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Method for co-expressing four structural proteins of African swine fever virus in insect cells and application thereof

An African swine fever virus and recombinant virus technology, applied in the fields of molecular biology and biology, can solve problems such as no method and system for expressing African swine fever virus, and achieve simple, convenient, high expression and good specificity. Effect

Active Publication Date: 2020-08-28
SHENZHEN CUSTOMS ANIMAL & PLANT INSPECTION & QUARANTINE TECH CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, although there are separate expressions or partial co-expression of the above four proteins in the prior art, there has been no co-expression of the four proteins of African swine fever virus pp62, p54, p30 and p72 in insect cells so far. method and system

Method used

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  • Method for co-expressing four structural proteins of African swine fever virus in insect cells and application thereof
  • Method for co-expressing four structural proteins of African swine fever virus in insect cells and application thereof
  • Method for co-expressing four structural proteins of African swine fever virus in insect cells and application thereof

Examples

Experimental program
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Effect test

example 1

[0101] Design and preparation of example 1pF4 carrier

[0102] In order to express African swine fever virus structural proteins pp62, p30, p54 and p72 simultaneously, the vector pFastBac-Dual was modified to have four cloning sites, and each cloning site had its own independent promoter. The present invention analyzes the full sequence of the carrier pFastBac-Dual, and finds that the 3510th base of the carrier is the Bsp1407 I restriction site and is unique, and the 3985th base is the SnaB I restriction site and is unique. There is no element between the 3510th base and the 3985th base, so it can be used as a modified region to modify the vector.

[0103]Since the original vector has two cloning sites, and the respective promoters are Polyhedrin and P10, the present invention cuts the vector pFastBac-Dual by Bsp1407 I and SnaB I, and excises the 3510th base and the 3985th base , and then insert a synthetic sequence F4 (the sequence contains the sequence between the 3510th ba...

example 2

[0104] Construction of Example 2 Recombinant Vector pF4-pp62-p54-p30-p72

[0105] Taking the Shenyang strain of African swine fever as the reference sequence (GenBank: MH766894.1), the pp62, p30, p54 and p72 gene sequences were synthesized by Shanghai Sangon Company (in order to facilitate the purification of expressed proteins, the 5' end of each gene sequence was added A His tag sequence), the gene sequence is shown in SEQ ID NO.2-5, respectively, to obtain the recombinant plasmid pUC-pp62 (the 5' and 3' ends of the pp62 sequence have restriction sites Sph I and Xho I respectively), pUC -p30 (the 5' and 3' ends of the p30 sequence have restriction sites Aat II and Eco81I, respectively), pUC-p54 (the 5' and 3' ends of the p54 sequence have restriction sites Sbf I and Nhe I, respectively), and pUC-p72 (the 5' and 3' ends of the p72 sequence have restriction sites BamH I and EcoR I, respectively).

[0106] According to the conventional molecular cloning technique, the pp62, p5...

example 3

[0114] Example 3 Extraction of recombinant bacmid Bacmid-pp62-p54-p30-p72

[0115] The recombinant plasmid pF4-pp62-p54-p30-p72 was transformed into the competent cell DH10Bac by means of thermal transformation, and a single white colony was picked after 48 hours and streaked and continued to culture for 48 hours. Pick a single white colony and carry out colony PCR identification to determine whether pp62-p54-p30-p72 transposition is successful. Positive results were identified, cultured in large quantities overnight, and analyzed with PureLink TM HiPure Plasmid Purification Kit is used to extract and purify recombinant bacmid Bacmid-pp62-p54-p30-p72 in large quantities.

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Abstract

The invention relates to a method for co-expressing four structural proteins of an African swine fever virus in insect cells and an application thereof. According to the method, four structural proteins pp62, p54, p30 and p72 genes of the African swine fever virus are inserted into a shuttle vector obtained after transforming pFastBac-Dual to prepare a recombinant shuttle plasmid pF4-pp62-p54-p30-p72, a recombinant baculovirus rBac-pp62-p54-p30-p72 is obtained, and efficient co-expression in the insect cells is achieved. The indirect ELISA method for detecting an African swine fever antibody is established based on the co-expressed mixed recombinant protein, and the method has the advantages of high sensitivity, high specificity, good repeatability and the like.

Description

technical field [0001] The application relates to the fields of molecular biology and biotechnology, in particular to an insect baculovirus expression system for simultaneously expressing four structural proteins of African swine fever virus pp62, p30, p54 and p72 and its application. Background technique [0002] African swine fever (African swine fever, ASF) is a kind of severe, highly contagious infectious disease of pigs caused by African swine fever virus (African swine fever virus, ASFV) infection. Its acute symptoms are characterized by high fever, reticuloendothelial hemorrhage, and high mortality. All breeds and ages of domestic and wild boars are susceptible. Ornithodoros soft ticks, especially O. moubata and O. erraticus are the storage and transmission vectors of African swine fever virus. Previously, the disease was limited to Africa, until it spread to Europe, South America and the Caribbean in the middle of the last century. It was introduced to Eastern Euro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/866C12N15/66C12N15/34G01N33/68G01N33/569G01N33/543
CPCC07K14/005C12N15/66C12N15/86C12N2710/12022C12N2710/12051C12N2710/14043C12N2800/105G01N33/54393G01N33/56983G01N33/6854G01N2333/01G01N2469/20
Inventor 黄超华花群义史卫军吴江林彦星曹琛福曾少灵刘建利阮周曦杨俊兴
Owner SHENZHEN CUSTOMS ANIMAL & PLANT INSPECTION & QUARANTINE TECH CENT
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