阅读说明：本技术 重组猪伪狂犬病毒及其制备方法和应用 (Recombinant porcine pseudorabies virus and preparation method and application thereof ) 是由 李群辉 林丽苗 周庆丰 曹雪珍 吴雨 李薇 蔡新斌 吴云燕 申翰钦 于 2021-04-19 设计创作，主要内容包括：本发明涉及一种重组猪伪狂犬病毒及其制备方法和应用,所述重组猪伪狂犬病毒为基因组中插入了猪非典型性瘟病毒E2基因的表达盒的猪伪狂犬病毒变异株GDR。本发明提供了一种重组猪伪狂犬病毒,通过将猪非典型性瘟病毒(APPV)E2基因与猪伪狂犬病毒变异株结合在一起,获得了一种全新的重组猪伪狂犬病毒rPRV-E2。将猪非典型性瘟病毒E2基因的表达盒插入到猪伪狂犬病毒变异株基因组中,导致缺失了部分US7和US8基因,得到的重组猪伪狂犬病毒不仅安全性好,而且免疫后既能产生针对伪狂犬病毒变异株的抗体,又能产生针对猪非典型性瘟病毒E2蛋白的抗体,为猪伪狂犬病毒-猪非典型性瘟病毒二联疫苗的研制奠定了基础。(The invention relates to a recombinant porcine pseudorabies virus, a preparation method and application thereof, wherein the recombinant porcine pseudorabies virus is a porcine pseudorabies virus variant GDR with a genome inserted with an expression cassette of a porcine atypical pestivirus E2 gene. The invention provides a recombinant porcine pseudorabies virus, which is a brand new recombinant porcine pseudorabies virus rPRV-E2 obtained by combining an atypical pestivirus (APPV) E2 gene of a pig with a porcine pseudorabies virus variant strain. The expression cassette of the porcine atypical pestivirus E2 gene is inserted into the genome of the porcine pseudorabies virus variant strain, so that part of genes US7 and US8 are deleted, the obtained recombinant porcine pseudorabies virus has good safety, and can generate an antibody aiming at the pseudorabies virus variant strain and an antibody aiming at the porcine atypical pestivirus E2 protein after immunization, thereby laying the foundation for the development of the porcine pseudorabies virus-porcine atypical pestivirus bivalent vaccine.)

1. The recombinant porcine pseudorabies virus is a porcine pseudorabies virus variant GDR which is deleted from 123210-124654 th nucleotides of a genome and inserted with an expression cassette of a porcine atypical pestivirus E2 gene at the deleted position.

2. The recombinant porcine pseudorabies virus according to claim 1, wherein the nucleotide sequence of the porcine atypical pestivirus E2 gene is represented by SEQ ID NO. 1.

3. The recombinant porcine pseudorabies virus according to claim 1, wherein the promoter used in the expression cassette is a CMV promoter.

4. The recombinant porcine pseudorabies virus according to claim 1, wherein the terminator used in the expression cassette is SV 40.

5. Use of the recombinant porcine pseudorabies virus according to any one of claims 1 to 4 for the preparation of a vaccine for the prevention of atypical swine fever and pseudorabies.

6. A vaccine for the prevention of atypical swine fever and pseudorabies, comprising the recombinant porcine pseudorabies virus of any one of claims 1 to 4 and an adjuvant.

7. A method for preparing the recombinant porcine pseudorabies virus according to any one of claims 1 to 4, comprising the following steps: constructing a transfer vector of an expression cassette containing the porcine atypical pestivirus E2 gene, and carrying out homologous recombination on the transfer vector and the porcine pseudorabies virus variant GDR to obtain the recombinant porcine pseudorabies virus.

8. The method according to claim 7, wherein the method for constructing the transfer vector comprising the expression cassette of E2 gene of atypical pestivirus comprises the steps of: and (2) respectively amplifying a left homologous arm of a US7 gene region and a right homologous arm of a US8 gene region by using the genome DNA of a porcine pseudorabies virus variant GDR as an amplification template, inserting the left homologous arm, an expression cassette of the porcine atypical pestivirus E2 gene and the right homologous arm into a plasmid, and enabling the expression cassette of the porcine atypical pestivirus E2 gene to be positioned between the left homologous arm and the right homologous arm to obtain the transfer vector.

9. The method according to claim 8, wherein the primer set for amplifying the left homology arm is shown in SEQ ID NO. 2 and SEQ ID NO. 3, and the primer set for amplifying the right homology arm is shown in SEQ ID NO. 4 and SEQ ID NO. 5.

10. The method of claim 7, wherein the homologous recombination comprises the steps of: and (3) transfecting the transfer vector and the porcine pseudorabies virus variant GDR to receptor cells, culturing the receptor cells until virus plaques appear, and then performing plaque purification.

Technical Field

The invention relates to the field of animal virology, in particular to a recombinant porcine pseudorabies virus and a preparation method and application thereof.

Background

Pestiviruses are single-stranded positive-strand RNA viruses with high variation in envelope height, belong to the genus pestivirus of the family Flaviviridae, and infect pigs, ruminants, wild animals, and the like. Porcine atypical pestivirus (APPV) is a novel pestivirus discovered for the first time in 2015 (Hause, B.M., et al,2015.Discovery of a novel viral pestivirus with a novel viral activity in pigs in the USA. the Journal of genetic virology 96, 2994. supple. 2998), so far, there are many cases of APPV infection in ten commercial pig groups in the world, and APPV strains isolated by scholars in different regions have great variability. APPV has been sequentially reported to be closely associated with congenital tremor in piglets. Congenital Tremor (CT) of piglets, commonly known as "piglet tremor disease" or "jumping disease", refers to a disease in which local or whole body muscles such as the head, limbs and the like of newly born piglets exhibit paroxysmal contracture, which can cause standing difficulty, milk sucking obstruction and even death of the piglets. APPV can be detected in various tissues of affected pigs, but the highest virus content is the mandibular lymph node, and the moderate virus content is the peripheral lymphoid organs (spleen, tonsil, and inguinal lymph node), central nervous system (brainstem, brain, cerebellum, etc.), and digestive system (duodenum). The pathological features of AII CT piglet tissue caused by APPV are brain white matter vacuolation, and the ultramicrostructure is observed to be cerebellum and spinal cord myelination reduction and destruction and brain white matter myelin disintegration. Furthermore immunohistochemistry showed a decrease in spinal oligodendrocytes in affected pigs, suggesting that APPV may have a deleterious effect on fetal oligodendrocytes, affecting the formation of myelin, leading to its loss of function. By inoculating APPV to pregnant sows in an intramuscular injection or uterus manner, the result shows that the newborn piglets inoculated with APPV powder show CT symptoms, and various tissue samples of the sick piglets detect the APPV, so that the APPV can be transmitted through placenta, and the central nerve of the fetal pigs is damaged by infecting the APPV in the fetal development stage, thereby leading the newborn piglets to show the CT symptoms. In addition, APPV was detected in the semen and foreskin of adult boars presenting CT symptoms at birth, indicating that APPV infected boars can be detoxified either transiently or persistently by semen. Studies have found that APPV is also detectable in the salivary, duodenal, colon and pancreatic organs of previously diseased pigs, suggesting that APPV may be transmitted by the fecal oral pathway (Groof A D, Deijs M, Guelen L, et al. anatomical Port viruses: A Possible Cable of genetic Tremor Type A-II in Newborn Piglets. viruses,2016,8(10): 271).

As a newly discovered pestivirus, APPV has a distant genetic relationship with Classical Swine Fever Virus (CSFV), and the nucleotide homology of APPV with CSFV is about 50%. The APPV genome is flanked by non-coding regions (UTRs), i.e. 5 '-UTR and 3' -UTR (from N to C), and in the middle by a large Open Reading Frame (ORF) that encodes a large polyprotein and is then cleaved into smaller proteins. The coding sequence of all viral proteins is: npro, C, Erns, E1, E2, P7, NS2, NS4A, NS4B, NS5A, NS5B (Hause, B.M., et al,2015.Discovery of a novel reactive particulate in samples in the USA. the Journal of general virology 96, 2994). The related research of APPV as one of the emerging pestiviruses in pigs is still in the preliminary stage, and no preventive biological product is available for the atypical pestiviruses of pigs at present.

Pseudorabies (PR) is an acute infectious disease caused by Pseudorabies virus (PRV) infection, and can cause high mortality of piglets, reproductive failure of sows, nervous and respiratory symptoms of fattening pigs, reduction or loss of boar performance, and the like, and is extremely harmful to the pig industry. The clinical symptoms and course of disease in pigs vary greatly with age. The suckling piglets are most sensitive, the piglets within 15 days of age are usually the most acute, the disease course does not exceed 72 hours, and the death rate is 100 percent, and the piglets are mainly characterized by rising body temperature, diarrhea, trembling, uncoordinated movement, salivation, stiff neck muscles, watery movement of limbs and finally coma and death. Most fattening pigs are accompanied by temperature rise and dyspnea, generally do not die, and become recessive infection with toxicity or expel toxin after being endured. Adult pigs often exhibit no visible clinical symptoms or exhibit only mild body temperature elevation and generally do not die. In the early pregnancy of sows, abortion may occur about 20 days after infection, and in the late pregnancy, dead and mummy fetuses are frequent, or weak and dead fetuses are produced. PRV genus herpes virus family herpesviridae subfamily, genus transient varicella, virions are oval or circular, the diameter of non-enveloped particles is 110-150 nm, and the diameter of enveloped mature virions is 180 nm. PRV is a highly resistant species of herpes virus, and is viable for 7 days on object surfaces and in liquids, and remains stable between pH 4-9. Under septic conditions, the virus in the pathogens lost infectivity over 11 days. PRV is sensitive to ether, chloroform and other lipid solvents, formalin, ultraviolet irradiation and the like, 5% of carbolic acid is inactivated for 2min, and 0.5% -1% of sodium hydroxide is used for inactivating the carbolic acid. PRV has strong resistance to heat, and can be inactivated only after 30-50 min at 55-60 ℃ and 3min at 80 ℃. PRV infected pigs do not show clinical symptoms, but infectious viruses exist in the pig body in a latent state for a long time, and the viruses cannot be separated, but the existence of viral genomic DNA can be detected by a polymerase probe method, and the viruses in the latent state can be converted into the viruses with infectivity when the immunity is weakened due to external adverse environmental stimulation and the like. Research shows that compared with the prior strains, the new PRV epidemic strains have obvious variation (Wu R, Bai C, Sun J, Chang S, Zhang X. organism of virus microorganisms infection in northern China. J Vet Sci.2013,14(3):363-365), and the pathogenicity is obviously enhanced, and the existing vaccines can not completely protect the infection of the new epidemic strains.

Therefore, the research on the atypical pestivirus and pseudorabies virus of the pig causes great economic loss to the pig industry, and the research on how to prevent the infection of the atypical pestivirus and the pseudorabies virus of the pig has important significance to the pig industry.

Disclosure of Invention

Based on the above, there is a need for a recombinant porcine pseudorabies virus that can produce antibodies against both a variant strain of pseudorabies virus and an atypical pestivirus of swine after immunization.

A recombinant porcine pseudorabies virus is a porcine pseudorabies virus variant GDR which is deleted from 123210-124654 th nucleotides of a genome and inserted with an expression cassette of a porcine atypical pestivirus E2 gene at the deleted position.

The invention provides a recombinant porcine pseudorabies virus, which is a brand new recombinant porcine pseudorabies virus rPRV-E2 obtained by combining an atypical pestivirus (APPV) E2 gene of a pig with a porcine pseudorabies virus variant strain. The expression cassette of the porcine atypical pestivirus (APPV) E2 gene is inserted into the genome of the porcine pseudorabies virus variant strain to cause deletion of part of the genes US7 and US8, and the obtained recombinant porcine pseudorabies virus (rPRV-E2) has good safety, can generate an antibody aiming at the pseudorabies virus variant strain and an antibody aiming at the porcine atypical pestivirus E2 protein after immunization, and lays a foundation for developing a porcine pseudorabies virus-porcine atypical pestivirus bivalent vaccine.

In one embodiment, the nucleotide sequence of the E2 gene of the swine atypical pestivirus is shown in SEQ ID NO. 1.

In one embodiment, the promoter used in the expression cassette is a CMV promoter.

In one embodiment, the terminator used in the expression cassette is SV 40.

The invention also provides application of the recombinant porcine pseudorabies virus in preparing a vaccine for preventing atypical swine fever and pseudorabies.

The invention also provides a vaccine for preventing atypical swine fever and pseudorabies, which comprises the recombinant porcine pseudorabies virus and an adjuvant.

The invention also provides a preparation method of the recombinant porcine pseudorabies virus, which comprises the following steps: constructing a transfer vector of an expression cassette containing the porcine atypical pestivirus E2 gene, and carrying out homologous recombination on the transfer vector and the porcine pseudorabies virus variant GDR to obtain the recombinant porcine pseudorabies virus.

In one embodiment, the method for constructing a transfer vector containing the expression cassette of the porcine atypical pestivirus E2 gene comprises the following steps: and (2) respectively amplifying a left homologous arm of a US7 gene region and a right homologous arm of a US8 gene region by using the genome DNA of a porcine pseudorabies virus variant GDR as an amplification template, inserting the left homologous arm, an expression cassette of the porcine atypical pestivirus E2 gene and the right homologous arm into a plasmid, and enabling the expression cassette of the porcine atypical pestivirus E2 gene to be positioned between the left homologous arm and the right homologous arm to obtain the transfer vector.

In one embodiment, the primer pair for amplifying the left homology arm is shown as SEQ ID NO. 2 and SEQ ID NO. 3, and the primer pair for amplifying the right homology arm is shown as SEQ ID NO. 4 and SEQ ID NO. 5.

In one embodiment, the method of homologous recombination comprises the steps of: and (3) transfecting the transfer vector and the porcine pseudorabies virus variant GDR to receptor cells, culturing the receptor cells until virus plaques appear, and then performing plaque purification.

Drawings

FIG. 1 is a schematic structural view of the recombinant plasmid pMD-E2 in example 1;

FIG. 2 is a schematic diagram of the construction process of the transfer vector PMD-US78-E2 in example 1;

FIG. 3 is the PCR identification electrophoretogram of rPRV-E2 recombinant virus in example 1;

FIG. 4 is a fluorescent microscopic observation result chart of the recombinant virus rPRV-E2 in example 1;

FIG. 5 is a graph showing the results of the genetic stability test of the recombinant virus rPRV-E2 in example 1;

FIG. 6 is a graph comparing the antibody levels of PRV after immunization of mice with the recombinant virus rPRV-E2 of example 1;

FIG. 7 is a graph comparing the antibody levels of APPV-E2 after immunization of mice with the recombinant virus rPRV-E2 of example 1.

Detailed Description

In order that the invention may be more fully understood, a more particular description of the invention will now be rendered by reference to specific embodiments thereof that are illustrated in the appended drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

The recombinant porcine pseudorabies virus is a porcine pseudorabies virus variant GDR which is deleted from 123210-124654 th nucleotides of a genome and inserted with an expression cassette of a porcine atypical pestivirus E2 gene at the deleted position. The porcine pseudorabies virus variant strain GDR is HN1201, and the whole gene sequence is shown in ACCESSION NO. KP722022.

The invention provides a recombinant porcine pseudorabies virus, which is a brand new recombinant porcine pseudorabies virus rPRV-E2 obtained by combining an atypical pestivirus (APPV) E2 gene of a pig with a porcine pseudorabies virus variant strain. The expression cassette of the porcine atypical pestivirus (APPV) E2 gene is inserted into the genome of the porcine pseudorabies virus variant strain to cause deletion of part of the genes US7 and US8, and the obtained recombinant porcine pseudorabies virus (rPRV-E2) has good safety, can generate an antibody aiming at the pseudorabies virus variant strain and an antibody aiming at the porcine atypical pestivirus E2 protein after immunization, and lays a foundation for developing a porcine pseudorabies virus-porcine atypical pestivirus bivalent vaccine.

In a specific example, the nucleotide sequence of the E2 gene of the swine atypical pestivirus is shown in SEQ ID NO. 1.

In one specific example, the promoter used in the expression cassette is a CMV promoter. In one specific example, the terminator used in the expression cassette is SV 40. It will be appreciated that the particular type of promoter and terminator is not limited thereto and may be selected as desired, and that other optional regulatory elements may also be included in the expression cassette.

The vaccine for preventing atypical swine fever and pseudorabies, which is an embodiment of the present invention, comprises the recombinant porcine pseudorabies virus as described above and an adjuvant.

In one particular example, the adjuvant comprises one or more of a stabilizer, a complexing agent, an immunopotentiator, and water.

Optionally, the immunopotentiator is selected from one or more of albumin polypeptide, scallop polypeptide, cefuroxime polypeptide, epimedium flavone, kumquat flavone, oldenlandia flavone, soybean flavone, seabuckthorn flavone, astragalus polysaccharide, ginseng polysaccharide, lentinan, calyx seu fructus physalis polysaccharide, ganoderma lucidum mycelium polysaccharide, isatis root polysaccharide, lycium barbarum polysaccharide, angelica polysaccharide, ganoderma lucidum polysaccharide, rhodiola polysaccharide, jujube polysaccharide and chitosan, and can improve the low immune function and accelerate the induction of immune response reaction. Optionally, the stabilizer is selected from one or more of soluble divalent manganese salt, divalent calcium salt, divalent zinc salt and divalent iron salt to protect the active ingredient. Optionally, the complexing agent is selected from one or more of ethylenediaminetetraacetic acid and sodium polyacrylate.

The preparation method of the recombinant porcine pseudorabies virus provided by the embodiment of the invention comprises the following steps: constructing a transfer vector of the expression cassette containing the porcine atypical pestivirus E2 gene, and carrying out homologous recombination on the transfer vector and the porcine pseudorabies virus variant GDR to obtain the recombinant porcine pseudorabies virus.

Homologous Recombination (homologus Recombination) refers to Recombination that occurs between non-sister chromatids or between or within DNA molecules containing Homologous sequences on the same chromosome. Viral homologous recombination is a technique based on homology arms for integrating a gene of interest into the viral genome. The expression cassettes are connected in series to a transfer vector, and recombinant viruses are constructed through homologous recombination, so that virus gene modification, foreign gene expression, recombinant live vector vaccine preparation and the like are realized. The construction of recombinant virus is established on the basis of in vitro homologous recombination, a replication nonessential fragment containing a parent strain is screened, and exogenous genes are introduced into a virus genome through homologous recombination between a plasmid transfer vector and virus genome DNA, so that the biological characteristics of the parent strain are retained, and the recombinant virus can be replicated and propagated.

The basic process of constructing recombinant virus through in vitro connection is to make the transfer vector containing exogenous gene and the parent strain genome DNA arm after enzyme digestion treatment undergo the process of in vitro homologous recombination, then make the connection product transfect the mammalian cell which has been infected with parent virus in advance, and the chimeric genome can be assembled into recombinant virus particle under the action of parent virus.

In one embodiment, the method for constructing a transfer vector comprising an expression cassette of the porcine atypical pestivirus E2 gene comprises the steps of: the genome DNA of the porcine pseudorabies virus variant GDR is taken as an amplification template, the left homologous arm of the US7 gene region and the right homologous arm of the US8 gene region are respectively amplified, the left homologous arm, the expression cassette of the porcine atypical pestivirus E2 gene and the right homologous arm are inserted into a plasmid, and the expression cassette of the porcine atypical pestivirus E2 gene is positioned between the left homologous arm and the right homologous arm, so that a transfer vector is obtained.

In one specific example, the primer pairs for amplifying the left homology arm are shown in SEQ ID NO. 2 and SEQ ID NO. 3, and the primer pairs for amplifying the right homology arm are shown in SEQ ID NO. 4 and SEQ ID NO. 5.

In one specific example, the method of homologous recombination comprises the steps of: and (3) transfecting the transfer vector and the porcine pseudorabies virus variant GDR to a receptor cell, culturing the receptor cell until virus plaques appear, and then performing plaque purification.

In one particular example, a method of transfecting a recipient cell comprises the steps of: when the cultured receptor cells grow into a 70-90% cell monolayer, inoculating a proper amount of porcine pseudorabies virus variant GDR, and adsorbing for 3-5 hours; diluting the transfer vector in opti-DMEM, and diluting Lipofectamine 2000 in opti-DMEM; dripping the Lipofectamine 2000 diluent into the carrier diluent respectively, uniformly mixing while adding, and incubating at room temperature for 15-25 min; and (3) washing the receptor cells by serum-free opti-DMEM, dropwise adding the Lipofectamine 2000/DNA compound, gently shaking to uniformly mix the compound, and culturing the compound at 37 ℃ for 3-5 hours.

In one particular example, a method of plaque purification includes the steps of: the expression of the E2 gene was detected using a monoclonal antibody against APPV-E2, and the procedure was repeated by limiting dilution until each plaque obtained was stained positive with the anti-APPV-E2 antibody.

The following are specific examples.

Example 1

Preparation of expression cassette containing gene of swine atypical pestivirus E2(APPV-E2)

The APPV-E2 gene expression cassette sequence was synthesized by Nanjing Kingsrey Biotech, Inc. The APPV-E2 expression cassette comprises a CMV promoter sequence, a porcine atypical pestivirus E2 gene and an SV40 termination sequence, and the APPV-E2 sequence is designed by referring to GD-DH01-2018 strain (ACCESSION NO. MH493895). The expression cassette prepared above was cloned into the pMD-18T vector, which was designated pMD-E2, and a schematic diagram thereof is shown in FIG. 1.

II, construction of recombinant virus rPRV-E2

(1) Construction of transfer vector PMD-US78-E2

Referring to the entire gene sequence of porcine pseudorabies virus variant HN1201 published in Genebank (ACCESSION NO. KP722022), the left and right homology arms of the US7 and US8 regions were designed and primers for amplifying the homology arms are shown in the following table. The genomic DNA of a porcine pseudorabies virus variant GDR (stored in a laboratory of the inventor) is used as a PCR amplification template, a left homologous arm is amplified by a primer US7u-F, US7u-R, the expected amplification size is 1241bp, and the gene sequence is shown as Seq ID No: 6. The right homology arm was amplified with the primer US8d-F, US8d-R, the expected amplification size was 1280bp, and the gene sequence was shown in Seq ID No: 7. The amplified target fragments are respectively connected with pMD-18T vectors to construct corresponding recombinant plasmids PMD-US7u and PMD-US8 d. The recombinant plasmids are respectively digested by Sal I and Hind III, and are inserted into pMD-E2 plasmid in sequence to obtain a transfer vector PMD-US 78-E2. The specific construction process is shown in FIG. 2.

(2) Homologous recombination

According to Lipofectamine^TM2000 transfection kit (Invitrogen corporation) instructions, transfer vector PMD-US78-E2 and porcine pseudorabies virus variant GDR were transfected into Vero cells. The method comprises the following specific steps: culturing Vero in a six-hole plate until a cell monolayer of 70-90% grows, inoculating a proper amount of porcine pseudorabies virus variant GDR, and adsorbing for 4 hours; diluting 2 mu g of the transfer vector in opti-DMEM to ensure that the final volume of the mixed solution is 100 mu L; gently mixing 5 μ L Lipofectamine 2000 and 100 μ L opti-DMEM; respectively dripping 100 mu L of Lipofectamine 2000 diluent into 100 mu L of plasmid diluent, and uniformly mixing while adding; incubate at room temperature for 20 min. During the period, after the cells in the six-well plate are lightly washed twice by serum-free opti-DMEM, 0.5mL of serum-free opti-DMEM is added into each well, 200 mu L of Lipofectamine 2000/DNA compound is dropwise added into the 6-well plate, the 6-well plate is gently shaken to be uniformly mixed, the 6-well plate is placed at 37 ℃ for culture for 4h, the supernatant is discarded, 10% of fetal bovine serum DMEM culture medium is added, the 10% of fetal bovine serum DMEM culture medium is placed at 37 ℃ for culture for 24h, the 1% of fetal bovine serum DMEM culture medium is used for changing the liquid, the culture is placed at 37 ℃ for 3-5 d, and the observation is carried out every day until virus plaques become visible.

(3) Plaque purification of recombinant viruses

The visible plaques include the recombinant virus as well as the parental wild-type virus, and thus the recombinant virus is purified from the wild-type virus by a series of limiting dilutions. At each purification, the expression of the E2 gene was confirmed using a monoclonal antibody against APPV-E2 (prepared and stored in the laboratory of the inventors), and the purification procedure was repeated until each plaque obtained was stained positive with anti-APPV-E2 antibody, and the purified recombinant virus was rPRV-E2.

(4) Identification of recombinant Virus rPRV-E2

Primers were designed on both sides of the APPV-E2 expression cassette, with the primer sequences shown in the following tables rPRV-E2-JD-F and rPRV-E2-JD-R. The primers are used for amplification by taking the obtained rPRV-E2 as a template, so that a 1763bp specific fragment can be amplified, as shown in figure 3, and the 1763bp fragment contains not only two pairs of primer sequences but also an APPV-E2 expression cassette sequence of 1568bp without containing gene sequences of US7 and US8 through sequencing verification. Therefore, the obtained recombinant virus rPRV-E2 is successfully inserted with an expression cassette containing the E2 gene of the porcine atypical pestivirus, and a part of the genes of US7 and US8 are deleted.

Primer name	Primer sequences
		rPRV-E2-JD-F	ctggcagcgtatgacagctctg
rPRV-E2-JD-R	catgttattatgcacacagctg

(5) Indirect immunofluorescence identification of recombinant virus rPRV-E2

The recombinant virus rPRV-E2 and the parent porcine pseudorabies virus variant GDR thereof are respectively inoculated to Vero cells, and when the cytopathic effect reaches 60 percent, the expression condition of the E2 gene is detected by an indirect immunofluorescence method. The method comprises the following steps: discarding cell culture solution, washing the cell surface with PBS for 1 time, adding cold methanol into each hole, and fixing at room temperature for 10-15 minutes; washing with PBS for 1 time, adding APPV-E2 monoclonal antibody (prepared and stored in the laboratory of the inventor) diluted properly, and standing at 37 deg.C for 1 hr; washing with PBS 3 times; FITC-labeled anti-mouse IgG fluorescent antibody (purchased from SIGMA) was added to each well at an appropriate dilution and allowed to act at 37 ℃ for 1 hour; washing with PBS 3 times; when observed under an inverted fluorescence microscope, the cell plaques show specific green fluorescence, but no fluorescence is generated after the control group parent porcine pseudorabies virus variant GDR infects cells, as shown in figure 4, which indicates that the inserted APPV-E2 gene can be well and correctly expressed.

Third, stability test in vitro and in vivo of recombinant virus rPRV-E2

rPRV-E2 was passaged 20 times in CEF cells (chicken embryo fibroblasts), and the primers rPRV-E2-JD-F and rPRV-E2-JD-R were used to amplify every 5 passages, so that a 1763bp band could still be amplified, as shown in FIG. 5, indicating that the obtained recombinant virus rPRV-E2 remained stable after 20 passages in cells.

Fourth, animal experiment

BALB/C mice, 6 weeks old, were randomly divided into 2 groups of 5 mice each and weighed for injection of recombinant virus rPRV-E2 and DMEM, respectively. Each control group was injected with 100. mu.L of DMEM, and the other group was injected with 10. mu.L of hind limb muscle⁴TCID₅₀Dose (half tissue culture infectious dose) of recombinant virus rPRV-E2. After immunization, mice were observed daily for clinical symptoms of listlessness, anorexia, pruritus, tremor, etc., and were reweighed at 21d, blood was collected at 14d and 21d tail veins, serum was isolated, PRV antibody level in serum was measured using ELISA plates coated with PRV GDR strain whole virus, and APPV-E2 antibody level in serum was measured using ELISA plates coated with APPV-E2 protein (prepared and stored in the laboratory of the inventors).

The results show that the average weight gain of mice in 21d, rPRV-E2 group and DMEM control group after immunization is 4.6g and 4.8g respectively, the weight gain difference is not large, and the result shows that the recombinant virus rPRV-E2 has no inhibition effect on the weight gain of the mice. As shown in FIG. 6 and FIG. 7, PRV and APPV-E2 antibodies were detected 14d after immunization in the rPRV-E2 group, and the antibody level of the 21d recombinant virus rPRV-E2 group was significantly increased after immunization, and was not significantly changed in the control group. This shows that the recombinant virus rPRV-E2 can induce mice to generate obvious immune response, and not only can generate antibodies against pseudorabies virus variants, but also can generate antibodies against porcine atypical pestivirus E2 protein.

The relevant sequence information is as follows:

Seq ID No:1

ATGTCATGCCATGAGCGACAGGACTATTATAATGTCCAATTAGTCGTTGACGAAAAAACGGGCATAGAAAAACGGTCAATAATGGGCAAATGGACCGTAGTGACCAAAGAGGGTCGGGAGCCAAGATTAATGGAACAGATAAAAATGGTATCAAATAGCAGACTGACAGAAACTTACTGCTACAATAAACTAAACACCAGCAGCTGGGGGCGACACCCAATGAAACAGAGGGGGTGTGGGCAAACTGTACCCTATTGGCCTGGTGACAATGTTCTCGAAGAACAATACTTCAGCACGGGTTACTGGGTGAATGCCACAGGAGGTTGCCAGTTGAGGGAAGGTGTGTGGCTATCAAGAAAGGGCGGTGTCCAGTGCCAACGTAACGGCTCATCTTTGATCCTGCAGTTGGCAATCAAGGAGGAAAATGATACCATGGAGATACCGTGTGACCCGGTAGAAACCGAAAGCATGGGCCCGGTGGCACAGGGCACTTGCGTGTATAGTTGGGCAGTAGCTCCAAGAGGATGGTACTACAACAGGAAGGATGGCTATTGGCTCCAATATATAAAGAAAAATGACTATCAGTACTGGACAAAAATGCCCGCGGCTTCGTCCGCTGCAACAATGTACCGGCATTTACTACCTTTGTTAGTGGCTTGCCTTATGGGCGGTAGGATTTCAGTATGGATTGTAGCAATGCTTCTATCCCTACAGGTGGAGGCCAGCTAA

Seq ID No:2

GTCGACcgtgcggggtggtggcgctg

Seq ID No:3

GTCGACgcccctccagaaacagcagc

Seq ID No:4

AAGCTTgggcatcggcgactacctgc

Seq ID No:5

AAGCTTacatcaacaggcggttggcg

Seq ID No:6

CGTGCGGGGTGGTGGCGCTGATCTCCGACCCGCAGGTGGACCGGCTGCTGAACGAGGCGGTGGCCCACCGGCGGCCCACGTACCGCGCCCACGTGGCCTGGTACCGCATCGCGGACGGGTGCGCGCACCTGCTGTACTTTATCGAGTACGCCGACTGCGACCCCAGGCAGATCTTTGGGCGCTGCCGGCGCCGCACCACGCCGATGTGGTGGACCCCGTCCGCGGACTACATGTTCCCCACGGAGGACGAGCTGGGGCTGCTCATGGTGGCCCCGGGGCGGTTCAACGAGGGCCAGTACCGGCGCCTGGTGTCCGTCGACGGCGTGAACATCCTCACCGACTTCATGGTGGCGCTCCCCGAGGGGCAAGAGTGCCCGTTCGCCCGCGTGGACCAGCACCGCACGTACAAGTTCGGCGCGTGCTGGAGCGACGACAGCTTCAAGCGGGGCGTGGACGTGATGCGATTCCTGACGCCGTTCTACCAGCAGCCCCCGCACCGGGAGGTGGTGAACTACTGGTACCGCAAGAACGGCCGGACGCTCCCGCGGGCCTACGCCGCCGCCACGCCGTACGCCATCGACCCCGCGCGGCCCTCGGCGGGCTCGCCGAGGCCCAGGCCCCGGCCCCGGCCCAGGCCCCGGCCGAAGCCCGAGCCCGCCCCGGCGACGCCCGCGCCCCCCGGCCGCCTGCCCGAGCCGGCGACGCGGGACCACGCCGCCGGGGGGCGCCCCACGCCGCGACCCCCGAGGCCCGAGACGCCGCACCGCCCCTTCGCCCCGCCGGCCGTCGTGCCCAGCGGGTGGCCGCAGCCCGCGGAGCCGTTCCCGCCCCGGACCACCGCCGCGCCGGGCGTCTCGCGCCACCGCTCGGTGATCGTCGGCACGGGCACCGCGATGGGCGCGCTCCTGGTGGGCGTGTGCGTCTACATCTTCTTCCGCCTGAGGGGGGCGAAGGGGTATCGCCTCCTGGGCGGTCCCGCGGACGCCGACGAGCTAAAAGCGCAGCCCGGTCCGTAGCCTCCGCAGTACCGGCGTCGATGATGATGGTGGCGCGCGACGTGACCCGGCTCCCCGCGGGGCTCCTCCTCGCCGCCCTGACCCTGGCCGCCCTGACCCCGCGCGTCGGGGGCGTCCTCTTCAGGGGCGCCGGCGTCAGCGTGCACGTCGCCGGCAGCGCCGTCCTCGTGCCCGGCGACGCGCCCAACCTGACGATAGACGGGACGCTGCTGTTTCTGGAGGGGC

Seq ID No:7

GGGCATCGGCGACTACCTGCCGCCCGAGGTGCCGCGGCTCCGGCGCGAGCCGCCCATCGTCACCCCGGAGCGGTGGTCGCCGCACCTGAGCGTCCTGCGGGCCACGCCCAACGACACGGGCCTCTACACGCTGCACGACGCCTCGGGGCCGCGGGCCGTGTTCTTTGTGGCGGTGGGCGACCGGCCGCCCGCGCCGGCGGACCCGGTGGGCCCCGCGCGCCACGAGCCCCGCTTCCACGCGCTCGGCTTCCACTCGCAGCTCTTCTCGCCCGGGGACACGTTCGACCTGATGCCGCGCGTGGTCTCGGACATGGGCGACTCGCGCGAGAACTTTACCGCCACGCTGGACTGGTACTACGCGCGCGCGCCCCCGCGGTGCCTGCTGTACTACGTGTACGAGCCCTGCATCTACCACCCGCGCGCGCCCGAGTGCCTGCGCCCGGTGGACCCGGCGTGCAGCTTCACCTCGCCGGCGCGCGCGCGGCTGGTGGCGCGCCGCGCGTACGCCTCGTGCAGCCCGCTGCTCGGGGACCGGTGGCTGACCGCCTGCCCCTTCGACGCCTTCGGCGAGGAGGTGCACACGAACGCCACCGCGGACGAGTCGGGGCTGTACGTGCTCGTGATGACCCACAACGGCCACGTCGCCACCTGGGACTACACGCTCGTCGCCACCGCGGCCGAGTACGTCACGGTCATCAAGGAGCTGACGGCCCCGGCCCGGGCCCCGGGCACCCCGTGGGGCCCCGGCGGCGGCGACGACGCGATCTACGTGGACGGCGTCACGACGCCGGCGCCGCCCGCGCGCCCGTGGAACCCGTACGGCCGGACGACGCCCGGGCGGCTGTTTGTGCTGGCGCTGGGCTCCTTCGTGATGACGTGCGTCGTCGGGGGGGCCATCTGGCTCTGCGTGCTGTGCTCCCGGCGCCGGGCGGCCTCGCGGCCGTTCCGGGTGCCGACGCGGGCGCGGACGCACATGCTCTCTCCGGTGTACACCAGCCTGCCCACGCACGAGGACTACTACGACGGCGACGACGACGACGACGAGGAGGCGGGCGTCATCCGCCGGCGGCCCGCCTCCCCCAGCGGAGACAGCGGCTACGAGGGGCCGTACGCGAGCCTGGACCCCGAGGACGAGTTCAGCAGCGACGAGGACGACGGGCTGTACGTGCGCCCCGAGGAGGCGCCCCGCTCCGGCTTCGACGTCTGGTTCCGCGATCCGGAGAAACCGGAAGTGACGAATGGACCCAACTATGGCGTGACCGCCAACCGCCTGTTGATGT

the technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Sequence listing

<110> Win food group Ltd

<120> recombinant porcine pseudorabies virus, and preparation method and application thereof

<160> 7

<170> SIPOSequenceListing 1.0

<210> 1

<211> 729

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

atgtcatgcc atgagcgaca ggactattat aatgtccaat tagtcgttga cgaaaaaacg 60

ggcatagaaa aacggtcaat aatgggcaaa tggaccgtag tgaccaaaga gggtcgggag 120

ccaagattaa tggaacagat aaaaatggta tcaaatagca gactgacaga aacttactgc 180

tacaataaac taaacaccag cagctggggg cgacacccaa tgaaacagag ggggtgtggg 240

caaactgtac cctattggcc tggtgacaat gttctcgaag aacaatactt cagcacgggt 300

tactgggtga atgccacagg aggttgccag ttgagggaag gtgtgtggct atcaagaaag 360

ggcggtgtcc agtgccaacg taacggctca tctttgatcc tgcagttggc aatcaaggag 420

gaaaatgata ccatggagat accgtgtgac ccggtagaaa ccgaaagcat gggcccggtg 480

gcacagggca cttgcgtgta tagttgggca gtagctccaa gaggatggta ctacaacagg 540

aaggatggct attggctcca atatataaag aaaaatgact atcagtactg gacaaaaatg 600

cccgcggctt cgtccgctgc aacaatgtac cggcatttac tacctttgtt agtggcttgc 660

cttatgggcg gtaggatttc agtatggatt gtagcaatgc ttctatccct acaggtggag 720

gccagctaa 729

<210> 2

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

gtcgaccgtg cggggtggtg gcgctg 26

<210> 3

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

gtcgacgccc ctccagaaac agcagc 26

<210> 4

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

aagcttgggc atcggcgact acctgc 26

<210> 5

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

aagcttacat caacaggcgg ttggcg 26

<210> 6

<211> 1241

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

cgtgcggggt ggtggcgctg atctccgacc cgcaggtgga ccggctgctg aacgaggcgg 60

tggcccaccg gcggcccacg taccgcgccc acgtggcctg gtaccgcatc gcggacgggt 120

gcgcgcacct gctgtacttt atcgagtacg ccgactgcga ccccaggcag atctttgggc 180

gctgccggcg ccgcaccacg ccgatgtggt ggaccccgtc cgcggactac atgttcccca 240

cggaggacga gctggggctg ctcatggtgg ccccggggcg gttcaacgag ggccagtacc 300

ggcgcctggt gtccgtcgac ggcgtgaaca tcctcaccga cttcatggtg gcgctccccg 360

aggggcaaga gtgcccgttc gcccgcgtgg accagcaccg cacgtacaag ttcggcgcgt 420

gctggagcga cgacagcttc aagcggggcg tggacgtgat gcgattcctg acgccgttct 480

accagcagcc cccgcaccgg gaggtggtga actactggta ccgcaagaac ggccggacgc 540

tcccgcgggc ctacgccgcc gccacgccgt acgccatcga ccccgcgcgg ccctcggcgg 600

gctcgccgag gcccaggccc cggccccggc ccaggccccg gccgaagccc gagcccgccc 660

cggcgacgcc cgcgcccccc ggccgcctgc ccgagccggc gacgcgggac cacgccgccg 720

gggggcgccc cacgccgcga cccccgaggc ccgagacgcc gcaccgcccc ttcgccccgc 780

cggccgtcgt gcccagcggg tggccgcagc ccgcggagcc gttcccgccc cggaccaccg 840

ccgcgccggg cgtctcgcgc caccgctcgg tgatcgtcgg cacgggcacc gcgatgggcg 900

cgctcctggt gggcgtgtgc gtctacatct tcttccgcct gaggggggcg aaggggtatc 960

gcctcctggg cggtcccgcg gacgccgacg agctaaaagc gcagcccggt ccgtagcctc 1020

cgcagtaccg gcgtcgatga tgatggtggc gcgcgacgtg acccggctcc ccgcggggct 1080

cctcctcgcc gccctgaccc tggccgccct gaccccgcgc gtcgggggcg tcctcttcag 1140

gggcgccggc gtcagcgtgc acgtcgccgg cagcgccgtc ctcgtgcccg gcgacgcgcc 1200

caacctgacg atagacggga cgctgctgtt tctggagggg c 1241

<210> 7

<211> 1280

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

gggcatcggc gactacctgc cgcccgaggt gccgcggctc cggcgcgagc cgcccatcgt 60

caccccggag cggtggtcgc cgcacctgag cgtcctgcgg gccacgccca acgacacggg 120

cctctacacg ctgcacgacg cctcggggcc gcgggccgtg ttctttgtgg cggtgggcga 180

ccggccgccc gcgccggcgg acccggtggg ccccgcgcgc cacgagcccc gcttccacgc 240

gctcggcttc cactcgcagc tcttctcgcc cggggacacg ttcgacctga tgccgcgcgt 300

ggtctcggac atgggcgact cgcgcgagaa ctttaccgcc acgctggact ggtactacgc 360

gcgcgcgccc ccgcggtgcc tgctgtacta cgtgtacgag ccctgcatct accacccgcg 420

cgcgcccgag tgcctgcgcc cggtggaccc ggcgtgcagc ttcacctcgc cggcgcgcgc 480

gcggctggtg gcgcgccgcg cgtacgcctc gtgcagcccg ctgctcgggg accggtggct 540

gaccgcctgc cccttcgacg ccttcggcga ggaggtgcac acgaacgcca ccgcggacga 600

gtcggggctg tacgtgctcg tgatgaccca caacggccac gtcgccacct gggactacac 660

gctcgtcgcc accgcggccg agtacgtcac ggtcatcaag gagctgacgg ccccggcccg 720

ggccccgggc accccgtggg gccccggcgg cggcgacgac gcgatctacg tggacggcgt 780

cacgacgccg gcgccgcccg cgcgcccgtg gaacccgtac ggccggacga cgcccgggcg 840

gctgtttgtg ctggcgctgg gctccttcgt gatgacgtgc gtcgtcgggg gggccatctg 900

gctctgcgtg ctgtgctccc ggcgccgggc ggcctcgcgg ccgttccggg tgccgacgcg 960

ggcgcggacg cacatgctct ctccggtgta caccagcctg cccacgcacg aggactacta 1020

cgacggcgac gacgacgacg acgaggaggc gggcgtcatc cgccggcggc ccgcctcccc 1080

cagcggagac agcggctacg aggggccgta cgcgagcctg gaccccgagg acgagttcag 1140

cagcgacgag gacgacgggc tgtacgtgcg ccccgaggag gcgccccgct ccggcttcga 1200

cgtctggttc cgcgatccgg agaaaccgga agtgacgaat ggacccaact atggcgtgac 1260

cgccaaccgc ctgttgatgt 1280