CN1940063A - Pseudo-rabies gE/gI-gene loss poison strain, killed vaccine containing it and use - Google Patents

Abstract

The invention discloses the construction of a recombined pseudorabies virus (Pseudorabies virus, PrV) genetically engineered strain WKQ-001 with a preservation number of CCTCC-V200511 and an inactivated vaccine with a gene deletion marker of the pseudorabies virus prepared by the strain. The strain of the invention lacks the glycoprotein genes gI and gE of PrV, so it can be used as a marker gene for identifying and diagnosing artificially immunized pigs and naturally infected pigs. The invention also relates to the application of the recombinant pseudorabies virus strain and the gene deletion inactivated vaccine. Compared with similar foreign vaccines, it is more targeted and more suitable for the epidemic pig population of pseudorabies in my country; the strain of the present invention is derived from infected pigs, and the safety and protection of the inactivated vaccine are more suitable for porcine pseudorabies prevention. At the same time, the genetically engineered strain of the present invention does not contain any foreign gene, so it has higher biological safety.

Description

A pseudorabies gE-/gI-gene deletion strain, an inactivated vaccine containing the strain and application thereof

Technical field

The invention belongs to the technical field of animal virology and is related to genetic engineering. The present invention specifically relates to the construction of a recombinant pseudorabies virus (Pseudorabies virus, PrV) mutant strain (strain number: WKQ-001) and the use of the recombinant strain to prepare a pseudorabies gene deletion marker inactivated vaccine. The virus strain lacks the glycoprotein genes gI and gE of PrV, which leads to the reduction of PrV virulence, and thus can be used to prepare a pseudorabies safe vaccine.

The invention also relates to the application of the recombinant pseudorabies virus strain and gene deletion inactivated vaccine.

Background technique

Pseudorabies virus (PrV) belongs to Herpesviridae a herpesvirus subfamily, has the characteristics of rapid growth on cell culture, strong neurotropism and latent infectivity (Roizmorn B, Desrosiers R, Flecbenstein B, Lopez C, Minson Ad and studdert MJ. The family Herpesviridae; an update. Arch Virol. 1992, 123:425-449). The virus can infect pigs, cattle, sheep, dogs, cats, rabbits, mice, wild boars, mink, bears, foxes and other animals under natural conditions. Except for pigs, after other animals are infected with pseudorabies virus, they have typical symptoms such as fever, itching and acute encephalomyelitis, all of which are fatal infections. The disease was an outbreak in pigs. Pigs are the reservoir and source of infection of the virus. The main manifestations are reproductive impairment of sows and mass death of piglets, among which the mortality rate of piglets within 15 days of age is as high as 100%. Pseudorabies has caused huge economic losses to the pig industry.

In terms of research and application of pseudorabies vaccines, there are mainly two types of attenuated vaccines and inactivated vaccines. Due to the latent infection characteristics of the virus, the attenuated vaccine has the problems of unsafety and loose virus. It is generally believed that inactivated vaccines are safer than attenuated vaccines, but less effective. With the development of genetic engineering technology, people have developed some gene deletion vaccines for the prevention and control of pseudorabies, that is, using genetic engineering methods to insert or delete a sequence of the PrV genome, so that certain genes are not expressed, so as to attenuate PrV and without affecting its immunogenicity. In addition, the gene deletion vaccine can also reduce the virus shedding ability of immunized pigs after challenge, and its own latent infection ability is also greatly reduced. These artificially modified gene deletion vaccines are not only more reliable in terms of safety, but also facilitate the establishment of differential diagnosis methods. The combination of the two can not only prevent pseudorabies, but also distinguish between naturally infected pigs and immune pigs. By gradually eliminating naturally infected pigs, healthy pigs can be established, so that the ultimate goal of eradicating pseudorabies can be achieved.

The difficulty of porcine pseudorabies eradication lies in the long-term latency of pseudorabies virus and the immune failure caused by virus immune evasion. Studies have shown that the pseudorabies glycoprotein gene gE is the main factor causing the latency and immune evasion of pseudorabies virus. Glycoprotein gE is a protein that can be expressed by all PRV strains discovered so far (except some vaccine strains). It has group specificity and is of great significance in the differential diagnosis of PRV. Therefore, the prevention and control of pseudorabies by vaccination with inactivated vaccines that lack the gE gene of pseudorabies virus has become a generally accepted and popular method in the world.

Regarding the development of pseudorabies virus gE gene deletion vaccine, Huazhong Agricultural University developed a pseudorabies virus TK- ^/ gE- ^/ gI ^- gene deletion marker live vaccine in 2003, which has a better immune effect. The pseudorabies virus strain lacks the TK ^- /gE ^- /gI ^- gene, does not contain foreign genes, and belongs to a live vaccine constructed by genetic engineering (see Chinese invention patent publication, patent application number 03156706.1); Sichuan Agricultural University A pseudorabies TK ^- /gE ^- /gI ^- /LacZ ⁺ gene deletion marker attenuated live vaccine was reported. This strain lacks the TK ^- /gE ^- /gI ^- gene, but contains the expression cassette of the foreign gene LacZ (see , Guo Wanzhu, et al. Research on some biological characteristics of pseudorabies gene deletion vaccine strain (SA215), Chinese Journal of Preventive Veterinary Medicine, 2000, No. 9); the Harbin Veterinary Research Institute of Chinese Academy of Agricultural Sciences completed the gE/TK gene deletion mutant strain, the The mutant strain contains the expression cassette of the exogenous gene LacZ (see Tian Zhijun et al., Construction of pseudorabies virus gE/TK gene deletion mutant strain, Chinese Journal of Preventive Veterinary Medicine, 2004, No. 5); Nanjing Agricultural University reported a pseudorabies virus Shanghai strain (PRV-SH)/gE ^- /gI ^- /GFP ⁺ deletion strain (see Jiang Yan et al., Construction of pseudorabies virus Shanghai strain (PRV-SH)/gE ^- /gI ^- /GFP ⁺ deletion strain) Acta Microbiology, 2003 (2): 15-20), the deletion strain contains the expression cassette of the foreign gene green fluorescent protein GFP. Generally speaking, introducing exogenous genes into mutant strains is undoubtedly a shortcut for the screening of the mutant strains, because this can greatly improve the efficiency of screening. Some experts emphasized that LacZ, LUC and other reporter genes are foreign genes, which may affect their biological characteristics in the organism and have some negative effects on the organism; moreover, biological agents used in organisms are not allowed to be used casually. Those with foreign genes, so these gene-deleted vaccines inserted into the reporter gene can generally only be used as a means to study the latent infection of pseudorabies and the dynamics of its proliferation in the body, and cannot enter the market as commodities (Golden Scalenia etc., Research progress of pseudorabies gene deletion vaccine, Chinese Agricultural Sciences, 2002, 35(1): 89-93). Therefore, due to the defects mentioned above, the above three gene deletion vaccines may have biosafety problems once they enter the market. The applicant once developed a kind of pseudorabies gene vaccine (referring to Chinese Invention Patent Application Publication, Patent Application No. 03156706.1) that has lost TK/gE/gI gene. There is a great contribution of the vaccine, but as a weakened live vaccine, there may still be a risk of returning to strength. Therefore develop a kind of immune effect good, and safe pseudorabies virus gene deletion inactivated vaccine is the problem urgently needed to be solved in current production.

Contents of Invention

The task of the present invention is to overcome the defects in the prior art, obtain a recombinant pseudorabies genetically engineered virus strain by gene recombination method, and use the virus strain to prepare a kind of pseudorabies gE, gI with good control effect and high safety Gene deletion marks inactivated vaccines.

Based on the idea of the above invention, the present invention also includes a genetically engineered strain of pseudorabies virus lacking gE and gl genes, an inactivated vaccine prepared from the strain and its application.

The present invention is realized through the following technical solutions:

The key to the implementation of the present invention is that the applicant obtains a recombined Pseudorabies virus strain through gene recombination technology. The number of the strain is WKQ-001, and the strain was deposited on September 2, 2005. In China Center for Type Culture Collection (CCTCC), its deposit number is CCTCC-V200511.

The recombinant pseudorabies virus strain of the present invention has the following remarkable characteristics:

1) The recombinant strain has lost the gE and gI genes;

2) The recombinant strain does not contain foreign genes.

The applicant uses the preserved Pseudorabies virus strain WKQ-001, CCTCC-V200511 to prepare a pseudorabies virus deletion vaccine.

The gene deletion vaccine is an inactivated vaccine, which is prepared according to the following ratio:

By number of copies:

A. 3 parts of water phase

Each part of aqueous phase is prepared by adding 4 parts of sterilizing Tween 80 to the inactivated pseudorabies virus liquid prepared by 96 parts of pseudorabies virus strains described in claim 1 or 2;

B. 6 parts of oil phase

Each part of the oily phase is prepared by adding 94 parts in milliliters of No. 10 veterinary white oil, adding 2 parts of aluminum stearate in grams and 6 parts of Siben-80 in milliliters, and

C. Mix the water phase and oil phase described in A and B above to obtain the inactivated vaccine.

A more detailed technical solution is as follows:

The source strain that is used for gene recombination of the present invention is pseudorabies virus strain Ea strain (referring to Chen Huanchun etc., the isolation and identification of porcine pseudorabies virus E A strain, Journal of Animal Husbandry and Veterinary Medicine, 1998, 29 (2), 972104) as parent strain , partially deleting the coding regions of the glycoprotein genes gI and gE of PrV to obtain a deletion mutant strain, which is deposited in CCTCC with a deposit number of CCTCC-V200511.

The specific steps for preparing the pseudorabies virus gene deletion marker inactivated vaccine are:

1) Preparation of strains of recombinant rabies virus: use the intermediate transfer plasmid pIESE (this plasmid has been preserved in the China Center for Type Culture Collection (CCTCC) on September 2, 2005, and the preservation number is CCTCC NO: M205101), and pseudo Genomic DNA of rabies virus Ea strain (abbreviated as PrV Ea, the same below) was co-transfected into pig kidney passage cell IBRS-2, and the virus was harvested after pathological changes appeared. Obtained PrV ^gE- / ^gI- mutant strain through PCR screening and plaque screening, the number of this strain is WKQ-001, belongs to a kind of pseudorabies virus (Pseudorabies virus) strain, on September 2, 2005, preserved In China Center for Type Culture Collection (CCTCC), the deposit number is CCTCC-V200511.

2) Preparation of Pseudorabies virus gene deletion marker inactivated vaccine: add formaldehyde solution to the virus solution produced by the Pseudorabies virus strain (preservation number CCTCC-V200511) obtained in step 1) and mix well, then put it at 37°C for 36 hours , and then placed at room temperature (about 25°C) to continue to act for 12 hours to prepare the antigen for seedling production. In parts by volume: get 3 parts of water phase (each part of water phase is prepared by adding 4 parts of sterilized Tween 80 in the recombined pseudorabies virus liquid of 94 parts of inactivation: 6 parts of oil phase (each part of oil phase is used 94 parts of No. 10 veterinary white oil produced by Hangzhou Oil Refinery, Zhejiang Province, China, was prepared by adding 2 parts of aluminum stearate (in grams) and 6 parts of Siben-80.

The beneficial effects of the present invention are:

(1) The recombinant pseudorabies virus strain of the present invention is derived from the pseudorabies virus strain independently isolated and cloned by the applicant at home. Rabies is prevalent in swine herds, so its control is more targeted;

(2) The recombinant pseudorabies virus strain of the present invention is derived from infected pigs, compared with the pseudorabies virus strains isolated from other animals reported in the prior art, the security of the pseudorabies gene deletion marker inactivated vaccine prepared by the present invention It is more suitable for the prevention of swine pseudorabies and protective, and is conducive to the chemical treatment of pseudorabies in pig farms in my country.

(3) The recombinant pseudorabies virus strain of the present invention does not contain any exogenous gene, and compared with the pseudorabies gene vaccine containing marker genes such as LacZ in the same kind of pseudorabies virus gene vaccine reported in the prior art, it has higher biosafety sex.

Description of drawings

Fig. 1 shows the flow chart of pIESE plasmid construction, and the plasmid pIESE is a transfer plasmid containing part of the gE gene and gI gene of PRV Ea strain.

Fig. 2 shows the electrophoretic pattern identified by pIESE of pseudorabies virus gE, gI double gene deletion transfer vector. Figure 1: 15000Marker; 2: Stu I and BstEII double restriction plasmid pIESE. After double digestion with StuI+BstE II, a 6200bp fragment was produced instead of two electrophoresis bands of 1247bp and 6200bp. Figure 2 shows that these two restriction sites have been eliminated.

Figure 3 shows the construction process of the recombinant virus PrV gE ^- /gI ^- .

Figure 4 shows the electrophoretic pattern of recombinant virus PrV ^gE- /gI ^- PCR identification, in the figure, 1-18 is gE- ^/ gI ^- recombinant virus; 19 is water control; 20 is cell control; 21 is pIESE positive plasmid control 22 is the genome control of PRV EA strain.

As can be seen from Figure 4, the recombinant pseudorabies virus of the present invention can amplify the gE- ^/ gI ^- fragment of about 1400bp in size, while the traditionally isolated pseudorabies Ea strain (PRV EA strain) and the negative control cannot amplify this fragment, proving The gE and gI genes in the PrV genome DNA have been deleted.

Fig. 5 shows the genetic stability electrophoresis pattern of the recombinant virus PrV ^gE- / ^gI- , in the figure, 1-5 is the PCR amplification product of the fifth generation recombinant virus; 6-10 is the PCR amplification product of the tenth generation recombinant virus 11-15 is the PCR amplification product of the fifteenth generation recombinant virus; 16 is the water control; 17 is the cell control; 18 is the pIESE positive plasmid control.

Figure 6 shows the results of indirect immunofluorescence identification of recombinant viruses, in which 1 is the gE ^- /gI ^- mutant strain; 2 is the cell control; 3 is the traditional PRV Ea strain. From the results of indirect immunofluorescence, it can be seen that due to the deletion of the gE-gene, the pseudorabies mutant strain of the present invention (ie, the pseudorabies recombinant strain) cannot bind to the ^gE- monoclonal antibody, therefore, 1 and 2 in the figure are negative.

Figure 7 shows the protein dot hybridization identification pattern of recombinant pseudorabies virus PrV gE ^- /gI ^- , in the figure 1: parental virus strain control of pseudorabies virus Ea strain; 2: PK-15 cell control; 3: recombinant pseudorabies virus PrV gE ^- /gI ^- hybridization results. By Western dot blot; after gE gene deletion, PrV Ea gE ^- /gI ^- cannot express gE glycoprotein.

Below in conjunction with chart and embodiment the present invention will be further described.

Detailed ways

1. Primer design

According to the published Prv Ea strain gI (gene database accession number is AF306511); gE (gene database accession number is AF171937) gene sequence design and synthesis of a pair of specific primers referred to as P1, P2, amplified gI, gE double gene deletion The final DNA fragment size is 1400bp.

The primer sequences are as follows:

P1: 5'-TAGACGGGACGCTGCTGTTTCTGGAGG-3',

P2: 5'-CGGTCACGCCATAGTTGGGTCCATTCG-3'

The PCR amplification conditions are shown in Table 1:

Table 1 PCR conditions and primers of the present invention Primer PCR reaction conditions PCR reaction condition system gE ^- /gI ^- 94℃4min, 94℃1min51.8℃1min, 72℃1.5min35cycles, 72℃10min 10×buffer (without Mg ²⁺ ) 5.0μL, Template 5.0μL 25mmol/L MgCl ₂ 8.0μL 2mmol/LdNTP 3.0μL 10mmol/L P1 2.0μL 10mmol/L P2 2.0μL Taq 2.0μL DMSO 5.0μL H ₂ O 11μL 5006mmol.0μL kcl

2. Construction of the universal transfer vector pIESE

1. Plasmid pIECMV (including PRV Ea strain gD, gI, gE, partial coding sequence of 28k gene, 11k complete coding frame and multiple cloning site region of plasmid pcDNA3.1) was constructed by Dr. Fang Liurong from Animal Virus Laboratory of Huazhong Agricultural University. For the detailed preparation process, please refer to the Chinese invention patent application, application number: 200510012147.3) Digest plasmid pIECMV with restriction endonuclease STUI and BSTEII, remove its multi-cloning site region, recover the remaining fragments, connect with T4 DNA Ligase, 16°C Water bath overnight, transform Escherichia coli DH _5α , culture at 37°C for 16-20 hours, then pick a single clone colony, culture it in conventional LB medium for 12 hours, extract the plasmid, and store it at -20°C until identified by enzyme digestion use. The plasmid is deposited in CCTCC, and the deposit number is CCTCC NO: M205101

2. The sequence of the transfer vector was determined by the dideoxy terminal termination method (refer to: Molecular Cloning Handbook, China Science Press) sequencing and commonly used DNAclub, DNAsis2.5 software analysis. Its result is shown in accompanying drawing 1, accompanying drawing 2.

3. Construction of pseudorabies virus Ea strain gE gene deletion mutant

1. Extraction of the genome of pseudorabies virus Ea strain: transfer the detoxified and diseased PK-15 cells to a 500 mL centrifuge tube (5000 rpm, 5 min). Discard the culture medium, add PBS (PH7.4) to resuspend, transfer to a 50mL centrifuge tube (5000rpm, 5min), discard the supernatant, resuspend with 10mL LCM, ice-bath for 15min, add 1mL Freon (2500rpm, 8min), Take the supernatant. Ultracentrifuge at 24000rpm for 2 hours, discard the supernatant, and add 10mL TEN to resuspend. Add 10% SDS 500UL, act for 3min, extract 3 times with phenolform, 10000rpm, 10min each, precipitate with absolute ethanol for 30min, 8000rpm, 15min, resuspend, store at -20°C until use.

2 Screening and Purification of Pseudorabies Virus Mutants

1) Co-transfection of IBRS-2 cells: using liposome-mediated method (refer to Invitrogen’s Lipofectamine ^TM 2000 operating instructions), co-transfection of IBRS-2 cells with the transfer vector pIESE and pseudorabies virus Ea strain genome, lesions appeared Afterwards, the cell solution was collected, and 100 μL was inoculated into a 24-well cell culture plate of IBRS-2 cells growing into a single layer. After the lesion appeared, the poison was collected. Make a virus template with 200 μL, and screen positive mutants by PCR.

2) Plaque purification of pseudorabies virus mutant strains: Freeze and thaw the positive co-transfection products three times, dilute 10 ^-2 to 10 ^-7 , and inoculate 400 μL to grow in 6-well cell culture plates Adsorb the monolayer culture of PK-15 cells at 37°C for 1 hour, discard the culture medium, wash 3 times with PBS without calcium and magnesium, cover each well with 2.5mL of low melting point agarose, and culture in a CO ₂ incubator at 37°C , after plaque formation, stain with 1/10000 neutral red solution, incubate at 37°C for 1 hour, suck it out, and then incubate the 6-well cell culture plate at 37°C for 2 hours to make the residual neutral red The solution is fully evaporated. Plaques were picked with a capillary glass tube and placed in a 24-well plate covered with a monolayer of cells. After the lesion was complete, the virus was harvested and stored at -20°C. Then take out 200 μL to make virus template, pcr screening, plaque purification, and so on, until 100% positive mutants are obtained. See accompanying drawing 3, shown in accompanying drawing 4.

4. Biological characteristics of recombinant pseudorabies virus strain WKQ-001

The mutant strain belongs to porcine herpesvirus type I of the herpesviridae aherpesvirinae subfamily. Virus particles are round or oval in appearance, with a molecular weight of about 9.5×106 Daltons. The core diameter is 75nm. The length is about 12nm and the width is 9nm; the diameter of the unenveloped virion in the nucleus is about 110-150nm, and the diameter of the mature virion in the cytoplasm is about 180nm. The process of Pseudorabies virus entering the host cell can be divided into three stages, namely adsorption, membrane fusion, nucleocapsid release, etc., and it is passed on in a multiplication manner. Pseudorabies virus can proliferate on pig kidney passage cells, and the common medium is DMEM (pH7.0) growth solution containing newborn bovine serum. The virus can be stored at 4°C for a short period of time, and after freeze-drying, it can be stored at -70°C for a long time without loss of activity.

Table 2 Sensitivity test of recombinant pseudorabies virus strain WKQ-001 to ether, chloroform and trypsin group _TCID50 (0.1mL) group _TCID50 (0.1mL) group _TCID50 (0.1mL) ether control No CPE produces 10 ^-8.0 Trypsin control 10 ^-5.36 10 ^-7.96 Chloroform control 10 ^-1.36 10 ^-7.76

The data in Table 2 shows that the recombinant pseudorabies virus strain WKQ-001 of the present invention is sensitive to ether, chloroform and trypsin.

Table 3 Resistance of recombinant pseudorabies virus strain WKQ-001 to acid and alkali pH value Experimental group TCID ₅₀ (0.1mL) Control group TCID ₅₀ (0.1mL) 3.04.05.06.09.010.011.0 No CPE 10 ^-2.1 10 ^-7.9 10 ^-7.6 10 ^-7.6 10 ^-4.0 No CPE 10 ^-5.3 10 ^-7.7 10 ^-7.6 10 ^-7.6 10 ^-7.6 10 ^-7.6 10 ^-7.6

Table 3 shows that the recombinant pseudorabies virus strain WKQ-001 of the present invention remains stable between pH5.0-9.0.

Table 4 Resistance of recombinant pseudorabies virus strain WKQ-001 to 56°C Heating time (min) 15 20 30 45 control group TCID50 (0.1Ml) ^10-5.1 10 ^-4.0 10 ^-7.8 No CPE generated No CPE generated

Table 4 shows that the recombinant pseudorabies virus strain WKQ-001 of the present invention was completely inactivated by heating at 56° C. for 30 minutes.

4. The plaque test showed that the plaques formed by the recombinant pseudorabies virus strain WKQ-001 after gE and gI deletion were smaller than those formed by the traditional field strain pseudorabies virus Ea strain. The recombinant pseudorabies virus strain WKQ-001 was inoculated into pig kidney passage cell PK-15. After 15 generations of continuous inoculation, the gE-/gI- fragment of about 1400bp could still be expanded, indicating that the recombinant pseudorabies virus strain WKQ-001 inherited Stable and will not return to strength. See Attachment 5

5. Detection of pseudorabies virus Ea strain gE ^- /gI ^- mutant strain (ie WKQ-001)

1) Indirect immunofluorescence detection of deletion mutant strain WKQ-001: Wash the 6-well cell culture plate full of PK-15 cells with PBS solution for 3 times, air-dry naturally, fix with 100% formaldehyde for 5min (0.5mL/well), PBS The solution was washed 3 times and blotted dry. Add 200uL hyperimmune serum to each well, and incubate for 30min in a CO ₂ incubator at 37°C. Wash once with PBS and 5 times with ddH ₂ O. Add secondary antibody, 200uL/well. Incubate for 30 min in a CO ₂ incubator at 37°C. . Wash with PBS 3 times, add appropriate amount of PBS, observe with fluorescence microscope. See attached drawing 6.

2) Detection of deletion mutant strain WKQ-001 by protein dot hybridization Harvest PK-15 cells inoculated with PrV Ea gE ^- /gI ^- and PrVEa with CPE, freeze and thaw three times at -20°C, take 3цl of the freeze-thaw solution and drop it on the nitrocellulose membrane After air-drying, repeat the dropwise addition twice. Mouse anti-gE monoclonal antibody was used as the primary antibody, goat anti-mouse HRP-IgG was used as the secondary antibody, and the color was developed by diaminobenzidine. PrV Ea showed dark brown after staining, which was positive; while PrV Ea gE ^- /gI ^- and PK-15 cell control were negative. This indicated that after gE gene deletion, WKQ-001 could not express gE glycoprotein. See attached drawing 7.

5. Preparation of recombinant pseudorabies virus inactivated vaccine

1. Inoculum amount and culture of virus

Inoculate the recombinant pseudorabies virus strain WKQ-001 at 37°C for 24-36 hours in a monolayer of cells, and the amount of inoculation is 1/10 of the growth medium. Rotate and absorb for 1 hour at 37°C, add an appropriate amount of DMED Culture medium (containing 3% calf serum and 100 u/mL each of penicillin and streptomycin) was incubated at 37°C. 24-48 hours after inoculation, when the cytopathic effect (CPE) reaches more than 80%, the culture is terminated.

2. Virus inactivation

After repeated freezing and thawing of the diseased virus liquid three times, the virus liquid was collected. After the sterility test, and the TCID ₅₀ of PK-15 cells ≥ ^{10 -6} /0.1mL of the virus liquid mixed, with formaldehyde as inactivator, after adding formaldehyde solution and mixing well, the final concentration of formaldehyde solution is 1/1000 eight. Place it at 37°C for 36 hours, then place it at room temperature (around 25°C) for another 12 hours to prepare the antigen for seedling production. Take the inactivated virus liquid, inoculate in 3 flasks of PK-15 cell monolayer, culture at 37°C for 7 days and then blind pass the second generation. There should be no CPE.

Table 5 Examination results of the effect of formaldehyde on PRV inactivation Infection titer of PK-15 cells Batch number of virus solution Log ₁₀ TCID ₅₀ Before inactivation After inactivation Blind pass 1 generation Blind Pass 2 non-inactivated control 20050501200505102005052020050530 6.86.57.06.5 0000 0000 0000 6.86.57.06.5

After the 4 batches of virus suspensions listed in Table 5 were inactivated, the cells were inoculated and blindly passed for the second generation. The cells did not appear CPE, and the TCID ₅₀ was 0, which proved that the virus inactivation had completely lost the ability to replicate.

3. Research on emulsification process

(1) Preparation of oil phase: Take 94 parts (in milliliters) of No. 10 veterinary white oil (produced by Hangzhou Refinery in Zhejiang Province, China), add 2 parts of aluminum stearate (in grams), and add as you go. Stir until completely translucent. Then add 6 parts of Siben-80 (in milliliters), mix thoroughly, and then sterilize by autoclaving according to conventional methods (eg, sterilizing at 121°C for 30 minutes) for future use.

(2) Water phase preparation: by volume, 4 parts of sterilized Tween-80 were added to 96 parts of inactivated recombinant pseudorabies virus liquid, and shaken fully until the Tween-80 was completely dissolved.

(3) Emulsification: Put 6 parts of the oil phase into the colloid mill, start the machine to stir slowly, and slowly add 3 parts of the water phase at the same time, emulsify for 2.5 minutes at 8000-10000 rpm, then add 1 part of the water phase, continue Stir for 30 seconds to emulsify into an emulsion-type inactivated pseudorabies virus vaccine.

6. Inspection of Technical Indicators of Pseudorabies Virus Inactivated Vaccine

(1) Physical properties

Appearance This product is milky white homogeneous emulsion.

The dosage form is oil emulsion type. Take a clean straw to suck up a small amount of vaccine and drop it in cold water. It should be in the shape of oil droplets and not spread.

Viscosity: Use a straw with an outlet diameter of 1.2mm to draw 1mL of the vaccine and let it flow out vertically at a room temperature of about 25°C. If more than 0.4mL flows out within 8 seconds, it is considered qualified.

(2) The sterility test is carried out according to the appendix 169-171 of the "Chinese Veterinary Drug Code", and the growth should be sterile.

(3) Safety inspection Inoculate 5 mice with a weight of about 16-18 grams, inject 0.3 mL subcutaneously into each mouse, and observe for 14 days that the mice should be healthy and alive. Inoculate 2 healthy rabbits of 1.5-2kg, inject 5mL subcutaneously into each buttocks, observe for 14 days and they should be healthy and alive without adverse reactions.

(4) Efficacy test Inoculate 4 Prv antibody-negative and healthy weaned piglets with a body weight of about 10-20 kg, and inject 3 mL of the vaccine into each neck muscle. Blood was collected 28 days after injection, serum was separated, and antibody neutralization index was determined according to Appendix 315 of "Quality Standards for Veterinary Biological Products of the People's Republic of China". The neutralization index of immunized pig serum should be ≥ 316.

5 Determination of formaldehyde content According to the "Chinese Veterinary Pharmacopoeia" Appendix 177, should not exceed the specified amount.

Table 6 Inspection of various indexes of pseudorabies virus inactivated vaccine of the present invention Test items inspection date test result Judgment (S-conforms to regulations, U-does not meet regulations, I-no result, NI-not tested) 1 Exterior 2005.05.02 homogeneous milky white emulsion S 2 dosage form 2005.05.02 Does not spread in the form of oil droplets S 3 viscosity 2005.05.02 close to 8 seconds S 4 stability 2005.05.02 No delamination, no demulsification S 5 Sterility test 2005.05.02-2005.05.04 no bacterial growth S 6 Determination of formaldehyde content 2005.05.03 0.185% S 7 safety inspection 2005.05.04-2005.05.18 Rabbit 2/2 Healthy S 2005.05.04-2005.05.18 Mouse 5/5 healthy and alive S 8 Efficacy test (neutralizing antibody index) 2005.05.04-2005.05.31 316398398512 S

Description: (1) The gene deletion inactivated vaccine of the present invention is uniformly prepared into an oil emulsion (the same below)

(2) Testing basis: "Quality Standards of Veterinary Biological Products of the People's Republic of China", China Agricultural Science and Technology Press, 2001 edition

7. Tests on vaccine safety, immune efficacy, protection, immunity period and storage period

(1) Safety evaluation and biological test of the gene deletion vaccine of the present invention

1. Safety test on mice

Randomly take 3 bottles of 4 batches of vaccines prepared in the laboratory (20 bottles/batch, 100 ml/bottle), mix them evenly, inoculate 10 mice with a weight of about 18 grams, inject 0.3 ml subcutaneously into each, and observe for 14 days .

2. Newborn piglet safety test

4 batches of vaccines prepared in the laboratory (20 bottles/batch, 100 ml/bottle) were randomly taken from each batch of 3 bottles, after mixing evenly, healthy 1-day-old newborn piglets were selected, and 10 pigs were vaccinated in each batch, and each batch was injected intramuscularly with 2 Double the dose of 4mL pseudorabies oil emulsion vaccine, and set 4 other piglets as blank control. Before the vaccination and one week after the injection, all the piglets had their body temperature measured twice a day and the growth status, spirit and appetite of the piglets were observed. A total of 14 piglets were observed. sky.

3. Safety test of weaned piglets

Randomly take 3 bottles of 4 batches of vaccines prepared in the laboratory (20 bottles/batch, 100 ml/bottle), mix them evenly, and inoculate 4 healthy weaned piglets about 30 days old, each head is inoculated with 6 ml, and the dose is doubled. The immunization dose was inoculated, and another 4 pigs of the same age were used as blank control for observation for 14 days.

4. Pregnant sow safety test

Randomly take 3 bottles of 4 batches of vaccines (20 bottles/batch, 100 ml/bottle) prepared in the laboratory, mix them evenly, and inoculate 2 sows of about 80 days of pregnancy with 2 times the immune dose, and inject each head intramuscularly 10 ml. At the same time, 2 blank controls were set up, and they were raised under the same conditions until calving. The effect of vaccine on the reproductive performance of pregnant sows and the development of newborn piglets were observed.

The results are shown in Table 7

Table 7 Safety inspection results of the gene deletion inactivated vaccine of the present invention Vaccine batch inject animals 18 grams of white mice weaned piglets newborn piglet Sows around 80 days of gestation Dose (mL) only count Observation results Dose (mL) Head count Observation results Dose (mL) Head count Observation results Dose (mL) Head count Observation results sow fetus 0501051005200530 0.30.30.30.3 10101010 No Response No Response No Response 6666 4444 healthy healthy healthy healthy 4444 10101010 healthy healthy healthy healthy 10101010 2222 healthy healthy healthy healthy normal development normal development normal development normal

No adverse reaction occurred in the mice after inoculation. The 1-day-old newborn piglets and weaned piglets were vaccinated with double doses, and their mental state was good, their appetite was normal, and there were no adverse reactions such as swelling and fever at the inoculation site. The negative control group under the same conditions also had no adverse reactions. The sows injected with double doses for about 80 days of pregnancy did not suffer from abortion, and gave birth normally on schedule. The resulting piglets developed normally. There were no adverse reactions such as fever and swelling at the inoculation site, and the two control pregnant sows raised under the same conditions had no abnormal clinical reactions.

(2) Vaccine effectiveness test

1. Immune efficacy test of gilts

Randomly take 3 bottles of each batch of 4 batches of vaccines, mix them evenly, and inject 5 ml of neck intramuscular injection products into 4 gilts without mating, and set up 4 other gilts as non-immune controls. On days 21 and 28, blood was collected, serum was separated for microneutralization test, and Prv neutralizing antibody index was detected.

2. Immunity efficacy test of pregnant sows

Randomly take 3 bottles of 4 batches of vaccines for each batch, mix them evenly, select 4 sows, and inject 2 ml into each sow before mating, and set another 4 as non-immune control. On the 70th day of pregnancy, the same method and dosage were used to boost the immunization once, and the serum was collected before immunization, 28 days, 56 days, 80 days after immunization, and 14 days after the booster immunization for antibody detection. Two weeks after booster immunization, each pig was injected with 10 ^-7 TCID ₅₀ /0.1mL Prv E A strain virulent 2mL via ear vein, and 2mL nasally. Before the expected delivery date, that is, 14 days after the challenge, the immunized pigs and the control pigs were slaughtered, and the development of each litter was checked.

3. Immune efficacy test of weaned piglets

Randomly take 3 bottles of 4 batches of vaccines from each batch, mix them evenly, and inject 3 ml of the vaccines into 4 healthy weaned piglets at the age of 25 days respectively, and set up 4 blank controls. After 14 days of challenge, each pig was injected with 2mL of 10 ^-7 TCID ₅₀ /0.1mL Prv through the ear vein, and 1mL of Prv was injected into the nose for challenge, observed for 21 days, and the protection rate was calculated.

4. Immune efficacy test of newborn piglets

Randomly take 3 bottles of 4 batches of vaccines for each batch, mix them evenly, select 4 litters of healthy 1-day-old newborn piglets, 10 pigs in each litter, inject 8 piglets in 1 litter with 4 batches of vaccines, inject 2 mL each, and leave 2 head served as an unimmunized control. On the 14th day after immunization, each immunized pig and the control pig were injected with 10 ^-6 TCID ₅₀ /0.1mL Prv E A strain virulent 1mL through the ear vein, and challenged with 1mL intranasally, and observed for 21 days to calculate the protection rate. The results are shown in Table 8-

4.1 Gilts

Table 8 Inactivated vaccine of the present invention is to gilt immune effect test result Vaccine batch Immunization dose (mL) Number of test pigs (head) Antibody neutralization index Before immunization (days) After immunization (days) 0 14 twenty one 28 0501051005200530 control 2222 44444 11111 218-316251-398198-380258-3981 398-1000398-1258398-1258398-12581 398-1000398-1258308-1412398-12581

Note: The batch numbers of the vaccines in this table are 20050501-20050530, a total of 4 batches

Four batches of vaccines with batch numbers 20050501-20050530 were used to immunize gilts respectively. After 14 days of immunization, the neutralizing antibody index rose to 218-398. The neutralizing antibody index reached 1412 at 21-28 days.

4.2 After 28 days of immunization of pregnant sows, the test pigs with the highest neutralizing antibody index can reach 1412, 80 days after immunization, the serum neutralizing antibody index can still reach 1000, and 14 days after boosting immunization with the same dose, the serum neutralizing antibody index is high Those can reach 3548. 14 days after the challenge, the serum neutralizing antibody index was as low as 1258 and as high as 3548. 16 sows immunized with 4 batches of vaccines were slaughtered to count the number of fetuses. A total of 151 fetuses were taken, and 145 were healthy and live. There were 6 stillbirths and weak fetuses. A total of 38 fetuses were conceived in 4 non-immune control pigs, only 2 were healthy and alive, and 36 were stillborn and weak.

Table 9 Inactivated vaccine of the present invention is to pregnant sow immune effect test result Vaccine batch immune dose Number of test pigs Attack dose Neutralizing antibody index number of fetuses Before immunization (days) First exemption Second exemption total Healthy fetus stillbirth Weak tire 28 days after immunization 55 days after immunization 80 days after immunization 14 days after immunization 14 days after challenge Head count Head count Head count Head count 200505012005051020050520 555 444 444 000 389-1412380-1258316-1122 389-1122398-1000389-1000 389-1000316-1000316-639 1000-35481000-3548398-3548 1258-35481258-35481258-3548 383637 363536 110 101 20050530 control 5 44 44 0 316-1258 389-1000 316-1000 1000-3548 1258-3548 4038 382 131 15

4.3 14 days after the weaned piglets were immunized against the virus, the protection number of the piglets was 4/4, and the protection rate was 100%. During the observation period, the immunized pigs did not have any adverse reactions, while the unimmunized control piglets had 3 piglets with elevated body temperatures of 40°C, 41°C, and 41.8°C 2 days after the challenge, depression, trembling, vomiting, and diarrhea. One of the control pigs had neurological symptoms, manifested as ataxia, and died 7 days after the challenge; the symptoms of the three control pigs were gradually relieved during the observation period, and finally returned to normal.

Table 10 Inactivated vaccine of the present invention is to weaned piglet immune effect test result Vaccine batch Immunization dose (mL) Number of test pigs (head) Attack dose (mL) Immunization time (days) Antivirus protection number (head) Protection rate (%) 20050501200505102005052020050530 control 3333 44444 22222 14141414 4/44/44/44/41/4 10010010010025

4.4 Newborn piglets were challenged 14 days after immunization. During the observation period after challenge, one pig had a body temperature that rose to 40.5°C-41.8°C 24-72 hours after challenge. Adverse reactions. The total protection rate was 90.6%. Twenty-four hours after the challenge, the body temperature of the 8 non-immune control pigs rose to 40.5°C-42°C, the spirits were low, trembling, vomiting and diarrhea, and 6 of them died 36-50 hours after the challenge. The autopsy lesions of dead pigs showed upper respiratory tract mucosa and tonsil hemorrhage, pulmonary edema, large pinpoint hemorrhage points in the kidney, meningeal congestion, hemorrhage, and edema. The symptoms of the other two control pigs were relieved on the 10th day after the challenge, and the body temperature dropped to some extent, but they still maintained a slight fever of about 40°C during the observation period.

Table 11 Inactivated vaccine of the present invention is to newborn piglet immune effect test result Vaccine batch Immunization dose (mL) Number of test pigs (head) Attack dose (mL) Immunization time (days) The number of heads protected against poison (heads) Protection rate (%) Incidence control (head) 20050501200505102005052020050530 2222 8888 1111 14141414 7/87/87/87/8 87.587.587.5100 2/22/22/22/2

5 Detection of maternal neutralizing antibody levels in piglets after immunization of sows

Pick a head and inject 2mL of vaccine before mating, booster immunization (2mL) once a month before delivery, and take blood 2 days before delivery to test the neutralizing antibody as 1:16. On the first day postpartum, the antibody in milk reached 1:64-128. A total of 11 piglets were born, and blood was collected from 7 piglets at different ages, and the antibody level was completely tested. The results are shown in the table

Table 12 Detection of maternal neutralizing antibody level of piglets after immunization of sows with inactivated vaccine of the present invention

13 1:11 1:18.7 1:11 1:11 1:18.7 1:18.7 1:22 twenty one 1:9.4 1:11 1:11 1:8 1:8 1:11 1:8 30 1:5.6 1:9.4 1:9.4 1:9.4 1:5.6 1:5.6 1:56 40 1:4 1:4 1:8 1:2 1:4 1:5 1:2 50 1:2 - 1:4 - 1:2 - - 60 1:2 - - - - - -

According to the results of the experiment, the antibody levels of the colostrum milk of the vaccinated sows were higher than the antibody levels in the serum of the sows, so the antibody levels in the serum of the newborn piglets were also higher. However, as the age increases, the antibody level also declines rapidly, and it is already low by the age of 30 days. Therefore, in clinical practice, it is more appropriate to schedule the first immunization of piglets against pseudorabies at weaning.

(3) Immunization period of the vaccine

1 Antibodies began to appear in pigs 14 days after immunization. Although the neutralizing antibody index did not reach the positive standard of 316, it had increased significantly compared with non-immunized control pigs. Twenty-eight days after immunization, the neutralizing antibody indexes of the five batches of vaccines all reached above 398, reached a peak at 35-42 days after immunization, and gradually decreased thereafter, until the antibody level was equal to or close to the critical value at 180 days. According to the rise and fall of the antibody level, the immunity period of this vaccine is set as six months as a preventive use. For pregnant sows, in order to protect newborn piglets with a high level of maternal antibodies in colostrum, it is recommended to boost immunization once a month before delivery, which can completely protect piglets until weaning. Inject once for fattening piglets at weaning until slaughter. Inject the piglets for breeding once at weaning, and then boost the immunization once every 4-6 weeks as the basic immunization, and immunize once every six months thereafter. The specific results are shown in the table below.

Table 13 Detection of neutralizing antibodies after initial immunization of breeding pigs Vaccine lot number Number of immunized pigs (head) Serum anti-middle finger (average) serum and count 14 days 21 days 28 days 35 days 42 days 52 days 85 days 125 days 155 days 180 days 0501051005200530 control 44442 1992232512231 3543163163981 4573555013551 954125811228191 15131412125810961 14121122120310961 7588917948911 6303983163981 4073983163161 1991991121991

Determination results of antibody levels after secondary immunization of two breeds of pigs

The antibody level reached its peak at about 42 days after the first immunization, so the second immunization was carried out at 42 days, and the antibody level increased significantly 14 days after immunization, and at 180 days, the antibody level was significantly higher than that of the first immunization, and the antibody duration after immunization It is also longer, and the specific results are shown in the table below.

Table 14 Neutralization index after secondary immunization of breeding pigs Vaccine lot number Number of immunized pigs (head) Serum antibody neutralization index (average) 14 days 21 days 28 days 35 days 42 days 52 days 62 days 85 days 125 days 155 days 180 days 0501051005200530 control 44442 71627891701978911 68186511681868181 65116238651162381 59955584558455841 52585122541252581 49124309491240791 41414309430943091 31622511316231621 25112511281825111 79489110298911 6206308916301

(4) Shelf life of the vaccine

The vaccine was stored at 4-8°C for 12 months and 18 months; at 20-25°C for 1 month and 3 months, and the 1-month-old weaned piglets were immunized with a dose of 3mL, 21 days and 28 days after immunization, respectively. The blood was collected every day, and the level of neutralizing antibody was detected, all of which were between 316-398, and the level of antibody produced was within the normal range. After being challenged with a strong virus, all pigs were normal. no change. Therefore, the drafting standard is set to store at 4-8°C for 12 months, and store at 20-25°C for 1 month.

Table 15 Shelf life test of inactivated vaccine of the present invention (taking weaned piglet as example) Vaccine lot number Storage condition ℃ Storage time (month) Number of test pigs (head) Neutralization index (average) 1 day before immunization 21 days after immunization 28 days after immunization 14 days after challenge 0501051005200530 control 20-254-820-254-820-254-820-254-820-25 13121513121513121513121513 222222222222222224 111111111111111111 3983543463803803633542953723243803314783463803553803631 5014074364266024784894265375016025886164904784675014071 398436407416512467457489537436501575588380467446537436331

As shown in Table 15, after the inactivated vaccine of the present invention was stored at 4-8°C for 12 months and 15 months at 4-8°C, animals (such as pigs, mice, rabbits, etc.) were immunized respectively. The resulting neutralizing antibody index difference is not significant (P>0.05), illustrating that the inactivated vaccine of the present invention can be stored at 4-8°C for 12-15 months; at 20-25°C for 1 month and at 20-25°C After being stored at ℃ for 3 months, the neutralizing antibody index produced at 28 days was significantly different (0.01<P≤0.05). The vaccine of the present invention can be preserved for one month at 20-25°C.

Claims (7)

1, a kind of Pseudorabies virus Pseudorabies virus strain WKQ-001 of reorganization is deposited in CCTCC, and deposit number is CCTCC-V200511.

2, the pseudoabies poison strain of the described reorganization of claim 1 is characterized in that:

1) said strain has lacked gE, gI gene;

2) said strain does not contain foreign gene.

3, the gene-deleted vaccine that comprises claim 1 or 2 described recombinant pseudorabies virus strains.

4, the described gene-deleted vaccine of claim 3 is characterized in that, described gene-deleted vaccine is an inactivated vaccine.

5, claim 3 or 4 described gene-deleted vaccines by the proportioning of umber are:

3 parts of A, waters

Each part water adds the preparation of 4 parts of sterilization tween 80s by 96 parts of pseudoabies venom with the deactivation of claim 1 or 2 described pseudoabies strain preparations;

6 parts of B, oil phases

Each part oil phase is by 94 parts of No. 10 white oils for animals in milliliter, add in 2 parts of aluminum stearates of gram and in 6 parts of Si Ben-80 ratios preparations of milliliter and

C, the above-mentioned A of mixing and described water of B and oil phase.

6, claim 1 or the 2 described recombinant pseudorabies virus strains application in preparation Pseudorabies virus gene-deleted vaccine.

7, the application of each described gene-deleted vaccine of claim 3-5 in anti-system pseudoabies.

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