Pseudorabies virus gE/gI deletion mutant strain of double expression gC gene and construction and application thereof

A technology of pseudorabies virus and porcine pseudorabies virus, which is applied in the field of animal virology and genetic engineering, can solve the problems of ineffective resistance to infection by virulent mutant strains, and achieve good immune protection effect, good immune effect, and neutralizing antibodies high level effect

Pending Publication Date: 2019-05-14
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The technical problem to be solved by the present invention is to overcome the defect that the porcine pseudorabies virus vaccine in the existing market cannot effectively resist the infection of the

Method used

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  • Pseudorabies virus gE/gI deletion mutant strain of double expression gC gene and construction and application thereof
  • Pseudorabies virus gE/gI deletion mutant strain of double expression gC gene and construction and application thereof
  • Pseudorabies virus gE/gI deletion mutant strain of double expression gC gene and construction and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Example 1 Construction of homologous recombination vector (pMD-LA-RA; pMD-LA-RA-EGFP; pMD-LA-gC-RA)

[0064] 1. According to the gene sequence of the PRVZJ01 strain registered in GenBank (accession number: KM061380.1), using the PRV-AH strain of the pseudorabies virus variant as a template, design 2 pairs of primers LA-F / LA-R and RA-F / RA- R (Table 1) is used to amplify the homologous recombination arm fragments located on both sides of the gI gene and the gE gene (including part of the gI and gE genes), wherein the left arm fragment is referred to as LA, and the right arm fragment is referred to as RA.

[0065] 2. Connect the LA fragment and the pMD18-T vector by TA cloning, and then transform and screen to obtain the recombinant plasmid pMD-LA; the recombinant plasmid pMD-LA and RA fragments are double-digested with restriction endonucleases HindШ and PstI respectively , the digested product was ligated and transformed, and the recombinant plasmid pMD-LA-RA was obtaine...

Embodiment 2

[0073] Construction and purification of embodiment 2 recombinant virus

[0074] 1. Recombinant virus rPRV-AH-gI - / gE - / EGFP + the acquisition

[0075] (1) Inoculate monolayer BHK-21 cells with 0.1 MOI of pseudorabies virus mutant strain PRV-AH, and when 70% to 80% of the cells have lesions, it is repeatedly frozen and thawed 3 times, and the PRV-AH virus liquid is harvested;

[0076] (2) Transfection was performed on a 24-well cell culture plate, and the transfection was performed when the BHK-21 cells grew to 80%. refer to 2000Reagent transfection reagent instructions, such as figure 1 As shown in the construction strategy, the recombinant plasmid pMD-LA-RA-EGFP was transfected into BHK-21 cells. After 4 hours of transfection, 0.1 MOI of PRV-AH virus solution was inserted to allow homologous recombination of the plasmid and the viral genome in the cells. , and set up a transfection control group containing only the recombinant plasmid pMD-LA-RA-EGFP at the same time;...

Embodiment 3

[0084] The drawing of embodiment 3 recombinant virus one-step growth curves

[0085] 1. One-step virus growth curve drawing

[0086] rPRV-AH-gI - / gE - / gC + , rPRV-AH-gI - / gE - and PRV-AH strain were inoculated in the culture plate with BHK-21 cells at 1MOI respectively, at 37°C, 5% CO 2 After incubating in a cell culture box for 1 hour, discard the solution, wash twice with PBS, discard the solution, and add DMEM cell maintenance solution containing 2% fetal bovine serum. Collect the virus for the first time 1 hour after inoculation. The virus was harvested every 4 hours between 4 and 72 hours, and the collected virus was stored at -80°C. The virus titers were measured after repeated freezing and thawing of the viruses collected in all time periods three times, and the growth curves of the three viruses within 72 hours were drawn.

[0087] 2. Conclusion

[0088] Such as Figure 8 Shown, PRV-AH, rPRV-AH-gI - / gE - , rPRV-AH-gI - / gE - / gC + The three viruses h...

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Abstract

The present invention discloses a pseudorabies virus gE/gI deletion mutant strain rPRV-AH-gI-/gE-/gC+ of a double expression gC gene. The pseudorabies virus gE/gI deletion mutant strain is positionedat the position of missing gE/gI gene of recombinant pseudorabies virus rPRV-AH-gI-/gE and is constructed by inserting the gC gene; the nucleotide sequence of the gI/gE gene is shown as SEQ ID NO.1, and the nucleotide sequence of the gC gene is shown as SEQ ID NO.2. Compared with other PRV gene deletion mutant strains, the recombinant pseudorabies virus rPRV-AH-gI/gE-/gC+ inserts the gC gene innovatively by taking the gC gene as one of the main immunogenic proteins of PRV on the basis of the deletion of the gI/gE gene, and induces a body to produce a neutralizing antibody and a cellular immuneresponse. The invention further provides rPRV-AH-gI-/gE-/gC+ inactivated vaccine. The inactivated vaccine has better safety performance, and has better immunogenicity, higher neutralizing antibody level and better immune protection effect compared with an existing PRV mutant strain, can distinguish wild virus infected animals from vaccine immunized animals by an existing PRV gE differentiation diagnosis method, and is expected to be used as a genetic engineering inactivated vaccine for preventing and treating novel epidemic strains of porcine pseudorabies virus.

Description

technical field [0001] The invention relates to the technical fields of animal virology and genetic engineering, and more specifically relates to a pseudorabies virus gE / gI deletion mutant strain double-expressing gC gene and its construction and application. Background technique [0002] Pseudorabies is an acute infectious disease caused by pseudorabies virus (PRV). The main clinical symptoms include fever, nervous system symptoms, and respiratory symptoms. Pigs are the only natural host of PRV. PRV is characterized by high latent infection and is widespread in many countries. The control of pseudorabies is using widely effective inactivated and attenuated live vaccines. Since the 1970s, Bartha-K61 vaccine was introduced into China, and pseudorabies has been effectively controlled in China. However, since 2011, many large-scale pig farms immunized with live PRV vaccines have experienced sow abortion, stillbirth, and mummified fetuses, and piglets have clinical symptoms s...

Claims

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Application Information

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IPC IPC(8): C12N7/01C12N15/63C12N15/65C12N15/66C12N15/90A61K39/245A61P31/22C12R1/93
Inventor 琚春梅唐栋陈美静任小波颜志斌吴晓燕潘慧李艳华
Owner SOUTH CHINA AGRI UNIV
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