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Constructing method of high-fecundity hog cholera attenuated-virus marking vaccine carrying type 2 BVDV-E<rns> gene

A swine fever attenuated virus and construction method technology, which is applied in the field of construction of a high-productivity swine fever attenuated marker vaccine, can solve problems such as hidden dangers of biological safety, and achieve the effects of improving neutralization ability, good immunogenicity and good genetic stability.

Active Publication Date: 2019-10-15
ZHEJIANG UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, since the virus skeleton is BVDV, and the chimeric swine fever virus fragment comes from the virulent strain Alfort-187 that was once prevalent in Europe, there may be certain biological safety hazards if introduced

Method used

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  • Constructing method of high-fecundity hog cholera attenuated-virus marking vaccine carrying type 2 BVDV-E&lt;rns&gt; gene
  • Constructing method of high-fecundity hog cholera attenuated-virus marking vaccine carrying type 2 BVDV-E&lt;rns&gt; gene
  • Constructing method of high-fecundity hog cholera attenuated-virus marking vaccine carrying type 2 BVDV-E&lt;rns&gt; gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1: CSFV-C strain genome E rns Gene replacement with E of BVDV genotype 2 strain 890# rns Gene

[0038] According to BVDV2-890# strain E rns The gene sequence (GenBank accession number: NC_039237.1) was entrusted to Suzhou Jinweizhi Biotechnology Co., Ltd. to synthesize, and obtained strain E containing BVDV2-890# rn Gene sequence plasmid pUC57-B2E rns . with plasmid pUC57-B2E rns As the template, B2E0-fwd / B2E0-rev was used as the primer to carry out PCR amplification to obtain E containing BVDV2-890# strain rns Fragment A of the gene sequence. Using CSFV-C strain infectious cloning recombinant plasmid pA-FL22 as template, pA-B2E0-fwd / pA-B2E0-rev as primers, PCR amplification pA-FL22 except E rns All sequences except gene fragments (fragment B). In the two pairs of primers for amplifying fragment A and fragment B, the upstream primer of fragment A and the downstream primer of fragment B, and the downstream primer of fragment A and the upstream primer of f...

Embodiment 2

[0043] Example 2: Replacement of VR1 region of E2 gene of CSFV-C strain with VR1 fragment of E2 gene of CSFV epidemic strain QZ14

[0044] Using the genomic cDNA of CSFV type 2 strain QZ14 as a template, VR1-fwd and VR1-rev as primers, amplify the VR1 fragment of the E2 gene of the QZ14 strain (VR1 is the 7-126 nucleotide sequence of the E2 gene, as shown in the table 1) to obtain fragment C, and the primer sequences used are shown in Table 1. with recombinant plasmid pCSFV-C-B2E rns As template, pA-VR1-fwd and pA-VR1-rev as primers, amplify the infectious clone pCSFV-C-B2E rns Fragment D was obtained from all the genome sequences except the VR1 region of the CSFV-C strain E2 gene. In the two pairs of primers for amplifying fragment C and fragment D, the upstream primer of fragment C and the downstream primer of fragment D, and the downstream primer of fragment C and the upstream primer of fragment D respectively contain 20bp complementary sequences (shown as underlined in T...

Embodiment 3

[0046] Example 3: 7 specific site mutations on the genome of CSFV-C strain

[0047] Cloning of pCSFV-C-B2E with recombinant infectious rns -VR1 is the template, through 7 pairs of primers carrying mutation sites (as shown in Table 2), using the method of one-step directional cloning, the 3310th, 3433rd, 3531st, 4085th, Nucleotides at positions 8286, 10332 and 11836 were mutated to obtain an infectious clone pCSFV-Cm7-B2E 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.

[0048] Table 2: Primers required to introduce 7 mutation sites into the genome of strain C

[0049]

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Abstract

The invention relates to the technical field of biology, and aims to provide a constructing method of a high-fecundity hog cholera attenuated-virus marking vaccine carrying a type 2 BVDV-E<rns> gene.A molecular cloning and reverse heredity method is adopted, an E<rns> gene of a BVDV2-890# virus strain and the VR1 region of an E2 gene of a CSFV epidemic virus strain QZ14 are displaced with a genetic fragment corresponding to a CSFV-C strain, 7 specific mutation sites are introduced, and a recombinant virus rC-Marker2 is obtained. The obtained recombinant virus has a molecular marker (BVDV2 E<rns>), VR1 of the type 2 epidemic virus strain of the hog cholera virus and a specific mutation site, and a base is established for developing a marking vaccine adapted to an in vitro cell culture system. When the recombinant chimeric attenuated-virus making vaccine strain of the hog cholera virus, constructed through a technique, is applied, whether a hog with a positive hog cholera virus antibodyof a hog suffers from vaccine inoculation or wild virus infection can be judged by a serological method, the high-fecundity hog cholera attenuated-virus marking vaccine has better neutralization effects on the current epidemic gene type 2 hog cholera viruses; and the high-fecundity hog cholera attenuated-virus marking vaccine shows favorable growth capacity in an in vitro cell culture system, andthe production cost of the vaccine can be reduced.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a construction method of a high-productivity swine fever attenuated marker vaccine carrying type 2 bovine viral diarrhea virus Erns gene. Background technique [0002] Swine fever is a kind of infectious disease that must be notified in my country, which seriously threatens the healthy development of the pig industry. Swine fever is a highly contagious and lethal disease. Acute swine fever is characterized by a short course of disease, missed high fever, generalized spotty hemorrhage and splenic infarction. Chronic swine fever is mostly manifested as stunted growth of piglets, sow abortion, stillbirth, weak child, and button ulcers in the ileocecal loop at necropsy. Swine fever is caused by swine fever virus (CSFV). CSFV continues to evolve under the pressure of vaccine immunity, resulting in the virus still circulating in Eastern Europe, Asia, Latin America and other regions. The s...

Claims

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Application Information

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IPC IPC(8): C12N15/65C12N15/87C12N7/01C12Q1/70A61K39/187A61P31/14
CPCC12N15/65C12N15/87C12N7/00C12Q1/701A61K39/12A61P31/14C12N2710/12021C12N2770/24334A61K2039/552A61K2039/5254
Inventor 方维焕王作欢李肖梁曹统
Owner ZHEJIANG UNIV
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