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Recombinant adeno-associated virus transfer vector containing variant porcine pseudorabies virus gd protein gene, virus and preparation and application thereof

A porcine pseudorabies virus and porcine pseudorabies technology, applied in the field of genetic engineering, can solve the problems of loss of antigenic determinants, complex components of inactivated vaccines, and high cost, and achieve broad market application prospects, good immune protection, and good immunogenicity Effect

Active Publication Date: 2022-08-05
BRAINVTA (WUHAN) CO LTD +1
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AI Technical Summary

Problems solved by technology

Classical vaccines have poor protection against mutant strains. It is necessary to use the clinically isolated mutant pseudorabies virus as a gene sample to develop a more effective vaccine to prevent and control pseudorabies. However, the whole virus is used to prepare inactivated vaccines and gene-deleted attenuated vaccines. It is cumbersome and costly, so DNA vaccines, subunit vaccines and live vector vaccines will be the main directions for the development of PRV vaccines
[0006] The commercial vaccine used as a control in this experiment is the porcine pseudorabies inactivated vaccine commercialized by Keqian Biotechnology Company. This commercial vaccine plays an important role in the prevention and control of PR. The loss of antigenic determinants leads to poor immune efficacy, requiring a large vaccination dose and multiple immunizations to achieve a good immune effect, and the components of inactivated vaccines are complex, and allergic reactions often occur

Method used

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  • Recombinant adeno-associated virus transfer vector containing variant porcine pseudorabies virus gd protein gene, virus and preparation and application thereof
  • Recombinant adeno-associated virus transfer vector containing variant porcine pseudorabies virus gd protein gene, virus and preparation and application thereof
  • Recombinant adeno-associated virus transfer vector containing variant porcine pseudorabies virus gd protein gene, virus and preparation and application thereof

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Experimental program
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Effect test

Embodiment 1

[0088] 1. Primer design and PCR amplification

[0089] Using the genome of porcine pseudorabies virus wild virus strain PRV / JXFC / 2015 as the PCR template, the analysis software Oligo6.0 was used to design primers to amplify the gB and gD genes, and the amplified fragments were about 2718bp and about 1203bp, respectively. The primer sequences are as follows:

[0090] Table 1: PCR primers

[0091]

[0092] a: Sal I and Spe I restriction sites were designed at both ends. The left part of the primer sequence is the homology arm sequence, the italic part is the sequence of the Sal I and Spe I restriction sites, the rear of the restriction site is the transcription enhancer kozak sequence, and the underlined part is the gD target gene primer sequence.

[0093]b: XbaI and Sal I restriction sites were designed at both ends respectively. The left part of the primer sequence is the homology arm sequence, the italicized part is the XbaI, Sal I restriction site sequence, behind the ...

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Abstract

The invention belongs to the technical field of genetic engineering, and more particularly relates to a recombinant adeno-associated virus transfer vector containing the gD protein gene of mutated porcine pseudorabies virus, a recombinant adeno-associated virus, and its preparation and application. The recombinant adeno-associated virus (rAAV) prepared by the invention is used as a vector to express the gD protein of porcine pseudorabies virus, and the rAAV has various advantages such as safety, effectiveness and specificity. The genetic engineering strain prepared by the invention expresses the gD immunogenic protein of the circulating mutant porcine pseudorabies virus strain, and has good immune protection against multiple circulating mutant porcine pseudorabies virus strains. A single injection into the right lower limb tibialis anterior muscle in mice can induce sustained high levels of specific neutralizing antibodies and cellular immunity.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and more particularly, relates to a recombinant adeno-associated virus transfer vector containing a mutated porcine pseudorabies virus gD protein gene, a recombinant adeno-associated virus, and its preparation and application. Background technique [0002] Pseudorabies (PR) is caused by pseudorabies virus (PRV), and the main symptoms are fever, pruritus and encephalitis in livestock (pigs, cattle, sheep) and wild animals. Because the clinical symptoms are similar to rabies, Therefore, it is also called pseudorabies. PRV has a wide range of hosts in nature, with pigs being the main natural and storage host. PRV can infect pigs of different ages and cause different clinical symptoms, especially the most serious damage caused by infection of pregnant sows and suckling piglets. PRV causes reproductive disorders in pregnant sows, and neurological symptoms in suckling piglets lead to high...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/864C12N15/66C12N7/01A61K39/245A61P31/22C12R1/93
CPCC12N15/86C12N15/66C07K14/005C12N7/00A61K39/12A61P31/22C12N2710/16722C12N2750/14143C12N2750/14121C12N2750/14152A61K2039/552A61K2039/54C12N2710/16734
Inventor 张胜何晓斌潘杏方文晶冯诚
Owner BRAINVTA (WUHAN) CO LTD
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