 ^3.0 and 10 ^6.0 Mice with TCID ₅₀ died within 60 hours after inoculation. The LD ₅₀ of PRV HeN1 on BALB/c mice was calculated as 237TCID ₅₀ . The control mice injected with DMEM medium showed no abnormalities. After the SPF weaned piglets were inoculated with the PRV HeN1 strain, there was only a transient fever reaction, and the body temperature reached 41°C, which lasted for 3-5 days. In addition, there were no other clinical symptoms and autopsy changes.

There are antigenic differences between embodiment 2 PRV HeN1 strain and vaccine strain Bartha k61

1) Microneutralization test and sheep immune challenge test were used to confirm the antigenic difference between the newly isolated strain (PRV HeN1 strain) and the vaccine strain Bartha k61. Neutralization test operation method: 12 40-day-old SPF pigs, 5 of them were intramuscularly injected with 1 mL of inactivated PRV HeN1 strain containing 10 ^7.0 TCID ₅₀ , 5 were intramuscularly injected with 1 mL of Bartha k61 strain containing 10 ^7.0 TCID ₅₀ , and the other Two heads were injected with DMEM culture medium as a control, blood was collected before and after inoculation to separate serum, and 5 weeks after inoculation, they were killed. The neutralizing potency of the serum from Bartha k61-vaccinated pigs and HeN1-vaccinated pigs against two strains of viruses (Bartha k61 and HeN1) was determined by fixed virus dilution serum method. The amount of virus was 100TCID ₅₀ /well, and the serum was collected at different time points after inoculating SPF pigs with Bartha k61 and HeN1, and inactivated at 56°C for 30 minutes for later use. The results found that the level of neutralizing antibodies produced by Bartha k61-vaccinated pigs was generally lower than that of HeN1-vaccinated pigs, and the serum dilutions that could neutralize their own virus by 50% were all within 1:40, and the neutralizing ability of the newly isolated virus HeN1 Even lower, the serum dilution for 50% neutralizing virus was around 1:10 (Fig. 4A). HeN1-vaccinated pigs can produce high levels of neutralizing antibodies 2 weeks after infection, and the serum dilution of 50% neutralizing virus is above 1:80, and the neutralizing ability of both viruses is high (Figure 4B) .

2) The immune protection test of sheep: 14 sheep aged 6-8 months were tested negative for PRV neutralizing antibodies. They were randomly divided into four groups. Groups A and C (4 rats in each group) were intramuscularly injected with 0.2 doses of PRV Bartha k61 vaccine (the virus content in each dose was 10 ^5.0 TCID ₅₀ ), and groups B and D (3 rats in each group) were randomly divided into four groups. ) as a challenge control. Fourteen days after immunization, each sheep in groups A and B was intramuscularly injected with 1 mL of PRV HeN1 strain containing 1000 LD ₅₀ , and each sheep in groups C and D was intramuscularly injected with 1 mL of PRV Shuangcheng strain (PRV-S) containing 1000 LD ₅₀ . Blood sampling before challenge. A total of 8 sheep in groups A and C collected serum 14 days after immunization with PRV Bartha k61 vaccine, and used IDEXX kit to detect anti-PRV gB antibody (the nucleotide sequence of the gB gene is shown in SEQ ID NO.1), group A There were 3 antibodies positive, and 4 antibodies in group C were positive. Within 9 days after the challenge, all the control sheep became ill and died, and 2 sheep in the HeN1 challenge group became ill and died (Table 1). The affected sheep showed neurological symptoms such as itching and opisthotonus.

Table 1 The protective effect of Bartha k61 vaccine immunized sheep against different strains

group number of sheep Vaccine strain Attacking strain Antibody positive number before challenge Number of cases number of deaths protection number A 4 Bartha k61 HeN1 4/4 2/4 2/4 2/4 B 3 HeN1 3/3 3/3 0/3 C 4 Bartha k61 S 3/4 0/4 0/4 4/4 D. 3 S 3/3 3/3 0/3

Example 3 Construction of Gene Deletion Vaccine Strain rPRV-gE ^- -EGFP ⁺

The transfer vector plasmid pUCUsAB-EGFP was constructed by using the partial gI and Us2 genes of the PRV HeN1 strain as homology arms and the green fluorescent protein gene (EGFP) as a selection marker by gene cloning technology (the recombination site is shown in Figure 5, the transfer vector Enzyme digestion identification is shown in Figure 6), the PRV HeN1 genome and transfer vector were co-transfected into Vero cells, followed the instructions of the calcium phosphate transfection kit, and the Vero cells were pre-cultured in a 60mm dish to form a monolayer, before transfection Replace the fresh culture medium after 3 hours, mix 4 μg viral genomic DNA and 10 μg transfer vector pUCUsAB-EGFP, then add 2M CaCl ₂ to make the final concentration 0.24M, mix evenly, add this DNA-CaCl ₂ mixture to In a new tube containing an equal volume of 2XHBS, mix well and react at room temperature for 20 minutes, add it to a Vero cell culture dish, and place the transfected Vero cells in a 37°C, 5% CO ₂ incubator to continue culturing. After transfection Shock the transfected cells with 15% DMSO for 12 hours. When plaques with green fluorescence appeared, spread 1% low-melting point agarose, pick a single plaque and freeze-thaw once at -80°C, then The 10-fold dilution was inoculated into a six-well plate of Vero cells pre-cultured into a monolayer, and the plaques with green fluorescence were continued to be picked for repeated purification. After 4 rounds of plaque purification and identification, it was confirmed that the recombinant pseudorabies virus rPRV-gE ^- -EGFP ⁺ with deletion of gE gene and insertion of EGFP marker was obtained. The growth characteristics of recombinant pseudorabies virus rPRV-gE ^- -EGFP ^{+ were} studied by means of plaque test and one-step growth curve determination ^. There was no significant difference compared with the viral HeN1 strain, indicating that deletion and insertion did not affect the propagation speed of the recombinant virus in vitro. The recombinant virus rPRV-gE ^- -EGFP ⁺ was blindly passed on Vero cells for 12 generations, and the green fluorescent protein could still be stably expressed. The inserted foreign fragment and the viral gene sequence on both sides of the insertion site could be detected by PCR identification, indicating that the The obtained recombinant virus rPRV-gE ^- -EGFP ⁺ can be stably inherited. This strain (rPRV-gE ^- -EGFP ⁺ ) has been preserved in the General Microbiology Center of the China Committee for the Collection of Microbial Cultures, and the address is Beichen West Road, Chaoyang District, Beijing No. 1 Courtyard No. 3 Institute of Microbiology, Chinese Academy of Sciences, the strain preservation number is: CGMCC NO.6657, and the preservation date is October 12, 2012.

Example 4 rPRV-gE ^- -EGFP ⁺ immune efficacy evaluation

Twenty-two sheep aged 6-8 months were negative for PRV neutralizing antibody. They were randomly divided into six groups. Groups A and B (4 rats in each group) were intramuscularly injected with 0.2 doses of PRV Bartha k61 vaccine (the virus content in each dose was 10 ^5.0 TCID ₅₀ ), and groups C and D (4 rats in each group) were randomly divided into six groups. ) each rat was intramuscularly injected with 0.2 parts (10 ^5.0 TCID ₅₀ virus content per head) of rPRV-gE ^- -EGFP ⁺ , and groups E and F (3 rats in each group) were used as challenge control. 14 days after immunization, each sheep in groups A, C, and E was intramuscularly injected with 1 mL of PRV HeN1 strain containing 1,000 LD ₅₀ , and each sheep in groups B, D, and F was intramuscularly injected with 1 mL of PRV Shuangcheng strain (PRV-S) containing 1,000 LD ₅₀ . Blood sampling before challenge. Serum was collected from 16 sheep in groups A, B, C, and D 14 days after immunization with PRV Bartha k61 vaccine and rPRV-gE ^- -EGFP ⁺ . Anti-PRV gB antibodies were detected with IDEXX kits, and all sheep antibodies were positive. Within 7 days after the challenge, all the control sheep became ill and died, and the affected sheep showed neurological symptoms such as itching and opisthotonus. The morbidity and mortality of other groups are shown in Table 2. From the results of the challenge, it can be seen that Bartha k61 immunized sheep could not completely resist the challenge of the newly isolated strain HeN1 strain, while rPRV-gE ^- -EGFP ⁺ immunized to the Shuangcheng strain and HeN1 strain. All strains have good protective effects, so rPRV-gE ^- -EGFP ⁺ can be used as a vaccine candidate strain to deal with emerging outbreaks.

Table 2 The protective effect of rPRV-gE ^- -EGFP ⁺ immunized sheep against different strains

Claims (10)

1. A virulent strain of porcine pseudorabies virus (Porcine Pseudorabies Virus, PRV), named HeN1, is preserved in the General Microbiology Center of China Committee for the Collection of Microorganisms, and its strain preservation number is: CGMCC NO.6656. 2. a strain of porcine pseudorabies virus vaccine strain, is characterized in that described vaccine strain is on the basis of the porcine pseudorabies virus virulent strain described in claim 1, deletes gE gene and inserts EGFP mark and obtains by genetic engineering method of. 3. porcine pseudorabies virus vaccine strain as claimed in claim 2, is characterized in that insertion sequence and flanking nucleotide sequence thereof are shown in SEQ ID NO:3. 4. the porcine pseudorabies virus vaccine strain as claimed in claim 2 or 3, is characterized in that described porcine pseudorabies virus vaccine strain, named after rPRV-gE ^- -EGFP ⁺ , preserved in China Microorganism Culture Preservation Management Committee General Microorganism Center, its culture preservation number is: CGMCC NO.6657. 5. A vaccine composition for treating or preventing porcine pseudorabies, characterized in that it consists of the inactivated product of the virulent strain of porcine pseudorabies virus according to claim 1 and a pharmaceutically acceptable adjuvant. 6. A vaccine composition for treating or preventing porcine pseudorabies, characterized in that it consists of the gene-deleted vaccine strain according to any one of claims 2-4 and a pharmaceutically acceptable adjuvant. 7. The use of the virulent strain of porcine pseudorabies virus according to claim 1 in the preparation of medicines for preventing or treating porcine pseudorabies. 8. The use of the virulent strain of porcine pseudorabies virus according to claim 1 in the preparation of medicines for diagnosing or detecting porcine pseudorabies. 9. The use of the gene-deleted vaccine strain according to any one of claims 2-4 in the preparation of medicines for preventing or treating porcine pseudorabies. 10. Use of the gene-deleted vaccine strain according to any one of claims 2-4 in the preparation of medicines for diagnosing or detecting porcine pseudorabies.

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