Porcine pseudorabies virus strain and application thereof
阅读说明：本技术 猪伪狂犬病变异病毒毒株及其应用 (Porcine pseudorabies virus strain and application thereof ) 是由 陈义锋 王闯 林峰 陈智韡 杨宏富 刘阳阳 刘志昌 于 2021-06-21 设计创作，主要内容包括：本申请提供了猪伪狂犬病变异病毒毒株及其应用,属于病毒分子生物学和基因工程领域技术领域。通过在某猪场PRV病发体中分离的病毒,经PCR鉴定、测序和毒力测试,确认属于PRV强毒,命名为PRV SD2020株,以该毒株为亲本,利用基因编辑技术(CRISPR/CAS9系统)敲除其gI-gE基因和TK基因,构建了双基因所失株PRV SD2020ΔgI-gE和三基因缺失株PRV SD2020ΔgI-gE-TK。将这两个基因缺失株制备灭活疫苗和活疫苗,免疫攻毒保护试验结果表明所制备的疫苗具有良好的好安全性和免疫原性,表明该两个毒株适用于伪狂犬疾病防控和净化。(The application provides a porcine pseudorabies virus strain and application thereof, belonging to the technical field of virus molecular biology and genetic engineering. The virus separated from PRV disease in a pig farm is identified to be PRV virulent virus through PCR identification, sequencing and virulence test, and is named as PRV SD2020 strain, the gI-gE gene and TK gene of the virus are knocked out by using a gene editing technology (CRISPR/CAS9 system) by taking the strain as a parent, and a double-gene-deleted strain PRV SD2020 delta gI-gE and a triple-gene-deleted strain PRV SD2020 delta gI-gE-TK are constructed. The two gene deletion strains are used for preparing inactivated vaccines and live vaccines, and immune challenge protection test results show that the prepared vaccines have good safety and immunogenicity, and show that the two strains are suitable for preventing, controlling and purifying pseudorabies diseases.)
1. The porcine pseudorabies virus strain is characterized in that the strain is PRV SD2020 strain, and the gB, gC, gD, gI, gE and TK gene sequences of the strain are respectively shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO. 6.
2. The strain of claim 1, wherein the strain is PRV SD2020 strain gene deletion gI/gE double gene fragment.
3. The strain of claim 1, wherein the strain is PRV SD2020 strain gene deletion gI/gE/TK three gene fragment.
4. The strain of claim 2, wherein the strain is PRV SD2020 delta gI/gE strain, and the preservation number of the PRV SD2020 delta gI/gE strain is CCTCC NO: V202150.
5. The strain of claim 2, wherein the strain is PRV SD2020 delta gI/gE/TK strain, and the preservation number of the PRV SD2020 delta gI/gE/TK strain is CCTCC NO: V202149.
6. Porcine pseudorabies vaccine, characterized in that a strain according to any of claims 2 to 5 is used as antigen.
Technical Field
The application belongs to the technical field of virus molecular biology and genetic engineering, and more particularly relates to a porcine pseudorabies variant virus strain and application thereof.
Background
Pseudorabies is an important infectious disease of pigs caused by pseudorabies virus (PRV) and characterized by pruritus, fever, encephalomyelitis, neurosis and the like, is one of the most serious infectious diseases endangering the pig industry at present, is widely prevalent worldwide, and causes huge economic loss to the world pig industry every year. Since the virus can infect various domestic animals (pig, cow, sheep, goat, dog, rabbit, horse, etc.) and wild animals (fox, wolf, rat, bat, deer, bear, wild boar, etc.) at the same time, the disease is a typical natural epidemic disease. Because the pigs are the only natural carriers, some of the large pigs are infected with the latent virus for a long time and are discharged with the latent virus intermittently, so that the pigs become the infection sources of the swinery. The disease is mainly transmitted by contact, can be transmitted by air, water and polluted media, can be transmitted by placenta and semen, and can be transmitted by wild or domestic mammals such as mice, cats, dogs and the like. The disease was first reported in 1902, and was worldwide deposited after the 90 s, especially in areas with dense herds. The use of the Bartha-K61 vaccine strain was effective in controlling the viral transmission of RPV, but did not block infection. Most european countries, the united states and new zealand have cleared classical PRV infection in farm herds by immune decontamination.
PRV belongs to the order of herpesviridae, the family of herpesviridae, the subfamily of alphaherpesviridae, the genus varicella-zoster, is a double-stranded DNA virus, has a genome of about 150kb in overall length and can encode 7-100 genes, and virulence related genes include TK, glycoproteins gC, gI, gE, gG and the like. PRV only has one serotype at present, but the biological characteristics and the toxicity of different strains are different, PRV variant strains newly appeared in northeast, east and south China since 2011 cause serious economic loss to pig farms immunized with PRV classical vaccines, and the PRV variant strains are expressed as sow abortion, stillborn fetuses and mummy fetuses, central nervous symptoms, dyspnea and high mortality of piglets, respiratory diseases of growing and fattening pigs and occasionally death, PRV wild virus infection is diagnosed in laboratories, and epidemiological investigation results show that a plurality of site insertions or mutations occur in virulence genes gI, gE and the like of the PRV variant strains, and a plurality of site insertions, deletion mutations occur in main antigen genes gB, gC and gD, so that neutralization antibodies are mutated; the immune challenge protection test research shows that the traditional Bartha K61 vaccine can provide good immune protection for PRV classical strains (such as Ea strains) and the like, but can only provide partial protection for the variant strains of the epidemic viruses, which is consistent with the phenomenon of RPV infection frequently occurring in pigs immunized with the Bartha K61 vaccine clinically.
Therefore, vaccine candidate strains based on the variant strains are constructed, and the PRV variant strain invasion can be effectively controlled only by gradually establishing a purification swinery through enhancing immunity, differentiating infection and immunization strategy (DIVA), and finally purifying PRV infection.
Disclosure of Invention
The object of the present application includes, for example, providing a porcine pseudorabies variant virus strain and its use in the preparation of a porcine pseudorabies vaccine, to ameliorate at least some of the above problems.
The embodiment of the application can be realized as follows:
in one aspect, the embodiments of the present application provide a porcine pseudorabies variant virus strain, which is named as PRV SD2020 strain, and the gB, gC, gD, gI, gE and TK gene sequences of the PRV SD2020 strain are respectively shown as SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5 and SEQ ID No. 6.
On the other hand, PRV SD2020 strain is used as a base to be modified, and the gI/gE double gene segment on the PRV SD2020 strain is deleted, so that a new porcine pseudorabies virus isovirus genetic engineering strain is constructed.
In some embodiments, the deletion gI/gE fragment of PRV SD2020 strain is 2907bp, and a new porcine pseudorabies virus genetically engineered strain is constructed and named as PRV SD2020 delta gI/gE strain, and the preservation information of PRV SD2020 delta gI/gE strain is as follows:
the preservation number is: NO. V202150
And (3) classification and naming: pseudorabies virus PRV SD2020 delta gI/gE
Preservation day: 2021/5/25
The preservation unit: china center for type culture Collection (CCTCC for short)
The address of the depository: wuhan university of Wuhan, China
On the other hand, PRV SD2020 strain is used as a base to be modified, three gene segments of gI/gE/TK are deleted, and a new porcine pseudorabies virus isovirus genetic engineering strain is constructed.
In some embodiments, the deletion gI/gE fragment of PRV SD2020 strain is 2907bp, and the deletion TK fragment is 891bp, so as to construct a new porcine pseudorabies virus heterologous gene engineering strain named PRV SD2020 delta gI/gE/TK strain, wherein the preservation information of the PRV SD2020 delta gI/gE/TK strain is as follows:
the preservation number is: NO: V202149
And (3) classification and naming: pseudorabies virus PRV SD2020 delta gI/gE/TK
Preservation day: 2021/5/25
The preservation unit: china center for type culture Collection (CCTCC for short)
The address of the depository: wuhan university of Wuhan, China
On the other hand, the embodiment of the application also provides the application of the porcine pseudorabies variant virus strain in preparing the porcine pseudorabies vaccine, and the immune PRV SD2020 strain is used as an antigen, wherein the gI/gE segment is deleted or the genetic engineering strain of the gI/gE/TK segment is deleted.
The PRV virus is confirmed to belong to PRV through virulence test and PCR identification based on the virus separated from PRV disease in a pig farm, is named as PRV SD2020 strain, carries out gene sequencing and gene deletion strain construction aiming at the strain, and researches the vaccine performance of the constructed gene deletion strain.
The PRV inactivated vaccine prepared by using the genetic engineering strain of the PRV SD2020 deleted gI/gE segment or the gI/gE/TK segment has good immunogenicity and safety.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings that are required to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present application and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained from the drawings without inventive effort.
FIG. 1 is a VERO pathogram, wherein FIG. 1-a is a micrograph 24h after inoculation of PRV SD2020 strain, and FIG. 1-b is a micrograph 48h after inoculation of PRV SD2020 strain.
FIG. 2 shows the results of PCR detection before and after deletion of gI/gE in example 2.
FIG. 3 is a PCR electrophoretic detection map of PRV SD2020 strain after deletion of gI/gE in example 2.
FIG. 4 is a graph showing the expression of the gE gene identified by IFA after deletion of gI/gE from PRV SD2020 strain in example 2.
FIG. 5 is a PCR electrophoretic test chart before and after deletion of the PRV SD2020 strain gI/gE/TK in example 3.
FIG. 6 is a sequencing map of the PRV SD2020 strain in example 3 after deletion of gI/gE/TK.
FIG. 7 is a graph of daily body temperature monitoring after inoculation of each of the groups of reagents of example 6.
FIG. 8 is a graph showing the daily body temperature monitoring after various groups of patients in example 7
FIG. 9 is a graph showing the detection of the amount of gE antibody in blood before immunization, 4 weeks after immunization, and 2 weeks after challenge in each group of example 7.
FIG. 10 is a graph showing the detection of the content of neutralizing antibodies in blood before immunization, 4 weeks after immunization, and 2 weeks after challenge in each group in example 7.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application, and it is obvious that the described embodiments are some embodiments of the present application, but not all embodiments. The components of the embodiments of the present application, generally described and illustrated in the figures herein, can be arranged and designed in a wide variety of different configurations.
It should be noted that the features of the embodiments of the present application may be combined with each other without conflict.
Example 1: separation and identification of pseudorabies epidemic variant virulent strain
The detection of the porcine pseudorabies wild virus antigen is carried out on aborted fetuses from a certain pig farm in Shandong, the result is gE antigen positive, the supernatant of the positive material is inoculated with VERO cells for virus separation, a strain is obtained by separation, gene identification and specificity identification are carried out on the strain, the strain can be amplified to PRV corresponding genes (NO. 1-6), and the strain can be neutralized by pseudorabies virus specific positive serum, is identified as PRV virus and is named as PRV SD2020 strain.
The VERO cytopathic effect of the PRV SD2020 strain is shown in FIG. 1. FIG. 1-a and FIG. 1-b show the cases 24h and 48h after inoculation of PRV SD2020 strain, respectively, and it can be seen from FIG. 1 that PRV SD2020 strain specifically causes the typical lesion on regularity (CPE) of the infected cells.
The gB, gC, gD, gI, gE and TK gene sequences of PRV SD2020 strain are shown as Seq NO 1-6 in sequence.
The supernatant of PRV SD 2020F 7 cell culture was collected at 2mL × 106TCID50And respectively dripping 5 healthy pigs (gE antigen negative and gB antibody negative) of 55-60 days old into the solution per mL and 2mLPBS solution through the nose, continuously observing the clinical performance of the test pigs for 2 weeks, and monitoring the temperature change of the test pigs. The test result shows that PRV SD2020 strain is 2mL multiplied by 106TCID50the/mL nasal drip inoculation test pig can cause 5/5 disease of the test pig at the age of 55-60 days, shows obvious clinical symptoms, has high fever for more than 3 days at the temperature of more than 40.5 ℃, and has the symptoms of depression, appetite reduction, dyspnea or nerve symptoms and 1 death.
TABLE 1 PRV SD2020 Strain virulence assay
Group of
Strain
Dosage form
Number of animal heads
Route of attacking toxin
Onset of disease
Death was caused by death
Incidence of disease
Toxin counteracting group
PRV SD2020
2ml×106TCID50/ml
5
Nose drop
5
1
100％
Control group
PBS
2mL
5
Nose drop
0
0
0
Example 2: construction of porcine pseudorabies virus epidemic variation virulent strain genetic engineering strain (PRV SD2020 delta gI/gE strain)
The CRISPR/CAS9 technology is adopted to knock out gI/gE dual virulence genes on PRV, and the specific process is summarized as follows:
1. sgRNA sequence design and recombinant vector construction
The sgRNA sequences of gI and gE genes are designed and synthesized by referring to PRV whole genome sequences published in GenBank, the sgRNA sequences of the gI and gE genes are shown in Table 2, and the sgRNA sequences are inserted into BbsI enzyme cutting sites of a psgRNA vector to construct targeting plasmids.
Table 2 design gI/gE sgRNA sequences
The PCR amplification product was identified by electrophoresis, and the results of electrophoresis are shown in FIG. 2.
After obtaining PRV SD2020 delta gI/gE strain, obtaining gene sequence of upstream and downstream of gene deletion region through genome extraction and PCR amplification, as shown in SEQ ID NO.7, and then confirming as gI/gE gene deletion through sequencing and multiple comparison, the sequencing result is shown in figure 3, which shows that the 637 site is a demarcation point and 2907bp is deleted at the 637 site.
(2) Indirect immunofluorescence assay
BHK21 cells were digested at 1:5, passed through a six-well plate containing coverslips and added dropwise at 2 ml/well and placed at 37 ℃ in 5% CO2Culturing in a constant temperature incubator, inoculating PRV SD delta gI/gE strain at 0.01MOI when the strain grows to 80% fusion, and continuously culturing for 2 days; cells were collected and fixed for staining (procedure: discard culture medium supernatant, add cold PBS 1ml rinse 1 time gently, discard wash solution, fix cells with 80% cold acetone at room temperature for 6-8 minutes, then wash with cold PBS 3 times 5 minutes each, incubate with PBS containing 2% BSA for 30 minutes, incubate PRV gE monoclonal antibody, dilute primary antibody with PBS containing 2% BSA, add 100 μ l diluted primary antibody gently, incubate at room temperature for 60 minutes, wash with PBS 3 times 5 minutes each, dilute secondary antibody with PBS containing 2% BSA, add 100 μ l diluted secondary antibody gently, incubate at room temperature for 60 minutes, wash with PBS for minutes, wash with PBS 3 times 5 minutes each). The photographs were taken by observation under a fluorescent microscope, and as shown in FIG. 4, specific green fluorescence was detected by the gE monoclonal antibody after the cells were infected with the parent strain PRV E6. Whereas the PRV SD 2020. delta. gI/gE strain was used for a gE monoclonal antibody after infecting cellsThe monoclonal antibody did not detect specific fluorescence. Thus, PRV SD 2020. DELTA.gI/gE strain infected cells were deficient in gE protein expression.
Example 3: construction of porcine pseudorabies virus epidemic variation virulent strain genetic engineering strain (PRV SD2020 delta gI/gE/TK strain)
Three virulence genes gI/gE/TK on PRV are knocked out by using CRISPR/CAS9 technology, and the specific process is summarized as follows:
similarly, referring to the process of example 2, a TK gene target site (the sequence of the TK gene sgRNA is shown in Table 3) is further designed, a recombinant vector is constructed, transfection is carried out, homologous recombination is carried out on a plasmid and a virus genome, then a PRV SD2020 delta gI/gE/TK strain with gI, gE and TK three genes deleted is obtained through plaque purification, and the strain is identified.
TABLE 3 SgRNA sequence of TK gene
The strain PCR identification result is shown in FIG. 5, the sequenced TK sequence is shown in SEQ ID NO.8, and the peak map file
2. Transfection
The targeting plasmid is transfected according to standard transfection procedures (e.g., LTX or transfectamine2000 Protocol) and PRV E6 strain is infected at 0.01MOI 24 hours after transfection. Continuously observing for 3-5 days, and collecting cell culture when obvious cytopathic effect appears.
3. Plaque purification
(1) The VREO cells are evenly digested and spread on a 6-hole cell culture plate, the culture solution is sucked off after the cells grow into a monolayer, and the collected cell cultures are respectively subjected to 10-3、10-4Diluting, adding virus diluent into the culture plate, adsorbing at 37 ℃ for 2 hours, and then discarding residual liquid;
(2) mixing 2% low melting point agarose solution (stored at 42 deg.C) and 2 × cell maintenance solution (stored at 42 deg.C) at ratio of 1:1, adding into two culture wells at 2 ml/well standard, cooling and solidifying to obtain covering layer;
(3) inverting the culture plate, and placing at 37 ℃ with 5% CO2Culturing in a constant-temperature incubator;
(4) and continuously observing for 3-4 days, and when obvious plaques appear, respectively collecting the plaques (using a 1ml blue gun head, cutting off the tip part in advance, and then directly sucking the plaques by using a pipette), and repeating the steps for 3 times to obtain the single clone strain with the gI/gE gene deleted.
4. Identification of PRV SD2020 delta gI/gE strain
(1) gI/gE gene PCR identification
The cell culture under the picked plaque was inoculated into a culture plate that had grown into a monolayer of BHK-21 cells, and the culture was collected when significant lesions appeared. Mu.l of the culture was taken out and diluted to 200. mu.l, and DNA was extracted for PCR identification.
The identification process is roughly as follows:
PCR identification primer sequence:
F:5’-GCG TGT GCG TCT ACA TCT TCT-3’；
R:5’-GGT ATT TAA GCG GGG CGG GCA T-3’；
PCR reaction (50. mu.l): 2 XGC buffer 25. mu.l, dNTP mix 5. mu.l, upstream and downstream primers 2. mu.l each, Prime star 0.5. mu.l, cDNA template 2. mu.l, H2O 13.5.5. mu.l.
The reaction condition is that the pre-denaturation is carried out for 1 minute at 94 ℃; denaturation at 98 ℃ for 10 seconds, annealing at 56 ℃ for 15 seconds, and extension at 72 ℃ for 3 seconds for 35 cycles; extension at 72 ℃ for 5 minutes.
As shown in fig. 6. As can be seen from the figure, the TK gene of PRV SD 2020. delta. gI/gE/TK strain is 891bp deleted between the 70 th and 71 th base genes.
Example 4: preparation of inactivated vaccine of porcine pseudorabies virus gI/gE gene deletion strain (PRV SD2020 delta gI/gE strain)
(1) Taking PRV SD2020 delta gI/gE virus liquid qualified in inspection (the virus content is 10)8.5TCID50/ml) adding diethyl imine (BEI) with final concentration of 0.05% (V/V), mixing, inactivating in 30 deg.C constant temperature shaking table for 30 hr, adding 2% sodium thiosulfate solution, stopping in 37 deg.C shaking table for 2 hr, and standing at 2-8 deg.CAnd (5) storing.
(2) And taking out the antigen and the adjuvant, heating to 30 ℃, then inputting 1 part of the adjuvant into an emulsifying tank, stirring at a proper rotating speed (proper for forming vortex), slowly inputting 1 part of inactivated antigen with the same mass, and continuously stirring for 30-40 minutes at 350r/mim under the constant temperature condition of 30 ℃ to obtain the milky white or light pink oil emulsion inactivated vaccine.
(3) And (4) quantitatively subpackaging the vaccine, covering and sealing, sticking a label, and storing at 2-8 ℃.
Example 5: preparation of porcine pseudorabies virus gI/gE/TK gene deletion strain (PRV SD2020 delta gI/gE/TK strain) live vaccine
Uniformly mixing the qualified PRV SD2020 delta gI/gE/TK virus liquid with a certain volume (1: 1-1: 1.2) of freeze-drying protective agent (such as gelatin, sucrose and the like), subpackaging and freeze-drying to obtain the live vaccine (with the virus content of 10) containing the PRV SD2020 delta gI/gE/TK strain6TCID50)。
Example 6: vaccine safety test
Selecting 15 healthy susceptible piglets of 21-28 days old, dividing into 3 groups, including SD1 group, SD2 group and control group, respectively, and injecting PRV SD2020 delta gI/gE strain inactivated vaccine (content before inactivation is 10) into neck muscle of test pig of SD1 group8。5TCID504.0ml, SD2 group test pigs injected with PRV SD2020 delta gI/gE/TK strain live vaccine (10)7。0TCID50/ml)1.0ml, and the neck of the control group was injected with PBS diluent 1.0ml, continuously observed for 14 days, and continuously measured for 7 days after inoculation, and observed whether local or systemic adverse reaction was caused by the vaccine. The results of the various group temperature monitoring are shown in fig. 7.
TABLE 4 safety test
Test grouping
Vaccine
Number of immunizations
Immunological pathways
Immunization dose
SD1 group
PRV SD2020 delta gI/gE strain inactivated vaccine
5 heads
Neck intramuscular injection
2×108.5TCID50
SD2 group
PRV SD2020 delta gI/gE/TK strain live vaccine
5 heads
Neck intramuscular injection
2×107TCID50
Control group
PBS
5 heads
Neck intramuscular injection
/
Observations showed that all immunized pigs in groups SD1 and SD2 survived with good mental status, normal behavioral activities, and normal respiration, feeding, and defecation.
As can be seen from fig. 7, all the immunized pigs in SD1 and SD2 had no abnormality in body temperature compared with those before injection, and also compared with the blank control group; it was observed that the vaccine injection site (neck muscle) was well absorbed and there were no abnormalities such as red swelling, abscess, scab, etc. The PRV SD2020 delta gI/gE strain inactivated vaccine and the PRV SD2020 delta gI/gE/TK strain live vaccine have good safety.
Example 7: immunogenicity test of PRV SD2020 delta gI/gE strain inactivated vaccine and PRV SD2020 delta gI/gE/TK strain live vaccine
Selecting 15 PRV gB and gE antibody negative healthy piglets of 21-28 days old, randomly dividing into 3 groups, 5 piglets in each group, namely an SD1 group, an SD2 group and an offensive-toxin control group; SD1 group cervical intramuscular injection of vaccine 2ml (10) of example 18.5TCID50Perml), SD2 group cervical intramuscular injection of 1ml (10) of vaccine of example 16TCID50And/ml), the control group for counteracting toxic substances is not injected, and after immunization, the group is fed freely and kept separately. Challenge test was carried out 4 weeks later, and SD1, SD2 and control groups were inoculated with 2ml (10 ml) by nasal drip6TCID50/ml) PRV SD2020 strain F7 generation virus liquid, clinical performance of the test pig was observed every day, and body temperature of the test pig was recorded for 14 consecutive days. Test pig blood was collected before immunization, 28 days after immunization (before challenge) and 14 days after challenge, respectively, and serum was separated and tested for gE antibodies and neutralizing antibodies.
TABLE 5 immunogenicity assays
After the toxicity is attacked, the experimental pigs in the SD2 group are normal in mental state and behavior activity, normal in ingestion and defecation, and one example of the body temperature is increased (more than 40.5 ℃) in a transient way but not more than 3 times in temperature and is quickly reduced to the normal level; the SD1 group of experimental pigs have normal mental state, normal behavior and normal ingestion and defecation conditions, the body temperature of 1 experimental pig is increased by more than 40.5 ℃, 3 times of temperature are reached, the mental depression is caused, and the others are normal; and the control group of the offensive toxin starts on the 3 rd day after offensive toxin, all the pigs of the offensive toxin control test have high fever of more than 40.5 ℃ for more than 3 days (see figure 8), and the pigs have the disadvantages of depression, reduced appetite, incapability of touching with the head, dyspnea in 2 tests, and death due to the typical nervous symptoms of one end.
The immune challenge protection test result shows that test pigs in the SD2 group obtain 5/5 protection, test pigs in the SD1 group obtain 4/5 protection, and test pigs in the challenge control group obtain 5/5 disease. The detection results of the gE antibodies of the groups are shown in FIG. 9, and it can be seen from FIG. 9 that the test pigs of SD2 group did not undergo positive transformation of the gE antibodies, while one test pig of SD1 group underwent positive transformation, and all the test pigs of the challenge control group underwent positive transformation. The detection results of the neutralizing antibodies of the groups are shown in fig. 10, and as can be seen from fig. 10, the neutralizing antibody titers of the SD1 group and the SD2 group are 1: 8-1: 32, and after 2 weeks of challenge, the neutralizing antibody titer of the immune group rapidly reaches 1: 256-1: 1024, which is obviously higher than that of the challenge control group.
In conclusion, the test results show that the inactivated vaccine of the gI/gE double-gene deletion strain and the live vaccine of the gI/gE/TK triple-gene deletion strain of PRV SD2020 have good immunogenicity, so that the immunized pig can be well protected, and the positive conversion of the gE antibody can be effectively blocked, thereby providing a strong support for purifying the pseudorabies.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present application, and not to limit the same; although the present application has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; such modifications and substitutions do not necessarily depart from the spirit and scope of the corresponding technical solutions in the embodiments of the present application.
Sequence listing
<110> Jiangxi Zhengbang science and technology GmbH
<120> porcine pseudorabies virus strain and application thereof
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2745
<212> DNA
<213> porcine Pseudorabies virus (pseudorabiae virus)
<400> 1
atgcccgctg gtggcggtct ttggcgcggg ccccgcgggc atcggcccgg gcaccacggc 60
ggtgctggcc tcggacgtct ttggcctgct ccacaccacg ctgcagctgc gcggggcgcc 120
gtcgcgctag cgctgctgct gctggcgctc gccgcgaccc cgacgtgcgg cgcggcggcc 180
gtgacgcggg ccgcctcggc ctcgcccgcg cccgggacgg gcgccacccc agacggcttc 240
tccgcggagg agtccctcga ggagatcgac ggggccgtct cccccggccc ctcggacgcc 300
cccgacggcg agtacggcga cctggacgcg cgcacggccg tgcgcgcggc cgcgaccgag 360
cgggaccgct tctacgtctg cccgccgccg tccggctcca cggtggtgcg cctggagccc 420
gagcaggcct gccccgagta ctcgcagggg cgcaacttca cggaggggat cgccgtgctc 480
ttcaaggaga acatcgcccc gcacaagttc aaggcccaca tctactacaa gaacgtcatc 540
gtcacgaccg tgtggtccgg gagcacgtac gcggccatca cgaaccgctt cacggaccgc 600
gtgcccgtcc ccgtgcagga gatcacggac gtgatcgacc gccgcggcaa gtgcgtctcc 660
aaggccgagt acgtgcgcaa caaccacaag gtgaccgcct tcgaccgcga cgagaacccc 720
gtcgaggtgg acctgcgccc ctcgcgcctg aacgcgctcg gcacccgcgg ctggcacacc 780
accaacgaca cctacaccaa gatcggcgcc gcgggcttct accacacggg cacctccgtc 840
aactgcatcg tcgaggaggt ggaggcgcgc tccgtgtacc cctacgactc cttcgccctg 900
tccacggggg acatcgtgta catgtccccc ttctacggcc tgcgcgaggg ggcccacggg 960
gagcacatcg gctacgcgcc cgggcgcttc cagcaggtgg agcactacta ccccatcgac 1020
ctggactcgc gcctccgcgc ctccgagagc gtgacgcgca actttctgcg cacgccgcac 1080
ttcacggtgg cctgggactg ggcccccaag acgcggcgcg tgtgcagcct ggccaagtgg 1140
cgcgaggccg aggagatgat ccgcgacgag acgcgcgacg ggtccttccg cttcacgtcg 1200
cgggccctgg gcgcctcctt cgtcagcgac gtcacgcagc tcgacctgca gcgcgtgcac 1260
ctgggcgact gcgtcctccg cgaggcctcg gaggccatcg acgccatcta ccggcggcgc 1320
tacaacaaca cgcacgtgct ggccggcgac aagcccgagg tgtacctcgc ccgcgggggc 1380
ttcgtggtgg ccttccgccc gctgatctcg aacgagctgg cgcagctgta cgcgcgcgag 1440
ctcgagcgcc tcggcctcgc cggcgtcgtg ggccccgcgt cccccgcggc cgcccgtcgg 1500
gcccggcgct cccccggccc ggcggggacg cccgagccgc cggccgtcaa cggcacgggg 1560
cacctgcgca tcaccacggg ctcggccgag tttgcgcgcc tgcagttcac ctacgaccac 1620
atccaggcgc acgtgaacga catgctgagc cgcatcgcgg ccgcctggtg cgagctgcag 1680
aacaaggacc gcaccctgtg gggcgagatg tcgcgcctga accccagcgc cgtggccacg 1740
gccgcgctgg gccagcgcgt ctcggcgcgc atgctcggcg acgtgatggc catctcgcgg 1800
tgcgtggagg tgcgcggcgg cgtgtacgtg cagaactcca tgcgcgtgcc cggcgagcgc 1860
ggcacgtgct acagccgccc gctggtgacc ttcgagcaca acggcacggg cgtgatcgag 1920
ggccagctcg gcgacgacaa cgagctcctc atctcgcgcg acctcatcga gccctgcacc 1980
ggcaaccacc ggcgctactt taagctgggc ggcgggtacg tgtactacga ggactacagc 2040
tacgtgcgca tggtggaggt gcccgagacg atcagcacgc gggtgaccct gaacctgacg 2100
ctgctcgagg accgcgagtt cctgcccctc gaggtgtaca cgcgcgagga gctcgccgac 2160
acgggcctcc tggactacag cgagatccag cgccgcaacc agctgcacgc gctcaagttc 2220
tacgacattg accgcgtggt caaggtggac cacaacgtgg tgctgctgcg cggcatcgcc 2280
aacttcttcc agggcctcgg cgacgtgggc gccgccgtcg gcaaggtggt cctgggcgcc 2340
acgggggccg tgatctcggc cgtcggcggc atggtgtcct tcctgtccaa ccccttcggg 2400
gcgctcgcca tcgggctgct ggtgctggcc ggcctggtcg cggccttcct ggcctaccgg 2460
cacatctcgc gcctgcgccg caaccccatg aaggccctgt accccgtcac gacgaaggcg 2520
ctcaaggagg acggcgtcga agaggacgac gtggacgagg ccaagctgga ccaggcccgg 2580
gacatgatcc ggtacatgtc catcgtgtcg gccctcgagc agcaggagca caaggcgcgc 2640
aagaagaaca gcgggcccgc gctgctggcc agccgcgtcg gggcgatggc cacgcgccgc 2700
cggcactacc agcgcctcga gaacgaggac cccgacgccc cctag 2745
<210> 2
<211> 1464
<212> DNA
<213> porcine Pseudorabies virus (pseudorabiae virus)
<400> 2
atggcctcgc tcgcgcgtgc gatgctcgcg ctgctggcgc tctacacggc ggccatcgcc 60
gcggcgccgt cgtccacgac ggcgctcggc acgacgccca acgggggcgg gggcggcaac 120
agcagcgcgg gcgagctctc gccctcgccg ccctcgacgc ccgagcccgt ctcggggacg 180
acgggggccg cggcctccac gcccgccgcc gtctcgacgc cccgggtccc gccgccctcg 240
gtctcgcgcc ggaagcccca gcggaacggc aacaggacgc gcgtccacgg cgacaaggcc 300
acctcgcacg ggcgcaagcg catcgtgtgc cgcgagcggc tgttctcggc gagggtgggg 360
gacgcggtca gcttcgggtg cgccgtcgtc ccgcgcgccg gggagacctt cgaggtccgc 420
ttctgccgcc gcgggcgctt ccgctcgccc gacgccgacc ccgagtactt tgacgagccc 480
ccgcgcccgg agctcccgcg ggagcggctc ctcttcagct ccgccaacgc ctccctcgcc 540
cacgcggacg cgctcgcctc cgccgtcgtc gtcgagggcg agcgcgcgac cgtcgccaac 600
gtctcgggcg aggtgtccgt gcgcgtggcc gcggcggacg ccgagaccga gggcgtctac 660
acgtggcgcg tgctgtccgc caacggcacc gaggtccgca gcgccaacgt ctcgctcgtc 720
ctgtaccacc agcccgagtt cggcctgagc gcgccgcccg tcctcttcgg cgagcccttc 780
cgggcggtgt gcgtcgtccg cgactactac ccgcggcgca gcgtgcgcct gcgctggttc 840
gcggacgagc acccggtgga cgccgccttc gtgaccaaca gcaccgtggc cgacgagctc 900
gggcgccgca cgcgcgtctc cgtggtgaac gtgacgcgcg cggacgtccc gggcctcgcg 960
gccgcggacg acgcggacgc gctcgcgccg agcctgcgct gcgaggccgt gtggtaccgc 1020
gacagcgtgg cctcgcagcg cttctccgag gccctgcgcc cccacgtcta ccacccggcg 1080
gcggtctcgg tgcgcttcgt cgagggcttc gccgtctgcg acggcctctg cgtgcccccg 1140
gaggcgcgcc tcgcctggtc cgaccacgcc gccgacaccg tctaccacct cggcgcctgc 1200
gccgagcacc ccggcctgct caacgtgcgg agcgcccgcc cgctgtcgga cctcgacggg 1260
cccgtcgact acacctgccg cctcgagggc atgccctcgc agctgcccat cttcgaggac 1320
acgcagcgct acgacgcctc ccccacgtcc gtgagctggc ccgtcgtgac cagcatgatc 1380
accgtcatca ccggcatcgc catcctagcc atcgtgctgg tcatcatggc gacgtgcgtc 1440
tactaccgcc ggtccgcgct gtga 1464
<210> 3
<211> 1209
<212> DNA
<213> porcine Pseudorabies virus (pseudorabiae virus)
<400> 3
atgctgctcg cagcgctatt ggcggcgctg gtcgcccgga cgacgctcgg cgcggacgtg 60
gacgccgtgc ccgcgccgac cttccccccg cccgcgtacc cgtacaccga gtcgtggcag 120
ctgacgctga cgacggtccc ctcgcccttc gtcggccccg cggacgtcta ccacacgcgc 180
ccgctggagg acccgtgcgg ggtggtggcg ctgatctccg acccgcaggt ggaccggctg 240
ctgaacgagg cggtggccca ccggcggccc acgtaccgcg cccacgtggc ctggtaccgc 300
atcgcggacg ggtgcgcgca cctgctgtac tttatcgagt acgccgactg cgaccccagg 360
cagatctttg ggcgctgccg gcgccgcacc acgccgatgt ggtggacccc gtccgcggac 420
tacatgttcc ccacggagga cgagctgggg ctgctcatgg tggccccggg gcggttcaac 480
gagggccagt accggcgcct ggtgtccgtc gacggcgtga acatcctcac cgacttcatg 540
gtggcgctcc ccgaggggca agagtgcccg ttcgcccgcg tggaccagca ccgcacgtac 600
aagttcggcg cgtgctggag cgacgacagc ttcaagcggg gcgtggacgt gatgcgattc 660
ctgacgccgt tctaccagca gcccccgcac cgggaggtgg tgaactactg gtaccgcaag 720
aacggccgga cgctcccgcg ggcctacgcc gccgccacgc cgtacgccat cgaccccgcg 780
cggccctcgg cgggctcgcc gaggcccagg ccccggcccc ggcccaggcc ccggccgaag 840
cccgagcccg ccccggcgac gcccgcgccc cccggccgcc tgcccgagcc ggcgacgcgg 900
gaccacgccg ccggggggcg ccccacgccg cgacccccga ggcccgagac gccgcaccgc 960
cccttcgccc cgccggccgt cgtgcccagc gggtggccgc agcccgcgga gccgttcccg 1020
ccccggacca ccgccgcgcc gggcgtctcg cgccaccgct cggtgatcgt cggcacgggc 1080
accgcgatgg gcgcgctcct ggtgggcgtg tgcgtctaca tcttcttccg cctgaggggg 1140
gcgaaggggt atcgcctcct gggcggtccc gcggacgccg acgagctaaa agcgcagccc 1200
ggtccgtag 1209
<210> 4
<211> 1101
<212> DNA
<213> porcine Pseudorabies virus (pseudorabiae virus)
<400> 4
atgatgatgg tggcgcgcga cgtgacccgg ctccccgcgg ggctcctcct cgccgccctg 60
accctggccg ccctgacccc gcgcgtcggg ggcgtcctct tcaggggcgc cggcgtcagc 120
gtgcacgtcg ccggcagcgc cgtcctcgtg cccggcgacg cgcccaacct gacgatagac 180
gggacgctgc tgtttctgga ggggccctcg ccgagcaact acagcgggcg cgtggagctg 240
ctgcgcctcg accccaagcg cgcctgctac acgcgcgagt acgccgccga gtacgacctc 300
tgcccccgcg tgcaccacga agccttccgc ggctgcctgc gcaagcgcga gccgctcgcc 360
cggcgcgcgt ccgccgcggt ggaggcgcgc cggctgctgt tcgtctcgcg cccggcctcg 420
ggggacgcgg ggtcgtacgt gctgcgggtc cgcgtgaacg ggaccacgga cctctttgtg 480
ctgacggccc tggtgccgcc gagggggcgc cccgtcccca cgtcgccgcc cgcggacgag 540
tgccggcccg tcgtcggatc gtggcacgac agcctgcgcg tcgtggaccc cgccgaggac 600
gccgtgttca ccacccagcc cccgcccgag cccgagccgc cgacgacccc cgcgcccccc 660
cgggggaccg gcgccacccc cgagccccga tcggacgagg aggaggaggg tgacgcggag 720
acgacgacgc cgacgctgac cccggcgccc gggaccctgg acgcgaacgg cacgatggtg 780
ctgaacgcca gcgtcgtgtc gcgcgtcctg ctcgccgccg ccaacgccac ggcgggcgcc 840
cggagccccg ggaagatagc catggtgctg gggcccacga tcgtcgtcct cctgatcttc 900
ctgggcggga tcgcctgcgt ggcccggcgc tgcgcgcgga atcgcatcta ccggccgcga 960
cccgggcgcg gatcggcggt ccatgcggcg cccccgcggc gcccgccccc caaccccgtc 1020
gccggggcgc ccgtccccca gcccaagatg acgttggccg agctgcgcca gaagctcgcc 1080
accatcgcag aagaacaata a 1101
<210> 5
<211> 1740
<212> DNA
<213> porcine Pseudorabies virus (pseudorabiae virus)
<400> 5
atgcggccct ttctgctgcg cgccgcgcag ctcctggcgc tgctggccct ggcgctctcc 60
accgaggccc cgagcctctc cgccgagacg accccgggcc ccgtcaccga ggtcccgagt 120
ccctcggccg aggtctggga cgacctctcc accgaggccg acgacgatga cctcaacggc 180
gacctcgacg gcgacgaccg ccgcgcgggc ttcggctcgg ccctcgcatc cctgagggag 240
gcgcccccgg cccatctggt gaacgtgtcc gagggcgcca acttcaccct cgacgcgcgc 300
ggcgacggcg ccgtgctggc cgggatctgg acgttcctgc ccgtccgcgg ctgcgacgcc 360
gtgtcggtga ccacggtgtg cttcgagacc gcgtgccacc cggacctggt gctgggccgc 420
gcctgcgtcc ccgaggcccc ggagatgggc atcggcgact acctgccgcc cgaggtgccg 480
cggctccggc gcgagccgcc catcgtcacc ccggagcggt ggtcgccgca cctgagcgtc 540
ctgcgggcca cgcccaacga cacgggcctc tacacgctgc acgacgcctc ggggccgcgg 600
gccgtgttct ttgtggcggt gggcgaccgg ccgcccgcgc cggcggaccc ggtgggcccc 660
gcgcgccacg agccccgctt ccacgcgctc ggcttccact cgcagctctt ctcgcccggg 720
gacacgttcg acctgatgcc gcgcgtggtc tcggacatgg gcgactcgcg cgagaacttt 780
accgccacgc tggactggta ctacgcgcgc gcgcccccgc ggtgcctgct gtactacgtg 840
tacgagccct gcatctacca cccgcgcgcg cccgagtgcc tgcgcccggt ggacccggcg 900
tgcagcttca cctcgccggc gcgcgcgcgg ctggtggcgc gccgcgcgta cgcctcgtgc 960
agcccgctgc tcggggaccg gtggctgacc gcctgcccct tcgacgcctt cggcgaggag 1020
gtgcacacga acgccaccgc ggacgagtcg gggctgtacg tgctcgtgat gacccacaac 1080
ggccacgtcg ccacctggga ctacacgctc gtcgccaccg cggccgagta cgtcacggtc 1140
atcaaggagc tgacggcccc ggcccgggcc ccgggcaccc cgtggggccc gggcggcggc 1200
gacgacgcga tctacgtgga cggcgtcacg acgccggcgc cgcccgcgcg cccgtggaac 1260
ccgtacggcc ggacgacgcc cgggcggctg tttgtgctgg cgctgggctc cttcgtgatg 1320
acgtgcgtcg tcgggggggc catctggctc tgcgtgctgt gctcccggcg ccgggcggcc 1380
tcgcggccgt tccgggtgcc gacgcgggcg cggacgcaca tgctctctcc ggtgtacacc 1440
agcctgccca cgcacgagga ctactacgac ggcgacgacg acgacgacga ggaggcgggc 1500
gtcatccgcc ggcggcccgc ctcccccagc ggagacagcg gctacgaggg gccgtacgcg 1560
agcctggacc ccgaggacga gttcagcagc gacgaggacg acgggctgta cgtgcgcccc 1620
gaggaggcgc cccgctccgg cttcgacgtc tggttccgcg atccggagaa accggaagtg 1680
acgaatggac ccaactatgg cgtgaccgcc aaccgcctgt tgatgtcccg ccccgcttaa 1740
<210> 6
<211> 963
<212> DNA
<213> porcine Pseudorabies virus (pseudorabiae virus)
<400> 6
atgcgcatcc tccggatcta cctcgacggc gcctacggca ccggcaagag caccacggcc 60
cgggtgatgg cgctcggcgg ggcgctgtac gtgcccgagc cgatggcgta ctggcgcact 120
ctgttcgaca cggacacggt ggccggtatt tacgatgcgc agacccggaa gcagaacggc 180
agcctgagcg aggaggacgc ggccctcgtc acggcgcagc accaggccgc cttcgcgacg 240
ccgtacctgc tgctgcacac gcgcctggtc ccgctcttcg ggcccgcggt cgagggcccg 300
cccgagatga cggtcgtctt tgaccgccac ccggtggccg cgacggtgtg cttcccgctg 360
gcgcgcttca tcgtcgggga catcagcgcg gcggccttcg tgggcctggc ggccacgctg 420
cccggggagc cccccggcgg caacctggtg gtggcctcgc tggacccgga cgagcacctg 480
cggcgcctgc gcgcccgcgc gcgcgccggg gagcacgtgg acgcgcgcct gctcacggcc 540
ctgcgcaacg tctacgccat gctggtcaac acgtcgcgct acctgagctc ggggcgccgc 600
tggcgcgacg actgggggcg cgcgccgcgc ttcgaccaga ccgtgcgcga ctgcctcgcg 660
ctcaacgagc tctgccgccc gcgcgacgac cccgagctcc aggacaccct cttcggcgcg 720
tacaaggcgc ccgagctctg cgaccggcgc gggcgcccgc tcgaggtgca cgcgtgggcg 780
atggacgcgc tcgtggccaa gctgctgccg ctgcgcgtct ccaccgtcga cctggggccc 840
tcgccgcgcg tctgcgccgc ggccgtggcg gcgcaggcgc gcggcatgga ggtgacggag 900
tccgcgtacg gcgaccacat ccggcagtgc gtgtgcgcct tcacgtcgga gatgggggtg 960
tga 963
<210> 7
<211> 113
<212> DNA
<213> porcine Pseudorabies virus (pseudorabiae virus)
<400> 7
cctccgcagc tatggcgtga ccgccaaccg cctgttgatg tcccgccccg cttaaatacc 60
gggagaaccg gtccgcccgc attccgacat gcccggcgcc gcctccgtcg aca 113
<210> 8
<211> 72
<212> DNA
<213> porcine Pseudorabies virus (pseudorabiae virus)
<400> 8
atgcgcatcc tccggatcta cctcgacggc gcctacggca ccggcaagag cacgtcggag 60
atgggggtgt ga 72