CN102994458A - Porcine pseudorabies virus virulent strain, and gene deletion vaccine strain thereof and applications thereof - Google Patents
Porcine pseudorabies virus virulent strain, and gene deletion vaccine strain thereof and applications thereof Download PDFInfo
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Abstract
The invention discloses a porcine pseudorabies virus virulent strain, and a gene deletion vaccine strain thereof and applications thereof. The porcine pseudorabies virus virulent strain is named as HeN1, the microbial preservation number of the porcine pseudorabies virus virulent strain is CGMCC NO.6656, the deleted gE gene obtaines the gene deletion vaccine strain rPRV-gE-EGFP+ on the basis of the virulent strain HeN1, and the microbial preservation number is CGMCC NO.6657. The virulent strain can be prepared into inactivated vaccine (single vaccine or combined vaccine), the gene deletion vaccine strain rPRV-gE-EGFP+ can be prepared into activated vaccine or inactivated vaccine (single vaccine or combined vaccine) and the like, so that porcine pseudorabies can be effectively prevented or cured, or the gene deletion vaccine strain rPRV-gE-EGFP+ can be prepared into a diagnosis reagent for diagnosing the porcine pseudorabies. The gene deletion vaccine strain rPRV-gE-EGFP+ has the advantages of being good in safety, high in protection efficiency, beneficial to differential diagnosis.
Description
Technical field
The gene-deleted vaccine strain that the present invention relates to a strain virus virulent strain and obtain on its basis relates in particular to a strain porcine pseudorabies virus virulent strain HeN1, lacks the gene-deleted vaccine strain rPRV-gE that the gE gene obtains by this virulent strain HeN1
--EGFP
+, the invention still further relates to HeN1 and vaccine strain rPRV-gE
--EGFP
+Application in prevention or treatment porcine pseudorabies the invention belongs to biomedicine field.
Background technology
Pseudoabies (Pseudorabies, PR has another name called Aujeszky's disease, AD) be a kind of acute infectious disease of the multiple domestic animal, wildlife, companion animals and the laboratory animal that are caused by pseudorabies virus (Pseudorabies Virus, PRV).Under field conditions (factors), this disease is most commonly in pig, ox, sheep, dog, cat and muroid.Pseudorabies virus all is the lethality course of disease except to the pig to all susceptible animals.Be nervous symptoms, salivation, expiratory dyspnea, breeding difficulty, cessation of growth cessation, weightlessness and high mortality to the pig main manifestations.The clinical manifestation of sucking piglets infection pseudoabies is typical case, serious in consistent the most, nervous symptoms, paralysis, depleted dead all occur, and mortality ratio is almost up to 100%; Mortality ratio descends gradually with age, and becoming pig only is 2%, does not fall ill but become generally speaking pig to infect, and is stealthy process, rarely seenly sneezes, the light symptoms such as cough, fervescence; Pregnant sow infects Pseudorabies virus can cause miscarriage, stillborn foetus, the weak son of product and mummified fetus; Can cause vaginalitis to boar.This disease has the infectivity of height, in case in swinery, find, will be very fast by air with contact through respiratory infectious to other pigs and pig farm, the recent pseudorabies virus that studies show that can also be by milk and genital tract infection.
At large-scale pig farm, PR is routine immunization prevention and control objects.The seroepidemiological survey that utilizes the strong and weak malicious ELISA of discriminating to carry out finds still have a certain proportion of pig farm and pig to have wild malicious antibody after the gene-deleted vaccine immunity, and idol has the report of virus separation.Because the financial loss that causes is limited, does not cause enough attention.But in this year, have the large-scale pig farm of a plurality of use conventional vaccine immunity the popular phenomenon of doubtful PR to occur, main manifestations is that sow produces weak young, stillborn foetus, and nervous symptoms and death etc. appear in piglet.The tissue of censorship carried out PCR identifies and viral the separation, prove that there is the PRV wild virus infection really in the pig farm.This laboratory has detected the wild poison of PRV from the pathological material of disease of province's pig farm censorships such as Henan, Heilungkiang, Jilin, Liaoning, the Inner Mongol at present.Because the PRV genome is larger, about 145kb, we check order by the gE gene by pcr amplification PRV from each pig farm submitted sample, the result shows these two gene height homologies of each pig farm epidemic isolates, homology is more than 99%, minimum with PRVKaplan strain (Hungary, GenBank accession number JQ809328) homology, be 97.5%.
Immunization is the most effectual way of prevention and control PRV.China has introduced PRVBartha k61 strain the seventies in last century from Hungary, this virus is the good vaccine strain of generally acknowledging in the world, and domestic large-scale pig farm is mostly used this vaccine, and PR has also obtained good control.But present situation is, this vaccine is all crossed according to the conventional procedure immunity in PR morbidity pig farm, whether does new epidemic isolates exist antigenic variation to cause immune swine can not effectively resist this virus? we utilize the immunization trial of serum neutralization test and sheep to confirm, the neutralizing antibody that Bartha k61 immune swine produces can not the new isolated viral of efficient neutralization, and the sheep of immune Bartha k61 can not be resisted the attack of new virus fully.Therefore be badly in need of the new safely and effectively PRV vaccine of exploitation to tackle newfashioned strain.
Summary of the invention
One of the object of the invention provides a strain porcine pseudorabies virus virulent strain.
Two of the object of the invention provide a strain by this porcine pseudorabies virus virulent strain through gene engineering method disappearance gE gene and insert the attenuated vaccine strain of EGFP mark, the vaccine prepared with this attenuated vaccine strain has good protection effectiveness for the new epidemic isolates of PRV.
Three of the object of the invention provides the vaccine composition of a kind of prevention or treatment porcine pseudorabies.
Above-mentioned purpose of the present invention is achieved through the following technical solutions respectively:
Certain pig farm that doubtful PRV infects from China Henan Province gathers the cerebral tissue of disease pig, extracts tissue gene group DNA, utilize PRV gE Auele Specific Primer to carry out PCR and identify (Fig. 1), with positive brain tissue homogenate liquid carry out centrifugal, to cross leaching filtrate frozen stand-by.The Vero cell is after 37 ℃ of lower 48h of cultivation form individual layer, and the pathological material of disease suspension supernatant that every bottle graft kind 1ml filters behind the absorption 1h, changes the DMEM nutrient solution that contains 2% foetal calf serum and continues 37 ℃ of lower cultivations, observes to have or not cytopathy to produce (Fig. 2) in 3～4 days.When 60～70% cells CPE occurs and change, receive poison, will have the strain of pathology to pass that the Auele Specific Primer with PRV gE carries out pcr amplification and the evaluation of virus particle electron microscopic observation after 2～3 generations.Test shows and utilizes can the increase fragment of expection size of PRV gE Auele Specific Primer, with behind this sequencing fragment with GenBank on the sequence delivered compare, its homology is more than 97%, its sequence results is shown in the SEQ ID NO:2.Culture is carried out electron microscopic observation, can observe virus particle rounded, hollow and solid two kinds of features are arranged, cyst membrane has for having of having without cyst membrane (Fig. 3).Prove according to the above results, this isolated strain is PRV, called after HeN1 strain, the Classification And Nomenclature pseudorabies virus, this strain is deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center, No. 3 in the Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica in address, its culture presevation is numbered: CGMCC NO.6656, preservation date are on October 12nd, 2012.Typical pseudoabies symptom has appearred after the BALB/c mouse inoculation PRV HeN1 strain: with the mouth injection site that bites, cause local by hair come off, dermatorrhagia, severe patient hind leg bitten broken, injecting virus content is 10
3.0-10
6.0TCID
50Mouse dead in the 60h after inoculation.By calculating PRV HeN1 to the LD of BALB/c mouse
50Be 237TCID
50The contrast mouse of injection DMEM nutrient solution is without unusual performance.Only occur one after the SPF weanling pig inoculation PRV HeN1 strain and cross the heat pyrexia reaction, body temperature reaches 41 ℃, continues 3-5 days, in addition without other clinical onset symptoms with cut open inspection and change.
Utilize the Immunization test of microneutralization test and sheep to confirm to have antigenic difference between new isolated strain (PRV HeN1 strain) and the vaccine strain Bartha k61.The neutralizing antibody level that found that the generation of Bartha k61 Pigs Inoculated is compared generally lower with the HeN1 Pigs Inoculated, the serum dilution of its energy 50% neutralization self virus is all in 1:40, neutralising capacity to new isolated viral HeN1 is lower, in 50% and the serum dilution of virus about 1: 10.HeN1 Pigs Inoculated 2 weeks after infection just can produce high-caliber neutralizing antibody, in 50% with viral serum dilution all more than 1:80, and all higher to the neutralising capacity of two-strain.
By gene clone technology with the part gI of PRV HeN1 strain and Us2 gene as homology arm, and green fluorescence protein gene (EGFP) has made up metastasis transplanting physique grain pUCUsAB-EGFP as selection markers, with PRVHeN1 genome and transfer vector cotransfection Vero cell, take turns plaque purification and identify that confirmation has obtained to lack the recombinant pseudorabies virus rPRV-gE that the gE gene inserts the EGFP mark simultaneously through 4
--EGFP
+By methods such as plaque test and one step growth mensuration to recombinant pseudorabies virus rPRV-gE
--EGFP
+Growth characteristics study, found that recombinant virus rPRV-gE
--EGFP
+Rate of propagation in cell in vitro is cultivated is compared with parent's poison HeN1 strain does not have significant difference, illustrates that deletion and insertion does not affect recombinant virus in external reproduction speed.Recombinant virus rPRV-gE
--EGFP
+In 12 generations of blind passage on the Vero cell, green fluorescent protein still can stably express, identifies by PCR to detect the external source fragment of insertion and the virus gene sequence of insertion point both sides, shows the recombinant virus rPRV-gE that obtains
--EGFP
+Can genetic stability.
Therefore the invention allows for a strain porcine pseudorabies virus vaccine strain, it is characterized in that described vaccine strain is on the basis of described porcine pseudorabies virus virulent strain, by gene engineering method disappearance gE gene and insert that the EGFP mark obtains.
In a specific embodiment of the present invention, insertion sequence and flanking nucleotide sequence thereof are shown in the SEQ ID NO:3.
In a specific embodiment of the present invention, described porcine pseudorabies virus vaccine strain, called after rPRV-gE
--EGFP
+The Classification And Nomenclature pseudorabies virus, this strain is deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center, No. 3 in the Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica in address, its culture presevation is numbered: CGMCC NO.6657, preservation date are on October 12nd, 2012.。
By to PRV HeN1 strain and rPRV-gE--EGFP
+Immune efficacy estimate discovery, it is high that the HeN1 Pigs Inoculated is compared the neutralizing antibody level that Bartha k61 Pigs Inoculated produces; RPRV-gE
--EGFP
+After the immunity Shuangcheng strain and HeN1 strain are all had good protection effect, and Bartha k61 immune sheep can not be resisted the attack of new isolated strain HeN1 strain fully, so rPRV-gE
--EGFP
+Can be used as the vaccine candidate strain to tackle emerging epidemic situation.
Therefore, further, the invention allows for the vaccine composition of a kind for the treatment of or prevention porcine pseudorabies, it is characterized in that: formed by the product after the deactivation of described porcine pseudorabies virus virulent strain or the strain of porcine pseudorabies virus gene-deleted vaccine and pharmaceutically acceptable adjuvant.
Further, the invention allows for the purposes of described porcine pseudorabies virus virulent strain in preparation prevention or treatment pseudorabies medicine.And
The purposes of described porcine pseudorabies virus virulent strain in preparation diagnosis or detection pseudorabies medicine.And the purposes of described gene-deleted vaccine strain in preparation prevention or treatment pseudorabies medicine.And
The purposes of described gene-deleted vaccine strain in preparation diagnosis or detection pseudorabies medicine.
Virulent strain of the present invention can be prepared into inactivated vaccine (single seedling or connection seedling), gene-deleted vaccine strain rPRV-gE
--EGFP
+Can be prepared into living vaccine or inactivated vaccine (single seedling or connection seedling) etc., all can effectively prevent or treat porcine pseudorabies, also it can be prepared into the diagnostic reagent of diagnosis porcine pseudorabies.Gene-deleted vaccine strain rPRV-gE of the present invention
--EGFP
+Have that security is good, protection efficient is high, be beneficial to the advantage such as differential diagnosis.
Description of drawings
The PCR of Fig. 1 pig farm censorship brain tissue sample identifies;
1: negative control, 2-8: submitted sample 9: positive control, 10:DNA marker
The cytopathy that Fig. 2 brain tissue homogenate liquid vero cells infection produces;
A: normal Vero cell; B: infected Vero cell
The electron microscopic observation of Fig. 3 virus particle;
Fig. 4 two strain virus induce the neutralizing antibody level relatively;
A: the porcine blood serum of injection PRV Bartha k61 strain.
B: the porcine blood serum of injection PRV HeN1 strain.
Fig. 5 rPRV-gE
--EGFP
+The recombination site schematic diagram;
The enzyme of Fig. 6 pUCUsAB-EGFP is cut evaluation.
M:DL15000; 1.BamH I digested plasmid pUCUsAB-EGFP
Embodiment
Also the present invention will be further described in conjunction with the embodiments below by experiment, it should be understood that these embodiment only are used for the purpose of illustration, never limit protection scope of the present invention.Those of ordinary skills understand, and can carry out many changes to it in the spirit and scope that claim of the present invention limits, revise, even the equivalence change, but all will fall within the scope of protection of the present invention
The isolation identification of embodiment 1 porcine pseudorabies virus virulent strain (PRV HeN1 strain)
Certain pig farm that doubtful PRV infects from China Henan Province gathers the cerebral tissue of disease pig, extracts tissue gene group DNA, utilize PRV gE Auele Specific Primer to carry out PCR and identify (Fig. 1), with positive brain tissue homogenate liquid carry out centrifugal, to cross leaching filtrate frozen stand-by.The Vero cell is after 37 ℃ of lower 48h of cultivation form individual layer, and the pathological material of disease suspension supernatant that every bottle graft kind 1ml filters behind the absorption 1h, changes the DMEM nutrient solution that contains 2% foetal calf serum and continues 37 ℃ of lower cultivations, observes to have or not cytopathy to produce (Fig. 2) in 3～4 days.When 60～70% cells CPE occurs and change, receive poison, will have the strain of pathology to pass that the Auele Specific Primer with PRV gE carries out pcr amplification and the evaluation of virus particle electron microscopic observation after 2～3 generations.Test shows and utilizes can the increase fragment of expection size of PRV gE Auele Specific Primer, with behind this sequencing fragment with GenBank on the sequence delivered compare, its homology is more than 97%, its sequence results is shown in the SEQ ID NO:2.Culture is carried out electron microscopic observation, can observe virus particle rounded, hollow and solid two kinds of features are arranged, cyst membrane has for having of having without cyst membrane (Fig. 3).Prove according to the above results, this isolated strain is PRV, called after HeN1 strain, the Classification And Nomenclature pseudorabies virus, this strain is deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center, No. 3 in the Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica in address, its culture presevation is numbered: CGMCC NO.6656, preservation date are on October 12nd, 2012.Typical pseudoabies symptom has appearred after the BALB/c mouse inoculation PRV HeN1 strain: with the mouth injection site that bites, cause local by hair come off, dermatorrhagia, severe patient hind leg bitten broken, injecting virus content is 10
3.0-10
6.0TCID
50Mouse dead in the 60h after inoculation.By calculating PRV HeN1 to the LD of BALB/c mouse
50Be 237TCID
50The contrast mouse of injection DMEM nutrient solution is without unusual performance.Only occur one after the SPF weanling pig inoculation PRV HeN1 strain and cross the heat pyrexia reaction, body temperature reaches 41 ℃, continues 3-5 days, in addition without other clinical onset symptoms with cut open inspection and change.
There is antigenic difference between embodiment 2PRV HeN1 strain and the vaccine strain Bartha k61
1) utilize the Immunization test of microneutralization test and sheep to confirm to have antigenic difference between new isolated strain (PRV HeN1 strain) and the vaccine strain Bartha k61.The neutralization test working method: the SPF pig of 12 40 ages in days, wherein 5 intramuscular injection 1mL contain 10
7.0TCID
50The PRV HeN1 strain of deactivation, 5 intramuscular injection 1mL contain 10
7.0TCID
50Bartha k61 strain, in addition 2 injection DMEM nutrient solutions in contrast, the separation of serum of taking a blood sample weekly before inoculation and after the inoculation cutd open after inoculation extremely in 5 weeks.Adopt fixed virus diluted blood heat-clearing method mensuration vaccine Bartha k61 Pigs Inoculated serum and HeN1 Pigs Inoculated serum that the neutralization of two strain virus (Bartha k61 and HeN1) is tired.Virus quantity is 100TCID
50/ hole, serum are that Bartha k61 and HeN1 inoculate respectively behind the SPF pig in the different time points collection, and 56 ℃ of deactivation 30min are for subsequent use.The neutralizing antibody level that found that the generation of Bartha k61 Pigs Inoculated is compared generally lower with the HeN1 Pigs Inoculated, the serum dilution of its energy 50% neutralization self virus is all in 1:40, neutralising capacity to new isolated viral HeN1 is lower, in 50% and the serum dilution (Fig. 4 A) about 1: 10 of virus.HeN1 Pigs Inoculated 2 weeks after infection just can produce high-caliber neutralizing antibody, in 50% with viral serum dilution all more than 1:80, and to the neutralising capacity of two-strain all higher (Fig. 4 B).
2) immunoprotection of sheep test: 14 of 6-8 monthly age sheep, the PRV neutralizing antibody detects negative.Be divided at random four groups, (every part viral level is 10 to 0.2 part of A, C group (4 every group) every intramuscular injection
5.0TCID
50) PRV Bartha k61 vaccine, B, D group (3 every group) is as attacking poison contrast.Rear 14 days of immunity, every sheep intramuscular injection 1mL of A, B group contains 1000LD
50PRV HeN1 strain, C, D the group every sheep intramuscular injection 1mL contain 1000LD
50PRV Shuangcheng strain (PRV-S).Blood sampling detects before attacking poison.A, C group is totally 8 sheep 14 days collection serum after using PRV Bartha k61 vaccine immunity, utilize the IDEXX test kit to detect anti-PRV gB antibody (nucleotide sequence of gB gene is shown in SEQ ID NO.1), the A group has 3 antibody positives, and the C group has 4 antibody positives.Attack in malicious rear 9 days, the contrast sheep is morbidity and death all, and HeN1 attacks poison group 2 Mortalities (table 1).The morbidity sheep shows as the nervous symptoms such as itch, opisthotonus.
Table 1Bartha k61 vaccine immunity sheep is protected effect to the poison of attacking of different strains
| Group | The sheep number | Vaccine strain | The attack strain | Attack the front antibody positive number of poison | The morbidity number | Death toll | The protection number | |
| A | 4 | Bartha?k61 | HeN1 | 4/4 | 2/4 | 2/4 | 2/4 | |
| |
3 | ? | HeN1 | ? | 3/3 | 3/3 | 0/3 | |
| |
4 | Bartha? | S | 3/4 | 0/4 | 0/4 | 4/4 | |
| |
3 | ? | S | ? | 3/3 | 3/3 | 0/3 |
By gene clone technology with the part gI of PRV HeN1 strain and Us2 gene as homology arm, and green fluorescence protein gene (EGFP) has made up metastasis transplanting physique grain pUCUsAB-EGFP(recombination site as shown in Figure 5 as selection markers, the transfer vector enzyme is cut and is identified such as Fig. 6), with PRV HeN1 genome and transfer vector cotransfection Vero cell, specification sheets according to the calcium phosphate transfection test kit carries out, Cultivation of Vero makes it to form individual layer in the 60mm ware in advance, 3h changes fresh nutrient solution before transfection, with 4 μ g virus genom DNAs and 10 μ g transfer vector pUCUsAB-EGFP mixings, add again 2M CaCl
2, making its final concentration is 0.24M, evenly mixed, with this DNA-CaCl
2Miscellany joins in the new pipe that contains isopyknic 2XHBS again, and room temperature effect 20min behind the mixing joins in the Vero Tissue Culture Dish, and the Vero cell after the transfection is placed 37 ℃, 5%CO
2Continue in the incubator to cultivate, 12h suffers a shock to the cell of transfection with 15% DMSO after the transfection, when the plaque of green fluorescence to be had occurs, spread 1% low melting-point agarose, and the single plaque of picking is after-80 ℃ of freeze thawing once, be inoculated in Vero cell six orifice plates of cultivating into individual layer in advance with 10 times of doubling dilutions, the plaque that continues picking band green fluorescence repeats purifying.Take turns plaque purification and identify that confirmation has obtained to lack the recombinant pseudorabies virus rPRV-gE that the gE gene inserts the EGFP mark simultaneously through 4
--EGFP
+By methods such as plaque test and one step growth mensuration to recombinant pseudorabies virus rPRV-gE
--EGFP
+Growth characteristics study, found that recombinant virus rPRV-gE
--EGFP
+Rate of propagation in cell in vitro is cultivated is compared with parent's poison HeN1 strain does not have significant difference, illustrates that deletion and insertion does not affect recombinant virus in external reproduction speed.Recombinant virus rPRV-gE
--EGFP
+In 12 generations of blind passage on the Vero cell, green fluorescent protein still can stably express, identifies by PCR to detect the external source fragment of insertion and the virus gene sequence of insertion point both sides, shows the recombinant virus rPRV-gE that obtains
--EGFP
+Can genetic stability, this strain (rPRV-gE
--EGFP
+) be deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center, No. 3 in the Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica in address, its culture presevation is numbered: CGMCC NO.6657, preservation date are on October 12nd, 2012.
Embodiment 4rPRV-gE
--EGFP
+The immune efficacy evaluation
22 of 6-8 monthly age sheep, the PRV neutralizing antibody detects negative.Be divided at random six groups, (every part viral level is 10 to 0.2 part of A, B group (4 every group) every intramuscular injection
5.0TCID
50) PRV Bartha k61 vaccine, C, D group (4 every group) every intramuscular injection 0.2 part (every part viral level 10
5.0TCID
50) rPRV-gE
--EGFP
+, E, F group (3 every group) is as attacking the poison contrast.Rear 14 days of immunity, A, C, every sheep intramuscular injection 1mL of E group contain 1000LD
50PRV HeN1 strain, every sheep intramuscular injection 1mL of B, D, F group contains 1000LD
50PRV Shuangcheng strain (PRV-S).Blood sampling detects before attacking poison.A, B, C, D group totally 16 sheep are being used PRV Bartha k61 vaccine and rPRV-gE
--EGFP
+Immunity gathered serum in rear 14 days, utilized the IDEXX test kit to detect anti-PRV gB antibody, and all goat-anti bodies are all positive.Attack in malicious rear 7 days, the contrast sheep is morbidity and death all, and the morbidity sheep shows as the nervous symptoms such as itch, opisthotonus.Other group morbidities and death condition see Table 2, can find out from attacking malicious result, and Bartha k61 immune sheep can not be resisted the attack of new isolated strain HeN1 strain fully, and rPRV-gE
--EGFP
+After the immunity Shuangcheng strain and HeN1 strain are all had good protection effect, so rPRV-gE
--EGFP
+Can be used as the vaccine candidate strain to tackle emerging epidemic situation.
Table 2rPRV-gE
--EGFP
+Immune sheep is protected effect to the poison of attacking of different strains
Claims (10)
1. a strain porcine pseudorabies virus (Porcine Pseudorabies Virus, PRV) virulent strain, called after HeN1 is deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center, and its culture presevation is numbered: CGMCC NO.6656.
2. a strain porcine pseudorabies virus vaccine strain is characterized in that described vaccine strain is on the basis of porcine pseudorabies virus virulent strain claimed in claim 1, by gene engineering method disappearance gE gene and insert that the EGFP mark obtains.
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 the SEQ ID NO:3.
4. porcine pseudorabies virus vaccine strain as claimed in claim 2 or claim 3 is characterized in that described porcine pseudorabies virus vaccine strain, called after rPRV-gE
--EGFP
+, being deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center, its culture presevation is numbered: CGMCC NO.6657.
5. the vaccine composition for the treatment of or preventing porcine pseudorabies is characterized in that: be comprised of the product after the porcine pseudorabies virus virulent strain claimed in claim 1 deactivation and pharmaceutically acceptable adjuvant.
6. the vaccine composition for the treatment of or preventing porcine pseudorabies is characterized in that: be comprised of each described gene-deleted vaccine strain of claim 2-4 and pharmaceutically acceptable adjuvant.
7. the purposes of porcine pseudorabies virus virulent strain claimed in claim 1 in preparation prevention or treatment pseudorabies medicine.
8. porcine pseudorabies virus virulent strain claimed in claim 1 is in preparation diagnosis or detect purposes in the pseudorabies medicine.
9. the purposes of each described gene-deleted vaccine strain of claim 2-4 in preparation prevention or treatment pseudorabies medicine.
10. each described gene-deleted vaccine strain of claim 2-4 is in preparation diagnosis or detect purposes in the pseudorabies medicine.
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