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carry type 1 bvdv-e rns Construction method of genetically high fecundity classical swine fever attenuated marker vaccine

A technology of attenuated classical swine fever and its construction method, which is applied in the field of construction of attenuated classical swine fever vaccine with high fecundity, can solve the problems of early immune protection not as good as that of C strain, biological safety hazards, etc., and achieve good cell adaptability and strong replication ability , the effect of improving growth ability

Active Publication Date: 2021-09-17
ZHEJIANG UNIV
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  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the backbone of the virus is BVDV, and the early immune protection effect after vaccination may not be as good as that of the C strain
In addition, the recombinant virus is chimeric with the E2 protein of the European virulent strain Alfort-187, which has not yet appeared in China, and there may be certain biological safety hazards after introduction

Method used

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  • carry type 1 bvdv-e <sup>rns</sup> Construction method of genetically high fecundity classical swine fever attenuated marker vaccine
  • carry type 1 bvdv-e <sup>rns</sup> Construction method of genetically high fecundity classical swine fever attenuated marker vaccine
  • carry type 1 bvdv-e <sup>rns</sup> Construction method of genetically high fecundity classical swine fever attenuated marker vaccine

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Embodiment 1: CSFV-C strain genome E rns Gene replacement to E of BVDV genotype 1 strain VEDEVAC rns Gene

[0039] According to BVDV1-VEDEVAC strain E rns Gene sequence (GenBank accession number: KC695814.1), entrusted to Suzhou Jinweizhi Biotechnology Co., Ltd. to synthesize, obtained BVDV1-VEDEVAC strain E rns Gene sequence of plasmid pUC57-B1E rns . Plasmid pUC57-B1E rns as a template, and B1E0-fwd / B1E0-rev as primers for PCR amplification to obtain BVDV1-VEDEVAC strain E rns Fragment A of the gene sequence. Using the infectious cloning plasmid pA-FL22 of CSFV-C strain as template and pA-B1E0-fwd / pA-B1E0-rev as primers, PCR amplification of pA-FL22 except E rns All sequences except the gene fragment (fragment B). Among the two pairs of primers for amplifying Fragment A and Fragment B, the upstream primer of A fragment and the downstream primer of B fragment and the downstream primer of A fragment and the upstream primer of B fragment respectively comprise a c...

Embodiment 2

[0044] Embodiment 2: CSFV-C strain E2 gene VR1 region replacement is the E2 gene VR1 fragment of CSFV epidemic strain QZ14

[0045] According to the VR1 gene sequence of the CSFV epidemic strain QZ14 strain (VR1 is the 7-126 nucleotide sequence of the E2 gene, as shown in Table 1), design specific primers VR1-fwd and VR1-rev; according to the recombinant plasmid pCSFV-C -B1E rns For the gene sequence, design and amplify specific primers pA-VR1-fwd and pA-VR1-rev (primers are shown in Table 1) for all other sequences except the CSFV-C strain VR1 gene. Genomic RNA of the popular CSFV strain QZ14 was extracted, reverse-transcribed into cDNA, and PCR amplified to obtain the VR1 fragment (fragment C) of the E2 gene of the QZ14 strain. With the recombinant plasmid pCSFV-C-B1E rns As a template, PCR amplifies all genome fragments (fragment D) except the CSFV-C strain E2 gene VR1 region. Among the two pairs of primers for amplifying Fragment C and Fragment D, the upstream primer of...

Embodiment 3

[0047] Embodiment 3: Specific 7 site mutations on the CSFV-C strain genome

[0048] Cloning pCSFV-C-B1E with Recombinant Infectious rns -VR1 is a template, through 7 pairs of primers carrying mutation sites (as shown in Table 2), using a one-step directional cloning method, the 3310th, 3433rd, 3531st, 4085th positions of the chimeric CSFV genome in the recombinant plasmid , 8286, 10332 and 11836 nucleotides were mutated to obtain an infectious clone pCSFV-Cm7-B1E containing 7 specific mutation sites rns -VR1( figure 1 ). The mutation results were T3310G, C3433T, G3531T, A4085G, C8286A, A10332G and A11836C. The corresponding amino acid changes are: M979R, A1020V, V1053L, I1237M, L2638I, S3320G and K3821T.

[0049] Table 2: Primers required to introduce 7 mutation sites into the C strain genome

[0050]

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Abstract

The invention relates to the field of biotechnology and aims to provide a kind of BVDV-E carrying type 1 BVDV-E rns Construction method of genetically high-productivity swine fever attenuated marker vaccine. The present invention utilizes molecular cloning and reverse genetic methods to replace the Erns and VR1 regions of the E2 gene of the CSFV-C strain genome with the Erns gene of BVDV1 and the VR1 region of the CSFV epidemic strain QZ14 respectively, and 7 specific genes of the C strain genome are substituted. A mutation was introduced into the site to obtain the recombinant virus rC‑Marker1. The recombinant virus carries an exogenous marker (BVDV Erns), the VR1 region of the E2 gene of the CSFV epidemic strain and a specific mutation site, which lays the foundation for the development of a swine fever marker vaccine suitable for an in vitro cell culture system. The recombinant chimeric swine fever virus attenuated marker vaccine strain constructed by this technology can quickly determine whether the positive serum of swine fever virus is caused by wild virus infection or vaccination. The circulating strains have better neutralization ability, which can reduce the cost of vaccine production.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a type 1 BVDV-E carrier rns A method for constructing a genetically highly fecund attenuated vaccine for classical swine fever. Background technique [0002] Swine fever is one of the 17 first-class animal diseases stipulated in my country, which poses a great threat to the pig industry. Swine fever is a highly contagious and fatal infectious disease. The course of acute swine fever is short, manifested as persistent high fever, generalized capillary hemorrhage and splenic infarction. Chronic swine fever manifests as slow growth of piglets, reproductive disorders, abortion, stillbirth, and weak birth of sows. Necropsy revealed lymph node hemorrhage and splenic infarction. The disease is still prevalent in Asian countries including my country, Eastern Europe and South America. Classical swine fever is caused by classical swine fever virus (CSFV), which is a single-stranded positive...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/01C12N15/65A61K39/187A61P31/14
CPCA61K39/12A61K2039/5254A61K2039/552A61P31/14C12N7/00C12N15/65C12N2770/24321C12N2770/24334C12N2770/24351
Inventor 方维焕王作欢李肖梁曹统
Owner ZHEJIANG UNIV
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