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Method for constructing recombinant insect baculovirus expression vector of PVC2 (porcine circovirus type 2) Cap-labeled protein

A technology of baculovirus and labeled protein, which is applied in the direction of virus/bacteriophage, virus, virus peptide, etc., can solve the problems of time-consuming, laborious and cumbersome operation process, and achieve the effect of good experimental operation method

Inactive Publication Date: 2018-06-19
GUIZHOU UNIV
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Problems solved by technology

However, the fusion PCR method they used in introducing tags uses at least 3 pairs of primers, which is cumbersome and time-consuming

Method used

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  • Method for constructing recombinant insect baculovirus expression vector of PVC2 (porcine circovirus type 2) Cap-labeled protein
  • Method for constructing recombinant insect baculovirus expression vector of PVC2 (porcine circovirus type 2) Cap-labeled protein
  • Method for constructing recombinant insect baculovirus expression vector of PVC2 (porcine circovirus type 2) Cap-labeled protein

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[0023] 1 material

[0024] ■1.1 Virus strains, plasmids and cells

[0025] The virus PCV2GZ-RH1 strain isolated and preserved by the Institute of Animal Diseases, Guizhou University; DH10Bac Escherichia coli competent, Sf9 cells, pFastBacHTA, pFastBacDual plasmids are products of Invitrogen; Top10 Escherichia coli competent, pMD19-TSimpleVector are Bao Biological Engineering (Dalian ) Ltd. products.

[0026] ■1.2 Main Reagents

[0027] TaKaRa MinBEST Viral DNA / RNA Extraction Kit Ver.5.0 Nucleic Acid Extraction Kit, DNADL2000Marker, DNADL5000Marker, TaKaRaLATaq Enzyme, Prime Script One Step RT-PCRKitVer.2 Kit, T4DNA Ligase, Restriction Enzymes XhoI, SphI, Hind Ⅲ and BamH I, etc. are products of Bao Biological Engineering (Dalian) Co., Ltd.; E.Z.N.A. TM Gel Extraction Kit (50) is a product of Omega; Quick Pure Plasmid Mini kit is a product of Kangwei Century Biotechnology Co., Ltd.; Grace insect cell culture medium (without additives), Sf-900IISFM culture Base, liposome tran...

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Abstract

The invention discloses a method for constructing a recombinant insect baculovirus expression vector of PVC2 (porcine circovirus type 2) Cap-labeled protein. The method comprises the following steps:(1) cloning whole genes of a PCV2GZ-RH1 strain into a pMD19-T vector, designing a primer to introduce a V5Tag or Flag tag into the ORF2 tail end of PCV2 with a PCR technology on the basis of the recombinant T vector as a template; (2) subcloning the ORF2 carrying the tag into pFastBacHTA and pFastBacDual donor plasmid, and transforming the positive plasmid obtained by subcloning into DH10Bac competent cells for Bacmind bacmid preparation; (3) transfecting insect cells with Bacmind bacmid to obtain recombinant insect baculovirus and performing amplification on the recombinant insect baculovirusto P3-generation virus.

Description

technical field [0001] The present invention relates to the construction of a recombinant baculovirus strain, and also relates to the expression of a PCV2 Cap recombinant marker protein, so the present invention relates to the construction of a recombinant baculovirus strain and the recombinant marker protein expressed in PCV2 diagnosis , The application in prevention belongs to the development of veterinary biological products in the field of biotechnology. Background technique [0002] Porcine circovirus (PCV) is a small non-enveloped single-stranded circular DNA virus, a member of the family Circoviridae and the genus Circovirus, with a genome size of about 1800 bp. According to differences in pathogenicity, antigenicity and gene structure, etc., porcine circoviruses are generally divided into circovirus type 1 (PCV1) and circovirus type 2 (PCV2). PCV1 is not clinically pathogenic, while PCV2 infection can lead to a variety of clinical diseases, and can cause a series of...

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Application Information

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IPC IPC(8): C12N15/866C12N15/66C12N15/34
CPCC07K14/005C12N15/86C12N2710/14043C12N2750/10022
Inventor 徐国曾智勇梁海英代振江
Owner GUIZHOU UNIV
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