CN102994458B - 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, the gene-deleted vaccine strain rPRV-gE that relate in particular to a strain porcine pseudorabies virus virulent strain HeN1, is obtained by this virulent strain HeN1 disappearance gE gene
--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, except to pig, is all the lethality course of disease to all susceptible animals.To pig main manifestations, be nervous symptoms, salivation, expiratory dyspnea, breeding difficulty, cessation of growth cessation, weightlessness and high mortality.The clinical manifestation of sucking piglets infection pseudoabies is typical case, serious in consistent the most, all occurs nervous symptoms, paralysis, exhaustion death, and mortality ratio is almost up to 100%; Mortality ratio declines gradually with age, and becoming pig is only 2%, does not fall ill, and be stealthy process but generally become pig to infect, and 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; To boar, can cause vaginalitis.This disease has the infectivity of height, once in swinery, find, will be very soon by air with contact through respiratory infectious to other pigs and pig farm, recent research shows that pseudorabies virus 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 is found, still has a certain proportion of pig farm and pig to have wild malicious antibody after gene-deleted vaccine immunity, and the even report that has virus separation.Because the financial loss causing is limited, do not cause enough attention.But in this year, have the large-scale pig farm of a plurality of use conventional vaccine immunity to occur the phenomenon that doubtful PR is popular, main manifestations is that sow produces weak young, stillborn foetus, and nervous symptoms and death etc. appear in piglet.The tissue of censorship is carried out to PCR and identify with viral separated, prove that pig farm exists PRV wild virus infection really.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 PRV genome is larger, about 145kb, we check order by the gE gene by pcr amplification PRV from each pig farm submitted sample, 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 applied this vaccine, and PR has also obtained good control.But present situation is, this vaccine is all crossed according to conventional procedure immunity in PR morbidity pig farm, whether does new epidemic isolates exist antigenic variation to cause immune swine not to be highly resistant to 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 effectively neutralize new isolated viral, and the sheep of immune Bartha k61 can not be resisted the attack of new virus completely.Therefore be badly in need of the new vaccine of PRV safely and effectively of exploitation to tackle newfashioned strain.
Summary of the invention
One of the object of the invention is to provide a strain porcine pseudorabies virus virulent strain.
Two of the object of the invention be to 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 effect for the new epidemic isolates of PRV.
Three of the object of the invention is to provide 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, utilizes PRV gE Auele Specific Primer to carry out PCR evaluation (Fig. 1), by positive brain tissue homogenate liquid carry out centrifugal, to cross leaching filtrate frozen stand-by.Vero cell is cultivated 48h and is formed after individual layer at 37 ℃, the pathological material of disease suspension supernatant that every bottle graft kind 1ml filters, and after absorption 1h, the DMEM nutrient solution changing containing 2% foetal calf serum continues to cultivate at 37 ℃, observes and within 3～4 days, has or not cytopathy to produce (Fig. 2).When 60～70% cells occur that CPE changes, receive poison, with the Auele Specific Primer of PRV gE, carry out pcr amplification and the evaluation of virus particle electron microscopic observation after the strain that has pathology is passed to 2～3 generations.Test shows to utilize the PRV gE Auele Specific Primer big or small fragment of expection that can increase, by after this sequencing fragment with GenBank on the sequence delivered compare, its homology is more than 97%, its sequence results is shown in SEQ ID NO:2.Culture is carried out to electron microscopic observation, can observe virus particle rounded, have hollow and solid two kinds of features, cyst membrane has for having of having without cyst membrane (Fig. 3).According to the above results, prove, this isolated strain is PRV, called after HeN1 strain, Classification And Nomenclature pseudorabies virus, this strain is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, No. 3 in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, address Institute of Microorganism, Academia Sinica, its culture presevation is numbered: CGMCC NO.6656, preservation date is on October 12nd, 2012.After BALB/c mouse inoculation PRV HeN1 strain, there is typical pseudoabies symptom: 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 60h after inoculation.By calculating the LD of PRV HeN1 to BALB/c mouse
50for 237TCID
50.The contrast mouse of injection DMEM nutrient solution is without Novel presentation.SPF weanling pig only occurs that one crosses heat pyrexia reaction after inoculating PRV HeN1 strain, and body temperature reaches 41 ℃, continues 3-5 days, changes in addition without other clinical onset symptoms with cuing open to examine.
Utilize the Immunization test of microneutralization test and sheep to confirm to have antigenic difference between new isolated strain (PRV HeN1 strain) and vaccine strain Bartha k61.The neutralizing antibody level that found that the generation of Bartha k61 Pigs Inoculated is compared generally lower with 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 viral serum dilution 1: 10 left and right.HeN1 Pigs Inoculated just can produce high-caliber neutralizing antibody in 2 weeks after infection, 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, using the part gI of PRV HeN1 strain and Us2 gene as homology arm, and green fluorescence protein gene (EGFP) has built metastasis transplanting physique grain pUCUsAB-EGFP as selection markers, by PRVHeN1 genome and transfer vector cotransfection Vero cell, through 4, take turns plaque purification and obtained and lacked the recombinant pseudorabies virus rPRV-gE that gE gene inserts EGFP mark simultaneously with evaluation confirmation
--EGFP
+.By plaque test and the method such as 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 cultures is compared and be there is no significant difference with parent's poison HeN1 strain in vitro, illustrates that deletion and insertion does not affect recombinant virus reproduction speed in vitro.Recombinant virus rPRV-gE
--EGFP
+in 12 generations of blind passage on Vero cell, green fluorescent protein still can stably express, by PCR, is identified and the external source fragment of insertion and the virus gene sequence of insertion point both sides can be detected, shows obtained recombinant virus rPRV-gE
--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, lack gE gene and insert that EGFP mark obtains.
In a specific embodiment of the present invention, insertion sequence and flanking nucleotide sequence thereof are shown in SEQ ID NO:3.
In a specific embodiment of the present invention, described porcine pseudorabies virus vaccine strain, called after rPRV-gE
--EGFP
+classification And Nomenclature pseudorabies virus, this strain is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, No. 3 in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, address Institute of Microorganism, Academia Sinica, its culture presevation is numbered: CGMCC NO.6657, preservation date is on October 12nd, 2012.。
By to PRV HeN1 strain and rPRV-gE--EGFP
+immune efficacy evaluate discovery, it is high that HeN1 Pigs Inoculated is compared the neutralizing antibody level that Bartha k61 Pigs Inoculated produces; RPRV-gE
--EGFP
+after immunity, Shuangcheng strain and HeN1 strain are all had to good protection effect, and Bartha k61 immune sheep can not be resisted the attack of new isolated strain HeN1 strain completely, so rPRV-gE
--EGFP
+can be used as vaccine candidate strain to tackle emerging epidemic situation.
Therefore, further, the vaccine composition that the invention allows for a kind for the treatment of or prevention porcine pseudorabies, is characterized in that: the product after described porcine pseudorabies virus virulent strain deactivation or the strain of porcine pseudorabies virus gene-deleted vaccine and pharmaceutically acceptable adjuvant, consist of.
Further, the invention allows for the purposes of described porcine pseudorabies virus virulent strain in preparation prevention or treatment pseudorabies medicine.And
Described porcine pseudorabies virus virulent strain is in preparation diagnosis or detect the purposes in pseudorabies medicine.And the purposes of described gene-deleted vaccine strain in preparation prevention or treatment pseudorabies medicine.And
Described gene-deleted vaccine strain is in preparation diagnosis or detect the purposes in 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 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 efficiency is high, be beneficial to the advantages such as differential diagnosis.
Accompanying drawing explanation
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 induction neutralizing antibody level comparisons;
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
+recombination site schematic diagram;
The enzyme of Fig. 6 pUCUsAB-EGFP is cut evaluation.
M:DL15000; 1.BamH I digested plasmid pUCUsAB-EGFP
Embodiment
Below by experiment, also the present invention will be further described in conjunction with the embodiments, it should be understood that these embodiment, only for the object of illustration, never limit the scope of the invention.Those of ordinary skills understand, and in the spirit and scope that limit, can carry out many changes to it in the claims in the present invention, revise, and even 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, utilizes PRV gE Auele Specific Primer to carry out PCR evaluation (Fig. 1), by positive brain tissue homogenate liquid carry out centrifugal, to cross leaching filtrate frozen stand-by.Vero cell is cultivated 48h and is formed after individual layer at 37 ℃, the pathological material of disease suspension supernatant that every bottle graft kind 1ml filters, and after absorption 1h, the DMEM nutrient solution changing containing 2% foetal calf serum continues to cultivate at 37 ℃, observes and within 3～4 days, has or not cytopathy to produce (Fig. 2).When 60～70% cells occur that CPE changes, receive poison, with the Auele Specific Primer of PRV gE, carry out pcr amplification and the evaluation of virus particle electron microscopic observation after the strain that has pathology is passed to 2～3 generations.Test shows to utilize the PRV gE Auele Specific Primer big or small fragment of expection that can increase, by after this sequencing fragment with GenBank on the sequence delivered compare, its homology is more than 97%, its sequence results is shown in SEQ ID NO:2.Culture is carried out to electron microscopic observation, can observe virus particle rounded, have hollow and solid two kinds of features, cyst membrane has for having of having without cyst membrane (Fig. 3).According to the above results, prove, this isolated strain is PRV, called after HeN1 strain, Classification And Nomenclature pseudorabies virus, this strain is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, No. 3 in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, address Institute of Microorganism, Academia Sinica, its culture presevation is numbered: CGMCC NO.6656, preservation date is on October 12nd, 2012.After BALB/c mouse inoculation PRV HeN1 strain, there is typical pseudoabies symptom: 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 60h after inoculation.By calculating the LD of PRV HeN1 to BALB/c mouse
50for 237TCID
50.The contrast mouse of injection DMEM nutrient solution is without Novel presentation.SPF weanling pig only occurs that one crosses heat pyrexia reaction after inoculating PRV HeN1 strain, and body temperature reaches 41 ℃, continues 3-5 days, changes in addition without other clinical onset symptoms with cuing open to examine.
Between embodiment 2PRV HeN1 strain and vaccine strain Bartha k61, there is antigenic difference
1) utilize the Immunization test of microneutralization test and sheep to confirm to have antigenic difference between new isolated strain (PRV HeN1 strain) and vaccine strain Bartha k61.Neutralization test working method: the SPF pig of 12 40 ages in days, wherein 5 intramuscular injection 1mL are containing 10
7.0tCID
50the PRV HeN1 strain of deactivation, 5 intramuscular injection 1mL are containing 10
7.0tCID
50bartha k61 strain, in contrast, the separation of serum of taking a blood sample weekly before inoculation and after inoculating, cuts open and kills another 2 injection DMEM nutrient solutions for 5 weeks after inoculation.Adopt fixed virus diluted blood heat-clearing method mensuration vaccine Bartha k61 Pigs Inoculated serum and HeN1 Pigs Inoculated serum to tire to the neutralization of two strain virus (Bartha k61 and HeN1).Virus quantity is 100TCID
50/ hole, serum is that Bartha k61 and HeN1 inoculate respectively after SPF pig in different time points collection, 56 ℃ of deactivation 30min are standby.The neutralizing antibody level that found that the generation of Bartha k61 Pigs Inoculated is compared generally lower with 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 viral serum dilution 1: 10 left and right (Fig. 4 A).HeN1 Pigs Inoculated just can produce high-caliber neutralizing antibody in 2 weeks after infection, 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, PRV neutralizing antibody detects negative.Be divided at random four groups, A, (every part viral level is 10 to 0.2 part of every intramuscular injection of C group (4 every group)
5.0tCID
50) PRV Bartha k61 vaccine, B, D group (3 every group) are as attacking poison contrast.Latter 14 days of immunity, every sheep intramuscular injection 1mL is containing 1000LD for A, B group
50pRV HeN1 strain, every sheep intramuscular injection 1mL is containing 1000LD for C, D group
50pRV Shuangcheng strain (PRV-S).Before attacking poison, blood sampling detects.Totally 8 sheep of A, C group gathers serum for 14 days after using PRV Bartha k61 vaccine immunity, utilize IDEXX test kit to detect anti-PRV gB antibody (nucleotide sequence of gB gene is as shown in SEQ ID NO.1), A group has 3 antibody positives, and C group has 4 antibody positives.Attack in malicious latter 9 days, contrast sheep is morbidity death all, and HeN1 attacks poison group 2 Mortalities (table 1).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 | Sheep number | Vaccine strain | Attack strain | Attack the front antibody positive number of poison | Morbidity number | Death toll | 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, using the part gI of PRV HeN1 strain and Us2 gene as homology arm, and green fluorescence protein gene (EGFP) has built metastasis transplanting physique grain pUCUsAB-EGFP(recombination site as shown in Figure 5 as selection markers, transfer vector enzyme is cut and is identified as Fig. 6), by PRV HeN1 genome and transfer vector cotransfection Vero cell, according to the specification sheets of calcium phosphate transfection test kit, carry out, in 60mm ware, Cultivation of Vero makes it to form individual layer in advance, before transfection, 3h changes fresh nutrient solution, 4 μ g virus genom DNAs and 10 μ g transfer vector pUCUsAB-EGFP are mixed, add again 2M CaCl
2, making its final concentration is 0.24M, is mixed evenly, by this DNA-CaCl
2miscellany joins in the new pipe that contains isopyknic 2XHBS again, mixes rear room temperature effect 20min, joins in Vero Tissue Culture Dish, and the Vero cell after transfection is placed in to 37 ℃, 5%CO
2in incubator, continue to cultivate, after transfection, 12h suffers a shock to the cell of transfection with 15% DMSO, when the plaque that has green fluorescence occurs, spread 1% low melting-point agarose, and the single plaque of picking is after-80 ℃ of freeze thawing once, with 10 times of doubling dilutions, be inoculated in Vero cell six orifice plates of cultivating into individual layer in advance, the plaque that continues picking band green fluorescence repeats purifying.Through 4, taking turns plaque purification has obtained and has lacked the recombinant pseudorabies virus rPRV-gE that gE gene inserts EGFP mark simultaneously with evaluation confirmation
--EGFP
+.By plaque test and the method such as 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 cultures is compared and be there is no significant difference with parent's poison HeN1 strain in vitro, illustrates that deletion and insertion does not affect recombinant virus reproduction speed in vitro.Recombinant virus rPRV-gE
--EGFP
+in 12 generations of blind passage on Vero cell, green fluorescent protein still can stably express, by PCR, is identified and the external source fragment of insertion and the virus gene sequence of insertion point both sides can be detected, shows obtained recombinant virus rPRV-gE
--EGFP
+can genetic stability, this strain (rPRV-gE
--EGFP
+) be deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, No. 3 in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, address Institute of Microorganism, Academia Sinica, its culture presevation is numbered: CGMCC NO.6657, preservation date is on October 12nd, 2012.
Embodiment 4rPRV-gE
--EGFP
+immune efficacy evaluation
22 of 6-8 monthly age sheep, PRV neutralizing antibody detects negative.Be divided at random six groups, A, (every part viral level is 10 to 0.2 part of every intramuscular injection of B group (4 every group)
5.0tCID
50) PRV Bartha k61 vaccine, C, 0.2 part of every intramuscular injection of D group (4 every group) (every part viral level 10
5.0tCID
50) rPRV-gE
--EGFP
+, E, F group (3 every group) are as attacking poison contrast.Latter 14 days of immunity, A, C, every sheep intramuscular injection 1mL of E group are containing 1000LD
50pRV HeN1 strain, every sheep intramuscular injection 1mL is containing 1000LD for B, D, F group
50pRV Shuangcheng strain (PRV-S).Before attacking poison, blood sampling detects.A, B, C, D group totally 16 sheep are being used PRV Bartha k61 vaccine and rPRV-gE
--EGFP
+immunity gathers serum in latter 14 days, utilizes IDEXX test kit to detect anti-PRV gB antibody, and all goat-anti bodies are all positive.Attack in malicious latter 7 days, contrast sheep is morbidity death all, and morbidity sheep shows as the nervous symptoms such as itch, opisthotonus.Other group morbidities and death condition, in 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 completely, and rPRV-gE
--EGFP
+after immunity, Shuangcheng strain and HeN1 strain are all had to good protection effect, so rPRV-gE
--EGFP
+can be used as 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's 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, is lacked gE gene and is inserted that 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 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's common micro-organisms center, its culture presevation is numbered: CGMCC NO.6657.
5. a vaccine composition for treatment or prevention porcine pseudorabies, is characterized in that: the product after porcine pseudorabies virus virulent strain claimed in claim 1 deactivation and pharmaceutically acceptable adjuvant, consist of.
6. a vaccine composition for treatment or prevention porcine pseudorabies, is characterized in that: porcine pseudorabies virus vaccine strain and pharmaceutically acceptable adjuvant described in claim 2-4 any one, consist of.
7. porcine pseudorabies virus virulent strain claimed in claim 1 prevents or treats the purposes in pseudorabies medicine in preparation.
8. the purposes in pseudorabies medicine is diagnosed or detected to porcine pseudorabies virus virulent strain claimed in claim 1 in preparation.
9. the porcine pseudorabies virus vaccine strain described in claim 2-4 any one prevents or treats the purposes in pseudorabies medicine in preparation.
10. the purposes in pseudorabies medicine is diagnosed or detected to the porcine pseudorabies virus vaccine strain described in claim 2-4 any one in preparation.
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| CN111172321B (en) * | 2020-01-02 | 2021-04-23 | 中国检验检疫科学研究院 | Fluorescent PCR detection kit for identifying African swine fever infection and immunity |
| CN112626038B (en) * | 2021-02-04 | 2023-01-10 | 福建省农业科学院畜牧兽医研究所 | Construction of pseudorabies virus FB strain gE/gI gene deletion strain and application thereof as vaccine |
| CN113862230B (en) * | 2021-09-30 | 2023-08-08 | 中牧实业股份有限公司 | Porcine pseudorabies virus gene deletion strain, vaccine composition, preparation method and application thereof |
| CN116790509B (en) * | 2023-06-28 | 2024-02-02 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Monoclonal hybridoma cell strain secreting anti-porcine pseudorabies virus gB protein antibody and application thereof |
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| CN100354414C (en) * | 2005-09-29 | 2007-12-12 | 华中农业大学 | Pseudo-rabies gE/gI-gene loss poison strain, killed vaccine containing it and use |
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