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Recombinant porcine pseudorabies virus for expressing GP protein of porcine reproductive and respiratory syndrome virus, and application

A technology for porcine pseudorabies and respiratory syndrome, applied in the direction of viruses, antiviral agents, virus antigen components, etc., can solve the problems of serious harm and lack of prevention and control measures, and achieve strong pertinence, strong immune protection effect, and comprehensive antigen types. Effect

Active Publication Date: 2019-12-31
WUHAN KEQIAN BIOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disease is very harmful and currently lacks effective control measures

Method used

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  • Recombinant porcine pseudorabies virus for expressing GP protein of porcine reproductive and respiratory syndrome virus, and application
  • Recombinant porcine pseudorabies virus for expressing GP protein of porcine reproductive and respiratory syndrome virus, and application
  • Recombinant porcine pseudorabies virus for expressing GP protein of porcine reproductive and respiratory syndrome virus, and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Construction of recombinant porcine pseudorabies virus expressing porcine reproductive and respiratory syndrome virus GP protein

[0037] The present invention utilizes the advantages of the Crispr / Cas9 gene editing system and the Cre / lox recombinase system to quickly edit the viral genome, deletes the PRV SDN8 virulence genes TK, gI and gE, and uses the gene-deleted strain as a carrier, and in its gG gene Insert the antigen gene of PRRSV NADC30-like (including the non-transmembrane region coding sequence of GP3, GP4, GP5 and GP6 proteins), and develop live vaccines based on the GP gene of porcine pseudorabies virus recombinant porcine PRRS virus.

[0038] 1. sgRNA sequence design and recombinant vector construction

[0039] According to the TK, gE, gI and gG virulence gene sequences, online (http: / / crispr.mit.edu / ) design the single guide RNA (sgRNA) sequence targeting the deletion sequence, synthesize single-stranded DNA primers, anneal and ligate and trans...

Embodiment 2

[0061] Embodiment 2 recombinant porcine pseudorabies virus PRV SDN8-TK - / gE - / gI - / gG - -GP + Safety Tests on Mice

[0062] Purchase 30 6-8 week-old Kunming mice (gB, gE antibody and GP5 antibody are negative), randomly divided into A, B, C 3 groups, A group back subcutaneously injected 10 6 TCID 50 PRV SDN8-TK - / gE - / gI - / gG - -GP + Recombinant virus; group B back subcutaneously injected 10 6 TCID 50 For the PRV JS2012 wild-type strain, about an equal volume of serum-free DMEM medium was subcutaneously injected into the back of group C. Continuous observation for 7 days.

[0063] The results showed that the mice in groups A and C had no disease, and their spirit and appetite were good; the mice in group B all died on the third day after injection. Indicating that PRV SDN8-TK - / gE- / gI- / gG - -GP + The safety of the recombinant virus is good and can be used as a live vaccine.

Embodiment 3

[0064] Embodiment 3 recombinant porcine pseudorabies virus PRV SDN8-TK - / gE - / gI - / gG - -GP + Immunogenicity test on piglets

[0065] Select 20 21-day-old pseudorabies and PRRS-negative piglets (negative for gB, gE and GP5 antibodies), and randomly divide them into groups A, B, C and D, with 5 piglets in each group, and keep them in isolation. Intramuscular injection of PRV SDN8-TK in groups A and B - / gE - / gI - / gG - -GP + Strains (1.0×10 6.0 TCID 50 per head), C and D groups were injected with 1mL DMEM as unimmunized control. Piglets did not have any clinical symptoms after immunization. On the 21st day after immunization, blood was collected from the anterior vena cava in all four groups. Anti-gB ELISA (purchased from IDEXX Company) was used to detect gB antibody levels in groups A and C, and anti-GP5 ELISA (purchased from IDEXX Company) was used in groups B and D. Detect the level of GP5 antibody; and use PRVJS2012 strain and PRRSV NADC30-like strain to co...

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Abstract

The invention provides a recombinant porcine pseudorabies virus for expressing GP protein of a porcine reproductive and respiratory syndrome virus, and an application. A PRV virus strain genome is quickly edited through a Crispr / Cas9 gene editing technique and a Cre / lox recombination system, virulence genes gE, gI and TK of the PRV virus strain genome are subjected to fixedpoint deletion, and an antigenic gene of a NADC30-like strain is subjected to fixedpoint insertion at a gG position. According to the recombinant porcine pseudorabies virus for expressing GP protein of a porcine reproductiveand respiratory syndrome virus disclosed by the invention, non-transmembrane regional coding sequences of GP3 protein, GP4 protein, GP5 protein and GP6 protein of an epidemic PRRSV strain PRRSV NADC30-like are selected as antigenic genes for the first time, the kinds of antigens are more comprehensive, and the antigenic genes have higher applicability on current PRRSV epidemic situations, and arebetter in immunoprotection effects. A live vaccine provided by the invention can protect target animals from being invaded by PRV and PRRSV in a more pointed manner, and a powerful tool is provided for preventing and controlling epidemic situations of porcine pseudorabies and porcine reproductive and respiratory syndromes in China.

Description

technical field [0001] The invention belongs to the fields of biotechnology and preventive veterinary medicine, and in particular relates to a recombinant porcine pseudorabies virus expressing the GP protein of porcine reproductive and respiratory syndrome virus and its application. Background technique [0002] Pseudorabies virus (Pseudorabies virus, PrV) is the pathogen that causes pseudorabies in many animals. Pigs are the natural host of PRV, and many other animals can also be infected. The disease occurs in outbreaks in pigs, causing reproductive failure in sows and mass mortality in newborn piglets. At present, the prevention and treatment of pseudorabies mainly depends on vaccination. PRV Bartha-K61 strain is an artificially attenuated vaccine strain obtained by Bartha et al. in the 1960s by passing the isolated PRV wild strain through chicken embryo fibroblasts. Its virulence is greatly weakened, and its immunogenicity Well, it has been used for decades at home an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01C12N15/85C12N15/90A61K39/12A61K39/245A61P31/22A61P31/14C12R1/93
CPCA61K39/12A61K2039/5256A61K2039/552A61K2039/70A61P31/14A61P31/22C12N7/00C12N15/85C12N15/907C12N2710/16721C12N2710/16734C12N2710/16752C12N2770/10022C12N2770/10034C12N2800/107
Inventor 张华伟徐高原孙芳郝根喜周明光罗修鑫范金秀汤细彪
Owner WUHAN KEQIAN BIOLOGY CO LTD
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