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A kind of soluble protein of agv2 type circular virus vp3 and preparation method thereof

A circular virus and soluble technology, applied in the field of soluble proteins, can solve the problems of cumbersome cloning process and low efficiency, and achieve the effect of simplifying the cloning process

Inactive Publication Date: 2016-12-21
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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  • A kind of soluble protein of agv2 type circular virus vp3 and preparation method thereof
  • A kind of soluble protein of agv2 type circular virus vp3 and preparation method thereof
  • A kind of soluble protein of agv2 type circular virus vp3 and preparation method thereof

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Embodiment 1

[0033] 1) Design the primers for amplifying the pGEX-6p-1 linearized vector and the VP3 gene fragment of AGV2 virus: the upstream primer for amplifying the pGEX-6p-1 linearized vector is located at positions 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 the AGV2 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 AGV2 virus VP3 gene downstream primers include the 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 . The specific primer sequen...

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Abstract

The invention discloses an AGV2 (avian gyrovirus 2) type soluble VP3 (viral protein 2) and a preparation method thereof. The preparation method comprises the following steps: using a pGEX-6p-1 linearization carrier and a primer of an AGV2 VP3 gene segment; directly and quickly recombining and cloning VP3 in vitro by using recombinase ExnaseTM II without carrying out enzyme digestion ligation reaction; converting escherichia coli; carrying out inducible expression through IPTG, so as to achieve the fusion soluble expression of VP3 of AGV2 and GST and obtain purified soluble VP3. The AGV2 VP3 soluble expression and purified protein obtained through the preparation method can directly provide the soluble VP3 as an AGV2 diagnostic antigen and as an immunogen to obtain an anti-VP3 polyclonal antibody, provides an effective immunology reagent for carrying out AGV2 serology epidemiology researches and VP3 functional study, fills up the blank at home and abroad, and has an important meaning for further exploring VP3 biological function.

Description

technical field [0001] The invention relates to a soluble protein, in particular to an AGV2 type orbivirus VP3 soluble protein and a preparation method thereof. Background technique [0002] Chicken infectious anemia virus (CAV) has been considered the only member of the circovirus genus. 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 sequence of a new type of human circular virus HGyV in skin cotton samples of healthy people. Sequence analysis surprisingly found that the genome sequence homology between HGyV and AGV2 was as high as 96%. Recently, Maggi and Biagini et al. also detected DNA sequences of HGyV / AGV2 in serum samples of healthy people, organ transplant patients and HIV-positive patients. In China, Ye Jianqiang and others detected and reported the existence of AGV2 in chicken flocks and huma...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/01C12N15/70C12P21/02C12R1/19
CPCC07K14/01C12N15/70
Inventor 叶建强田晓彦施洋洋邵红霞秦爱建谢泉夏驰超周晓祥范中雷
Owner YANGZHOU UNIV
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