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African swine fever virus tandem gene, coexpression vector, construction method and application

A technology of African swine fever virus and co-expression vector, which is applied in the direction of virus/bacteriophage, virus, viral peptide, etc., can solve the problem of low co-integration efficiency of screening marker gene and target gene, integration efficiency, co-expression efficiency and expression stability of target gene Differences and other issues, to achieve the effect of high-efficiency immune protection

Active Publication Date: 2019-09-10
YANAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in the construction of stable expression cell lines in scientific research, if it is necessary to achieve stable co-expression of two or more genes in the field of antibody engineering and cell engineering, it usually needs to be achieved through the screening method of co-transfection of two vectors, and in practice, the following are often faced Questions: ① Two vectors need two screening markers for co-transfection; ② There are differences in the co-transfection efficiency and integration efficiency of the two vectors, as well as the co-expression efficiency and expression stability of the target gene; ③ The co-transfection efficiency and expression stability of the screening marker gene and the target gene Low integration efficiency
The above problems pose challenges for screening suitable stable expression cell lines in terms of efficiency, time and labor costs.

Method used

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  • African swine fever virus tandem gene, coexpression vector, construction method and application
  • African swine fever virus tandem gene, coexpression vector, construction method and application
  • African swine fever virus tandem gene, coexpression vector, construction method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Construction of co-expression vector of tandem genes of classical swine fever virus

[0039] S1, look up the P32, CD2V, and G6L gene sequences of the FN557520.1 strain in the NCBI database, analyze and intercept the position of the CD2V antigen peptide as the gene sequence of CD2V in the tandem gene by using the software DNAMAN, and analyze the three fragments. Restriction site and determine the restriction site to be introduced into the fusion protein to be synthesized, and optimize the codon of the prokaryotic expression system for its nucleic acid sequence. The same method is used to search for the I329L gene sequence of the FR682468.1 strain (accession number: ), by using the software DNAMAN to analyze and intercept the position of the I329L antigenic peptide as the gene sequence of I329L in the tandem gene, analyze the enzyme cleavage sites and determine The enzyme cutting site to be introduced into the tandem gene to be synthesized, and its codon optimization;

...

Embodiment 2

[0055] Vaccine preparation and expression and purification of target protein

[0056] The multi-gene co-expression vector of African swine fever was transformed into Escherichia coli and Lactobacillus for induced expression and purification. The specific steps are as follows:

[0057] a. Take 100 μL of competent cells melted on an ice bath, add 1 μL of target DNA, mix gently, and place in an ice bath for 30 minutes.

[0058] b. Heat shock in a water bath at 42°C for 90 seconds, then quickly transfer the tube to an ice bath for 2 minutes, and do not shake the centrifuge tube during this process.

[0059] c. Add 500 μL of sterile LB medium (without antibiotics) to each centrifuge tube, mix well, place at 37°C, and incubate at 150 rpm for 1 hour to recover the bacteria.

[0060] d. Pipette 200 μL of transformed competent cells and add them to the LB agar medium with kana, spread the cells evenly, place the plate at 37°C until the liquid is absorbed, invert the plate and culture...

Embodiment 3

[0064] African swine fever immune protection experiment

[0065] 1) Vaccine preparation

[0066] Collect a large number of plasmid DNA, express the target protein, measure the protein concentration with a spectrophotometer, and store at -80°C. Carry out stability and safety tests of DNA vaccines and subunit vaccines.

[0067] 2) Immunization program

[0068] Analysis of expression effect of multivalent DNA vaccine in vitro. Through prokaryotic expression, whether the corresponding antigen is detected at the cellular level (such as Figure 4 shown):

[0069] a. Transform the constructed mammalian tandem expression shuttle plasmid vector into Escherichia coli by electroporation or conjugative transfer, add kanamycin after 4 hours to kill the uninfected Escherichia coli cells, at 24h, 36h, 48h, 72h, The cells were harvested after 96 hours, and the gene expression effects in vitro were detected at the cellular level, mRNA and protein expression levels by fluorescence microsco...

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Abstract

The invention belongs to the technical field of genetic engineering vaccines, and particularly relates to an African swine fever virus tandem gene, a coexpression vector, a construction method and application. The recombinant vector is obtained through the steps that an artificially synthesized polygene clone expression element is connected to the portion between the BglII and BamHI restriction enzyme cutting sites in a pIRES2-egfp vector, and oriR101 is inserted in the portion behind the XbaI site of 1986 nt of the pIRES2-egfp vector, wherein the nucleotide sequence of the gene clone expression element is shown in SEQ ID NO: 1; the recombinant vector is transformed into escherichia coli competent cells, screening and cultivation enlargement are conducted, then polygenic protein is collected and purified, and a subunit vaccine against the African swine fever disease is obtained; the recombinant vector is transformed into lactobacillus competent cells, and after screening and cultivation enlargement are conducted, a lactobacillus vaccine is obtained. The prepared vaccine stimulates the body to generate an antibody in different levels and different gradations, stimulates the body toproduce a strong and effective specific immune response, exerts high-efficiency immune protection, and makes an immune response against the African swine fever virus.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering vaccines, in particular to an African swine fever virus tandem gene, co-expression vector, construction method and application. Background technique [0002] African swine fever (ASF) is an acute, hemorrhagic, severe infectious disease caused by African swine fever virus (ASFV) infecting domestic pigs and various wild boars (African wild boar, European wild boar, etc.). The World Organization for Animal Health (OIE) listed it as a legally notifiable animal disease, and this disease is also a class of animal epidemics that my country focuses on preventing. It is characterized by a short course of disease and a mortality rate of up to 100% in the most acute and acute infections. African swine fever virus is an important member of the African swine fever virus genus in the African swine fever family. Some characteristics of the virus are similar to those of the iridoviridae and poxvirida...

Claims

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Application Information

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IPC IPC(8): C12N15/62C12N15/74A61K39/12A61P31/20
CPCC07K14/005C12N15/74A61K39/12A61P31/20C12N2710/12022C12N2710/12034A61K2039/552A61K2039/53Y02A50/30
Inventor 岳昌武吕玉红崔相宜赵雯党冬梅孙心悦田红英
Owner YANAN UNIV
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