Construction, amplification and purification method of porcine CD40L/GMCSF/PCV2Cap recombinant adenovirus

A recombinant adenovirus, CD40L technology, applied in the direction of recovery/purification, double-stranded DNA virus, single-stranded DNA virus, etc., can solve the problem of low immunogenicity, improve immunogenicity, increase the level of proliferation, and increase cytokines horizontal effect

Inactive Publication Date: 2016-08-10
NORTHWEST A & F UNIV
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Problems solved by technology

[0008] Recombinant adenovirus PCV2 has been studied, but its immunogenicity is relatively low when used as a vaccine

Method used

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  • Construction, amplification and purification method of porcine CD40L/GMCSF/PCV2Cap recombinant adenovirus
  • Construction, amplification and purification method of porcine CD40L/GMCSF/PCV2Cap recombinant adenovirus
  • Construction, amplification and purification method of porcine CD40L/GMCSF/PCV2Cap recombinant adenovirus

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Embodiment Construction

[0054] The present invention will be further described below in conjunction with embodiment.

[0055] A method for constructing, amplifying and purifying porcine CD40L / GMCSF / PCV2Cap recombinant adenovirus, comprising the following steps:

[0056] S1 sequentially connected the coding genes of CD40L, GM-CSF and PCV2Cap proteins to the vector pUC57, which was identified by sequencing and named pUC-CD40L-Cap-GMCSF;

[0057] S2 Link CD40L, Cap, and GM-CSF to pShuttle-CMV: Using pUC-CD40L-Cap-GMCSF as a template, perform PCR amplification with upstream primers with Xho I and downstream primers with EcoR V to obtain the target gene CD40L-Cap- GMCSF, connected to pShuttle-CMV after double enzyme digestion, transformed into DH5a, picked bacteria, shaken bacteria, extracted plasmid, after PCR identification and single and double enzyme digestion identification were correct, sent for sequencing, after sequencing was correct, named PS-CD40L-Cap- GMCSF;

[0058] S3 Linearize the construc...

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Abstract

The invention relates to a construction, amplification and purification method of a porcine CD40L / GMCSF / PCV2Cap recombinant adenovirus. The method comprises the steps of: S1. connecting CD40L, GM-CSF and Cap to a vector pUC57 in order, and naming the product as pUC-CD40L-Cap-GMCSF; S2. connecting CD40L, Cap and GM-CSF to pShuttle-CMV, converting DH5a, conducting bacteria picking, bacteria shaking and plasmid extraction, and naming the product as PS-CD40L-Cap-GMCSF; S3. linearizing the constructed PS-CD40L-Cap-GMCSF, then conducting electric transformation on BJ5183 with the linearized PS-CD40L-Cap-GMCSF and skeleton plasmid pAdEasy-1, and then carrying out bacteria picking, bacteria shaking and plasmid extraction, and performing single enzyme digestion identification; S4. when the identification result is right, using a kit to extract plasmid, transfecting HEK293A cell, when cell lesion appears, collecting cells, performing centrifugation, then resuspending the precipitate in autoclaving PBS; S5. carrying out repeated freezing and thawing, performing centrifugation to extract the supernatant, thus obtaining recombinant adenovirus; 6. amplifying the obtained recombinant adenovirus; and 7. purifying the recombinant adenovirus. The method provided by the invention for the first time adds porcine tumor necrosis factor related activation protein gen and porcine granulocyte-macrophage colony stimulating factor into the adenovirus vector simultaneously to improve expression of the PCV2 Cap recombinant adenovirus immunogenicity.

Description

technical field [0001] The invention relates to the technical field of animal virus genetic engineering, in particular to a method for constructing, amplifying and purifying porcine CD40L / GMCSF / PCV2Cap recombinant adenovirus. Background technique [0002] Adenovirus has the advantages of strong susceptibility, low pathogenicity, wide host range, good stability, easy operation, high virus titer, easy concentration and storage, high efficiency of mediating gene transfer, and ability to accommodate large gene fragments. Moreover, its biological background has been studied quite clearly, so adenovirus vectors are widely studied and applied in vaccine preparation, gene therapy of cancer, infectious diseases and genetic diseases, and are considered to be one of the most promising vectors in gene transfer. . Adenoviral vectors can efficiently express foreign genes, and can perform reactions such as cleavage, glycosylation, and phosphorylation of foreign proteins. The expressed pro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01C12N7/02C12N15/861C12Q1/70
CPCC12N7/00C07K14/005C07K14/535C07K14/70578C12N15/86C12N2710/10043C12N2710/10051C12N2750/10022C12Q1/06
Inventor 童德文黄勇李德龙赵晓民
Owner NORTHWEST A & F UNIV
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