carry type 2 bvdv-e rns Construction method of genetically high fecundity classical swine fever attenuated marker vaccine

A technology of attenuated classical swine fever virus and construction method, which is applied in the field of construction of attenuated classical swine fever vaccine with high fecundity, can solve biological safety hazards and other problems, and achieve the effects of strong replication ability, good cell adaptability, and good neutralization ability

Active Publication Date: 2022-05-13
ZHEJIANG UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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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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  • carry type 2 bvdv-e <sup>rns</sup> Construction method of genetically high fecundity classical swine fever attenuated marker vaccine
  • carry type 2 bvdv-e <sup>rns</sup> Construction method of genetically high fecundity classical swine fever attenuated marker vaccine
  • carry type 2 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

[0037] Embodiment 1: CSFV-C strain genome E rns Gene replacement into 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 synthesized by entrusting Suzhou Jinweizhi Biotechnology Co., Ltd. to obtain the BVDV2-890# strain E rn Gene sequence of plasmid pUC57-B2E rns . Plasmid pUC57-B2E rns As a template, B2E0-fwd / B2E0-rev is used as primers for PCR amplification to obtain BVDV2-890# strain E rns Fragment A of the gene sequence. Using CSFV-C strain infectious clone recombinant plasmid pA-FL22 as template, pA-B2E0-fwd / pA-B2E0-rev as primers, PCR amplified 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 comp...

Embodiment 2

[0043] Embodiment 2: CSFV-C strain E2 gene VR1 region replacement is the E2 gene VR1 fragment of CSFV epidemic strain QZ14

[0044] In order to obtain the E2 gene VR1 fragment of CSFV epidemic strain QZ14 (VR1 is the 7th-126 nucleotide sequence of E2 gene, as shown in SEQ ID NO.5 in the sequence listing), the genome cDNA of CSFV type 2 strain QZ14 As a template, VR1-fwd and VR1-rev were used as primers to amplify to obtain Fragment C. The sequences of the primers used are shown in Table 1. With the recombinant plasmid pCSFV-C-B2E rns As a template, pA-VR1-fwd and pA-VR1-rev are used as primers to amplify the infectious clone pCSFV-C-B2E rns Fragment D was obtained for all genome sequences except the VR1 region of the E2 gene of the CSFV-C strain. Among the two pairs of primers for amplifying Fragment C and Fragment D, the upstream primer of C fragment and the downstream primer of D fragment and the downstream primer of C fragment and the upstream primer of D fragment respect...

Embodiment 3

[0046] Embodiment 3: Specific 7 site mutations on the CSFV-C strain genome

[0047] Cloning pCSFV-C-B2E with Recombinant Infectious rns -VR1 is used as a template, and the 3310th, 3433rd, 3531st, 4085th, 4085th, 3433rd, 3531th, 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 C strain genome

[0049]

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Abstract

The invention relates to the field of biotechnology and aims to provide a method for constructing a high-fecundity attenuated swine fever marked vaccine carrying type 2 BVDV-Erns gene. The present invention utilizes molecular cloning and reverse genetic method, the E of BVDV2‑890# strain rns Gene and CSFV epidemic strain QZ14 E2 gene VR1 region replaced the corresponding gene fragment of CSFV-C strain, and introduced 7 specific mutation sites, and obtained the recombinant virus rC-Marker2. The obtained recombinant virus carries a molecular marker (BVDV2 E rns ), VR1 and specific mutation sites of classical swine fever virus type 2 strains, laid the foundation for the development of labeled vaccines adapted to in vitro cell culture systems. The recombinant chimeric CSFV attenuated labeled vaccine strain constructed by this technology can determine whether the pigs positive for CSFV antibodies are caused by vaccination or wild virus infection by serological methods; Good neutralizing effect; good growth ability in in vitro cell culture system, which can reduce the production cost of the vaccine.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a method for constructing a high-fecundity swine fever attenuated labeled 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 pig industry. Swine fever is a highly contagious and fatal disease. Acute swine fever manifests as short course of disease, persistent high fever, generalized spotting of the whole body, and splenic infarction. Chronic swine fever is mostly manifested as stunted growth of piglets, miscarriage, stillbirth, and weak birth of sows, and button-shaped ulcers in the ileocecal loop can be seen in necropsy. Swine fever is caused by swine fever virus (CSFV). CSFV continues to evolve under the pressure of vaccine immunity, causing the virus to still circulate in Eastern Europe, Asia, Latin America ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/65C12N15/87C12N7/01C12Q1/70A61K39/187A61P31/14
CPCC12N15/65C12N15/87C12N7/00C12Q1/701A61K39/12A61P31/14C12N2710/12021C12N2770/24334A61K2039/552A61K2039/5254
Inventor 方维焕曹统李肖梁王作欢
Owner ZHEJIANG UNIV
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