Preparation method of soluble protein of GyV8 gyrovirus VP3

A circular virus, soluble technology, applied in the field of preparation of GyV8 new circular virus VP3 soluble protein, can solve problems such as low efficiency and cumbersome cloning process

Inactive Publication Date: 2017-08-01
YANGZHOU UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But sometimes the cloning process is cumbersome and inefficient due to the inability to find a suitable restriction site

Method used

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  • Preparation method of soluble protein of GyV8 gyrovirus VP3
  • Preparation method of soluble protein of GyV8 gyrovirus VP3
  • Preparation method of soluble protein of GyV8 gyrovirus VP3

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Embodiment

[0033] 1) Design and amplify the pGEX-6p-1 linearized vector and GyV8 virus VP3 gene fragment primers: the upstream primer for amplifying the pGEX-6p-1 linearized vector is located at position 1022-1047 of the pGEX-6p-1 plasmid; and at the 5' end With an extra TAA base; the downstream primer for amplifying the pGEX-6p-1 linearization vector is located at positions 916-941 of the pGEX-6p-1 plasmid; and with an extra CAT base at the 5' end. The upstream primers for amplifying the VP3 gene of GyV8 virus include the VP3 gene start codon ATG and the following 14 bases, and there are 15 additional bases at the 5' end that are reverse complementary to the downstream primers of the pGEX-6p-1 linearized vector ; Amplify GyV8 virus VP3 gene downstream primers include VP3 gene stop codon TAA and its first 14 bases, and have 15 additional bases reverse complementary to the upstream primer of the pGEX-6p-1 linearized vector at the 5' end . See attached table 1 for specific primer sequence...

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Abstract

The invention relates to a preparation method of soluble protein of novel GyV8 gyrovirus VP3. The preparation method comprises the following steps of using a pGEX-6p-1 linear carrier and a primer of a GyV8 virus VP3 gene segment to directly and quickly recombine and clone VP3 in vitro through recombinase ExnaseTMII without enzyme-cut and link up reaction, converting colibacillus coli, and performing IPTG (isopropyl-beta-d-thiogalactoside) inducing expression to realize the fusion soluble expression of GyV8 VP3 protein and GST (glutathione transferase), and obtain purified VP3 soluble protein. The preparation method has the advantages that the GyV8 virus VP3 gene is cloned by the recombinase ExnaseTM II in-vitro recombinant technique without relying on the enzyme cutting site and restriction endonuclease, the cloning process is simplified, and the quick cloning and expression of VP3 gene is realized.

Description

technical field [0001] The invention relates to a preparation method of VP3 soluble protein of GyV8 novel circular virus. This invention can directly provide VP3 soluble protein as GyV8 diagnostic antigen; obtain anti-VP3 polyclonal antibody as immunogen; provide effective immunological reagents for GyV8 serological epidemiological investigation and VP3 function research, filling the gap at home and abroad. Background technique [0002] Gyrovirus belongs to the genus Circovirus of the family Orbiviridae, which is a small non-enveloped icosahedral symmetrical single-stranded circular DNA virus with a size of more than 2 KB. Chicken Infectious Anemia Virus (CAV) has been considered the only member of the genus Gyrovirus in the family Circoviridae. Until 2011, Rijsewijk et al. detected a new type of Circovirus sequence, the second member of the Circovirus genus, named AGV2 from serum samples of infected chickens. In the same year, Sauvage et al. detected the first human circu...

Claims

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Application Information

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IPC IPC(8): C07K14/01C12N15/70C12N15/64C07K16/08C12R1/19
Inventor 叶建强刘敏邵红霞秦爱建季星妤施洋洋韦芊含
Owner YANGZHOU UNIV
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