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Building method and application of tsh and iss double-gene coexpression strain

A construction method and co-expression technology, applied in the field of genetic engineering, can solve the problems of restricting the widespread use of colibacillosis vaccines and lack of cross-immunity, and achieve the effects of suitable selection, simple process and simple method.

Inactive Publication Date: 2018-01-09
HEBEI KEXING PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the large number of Escherichia coli serotypes and the lack of cross-immunity between different serotypes or even different strains of the same serotype, it has brought great challenges to the use of vaccines to prevent and treat this disease, and it is also an important reason that limits the widespread use of E. coli disease vaccines

Method used

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  • Building method and application of tsh and iss double-gene coexpression strain
  • Building method and application of tsh and iss double-gene coexpression strain
  • Building method and application of tsh and iss double-gene coexpression strain

Examples

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Effect test

Embodiment 1

[0081] Example 1 tsh and iss Construction method of double-gene co-expression strain

[0082] This embodiment is carried out according to the following steps:

[0083] 1. Double gene cloning

[0084] Using the plasmid of avian pathogenic Escherichia coli as a template, the temperature-sensitive hemagglutinin tsh Genes and Enhanced Serum Resistance iss The gene is amplified. During the PCR amplification process, tsh Gene primer sequences and iss The primer sequences of the genes were designed according to the tsh (NCBI query number NC_007675.1) and iss genes (NCBI query number AF042279.1) published in the NCBI database.

[0085] I. tsh gene and iss Gene primer sequence design

[0086] According to the design of tsh (NCBI query number NC_007675.1) and iss gene (NCBI query number AF042279.1) published in the NCBI database, the details are as follows:

[0087] tsh The primer sequences of the genes are as follows:

[0088] Upstream primer: 5'-CCGGAATTCATGAACAGAATTTA...

Embodiment 2-4

[0201] Example 2-4 tsh and iss Construction method of double-gene co-expression strain

[0202] Embodiment 2-4 is respectively a kind of tsh and iss The construction method of the double-gene co-expression strain, the implementation steps are similar to Example 1, the only difference is:

[0203] Step 3 transform the expression bacteria and the co-expression of the target gene, pETDuet-1 / tsh / iss The plasmid was transformed into BL21(DE3) competent cells, and after culturing to the logarithmic phase, lactose was added to induce protein expression at different temperatures and times, specifically:

Embodiment 2

[0204] Example 2, at an induction temperature of 18°C ​​and an induction time of 10 hours, the expression of the protein was induced;

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Abstract

The invention discloses a building method of a tsh and iss double-gene coexpression strain. The method sequentially comprises the steps of double-gene clone, connection of tsh gene and iss gene with pETDuet-1 plasmids, expression strain conversion, target gene coexpression and the like. A microbial cell model based on tsh and iss is built; the biotransformation is performed; the method is simple;the process can be easily controlled; the double genes of tsh and iss are contained in the coexpression strain; the success ratio of the double-gene expression is high. The invention also provides application of the strain to prevention and treatment of avian colibacillosis. The method provided by the invention is applicable to the building of the tsh and iss double-gene coexpression strain; a gene engineering bacterium vaccine prepared from the strain is applied to the prevention and treatment of avian colibacillosis.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and relates to a method for constructing a double-gene expression bacterial strain and its application, in particular to a tsh and iss Construction method and application of double-gene co-expression strain. Background technique [0002] Avian colibacillosis (AC) is caused by avian pathogenic Escherichia coli (Avianpathogenic Escherichia coli , APEC) is a general term for local or systemic extraintestinal infection in chickens, turkeys and other poultry, clinically manifested as Escherichia coli omphalitis, cellulitis, salpingitis, peritonitis and swollen head syndrome and sepsis (pericarditis, liver Zhou Yan, air sac disease) etc. are relatively common, the mortality rate of infected poultry flocks is increased, the carcass waste rate is increased, the growth rate and feed conversion rate are reduced, and it is currently the most serious bacterial disease that hinders the healthy developme...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N15/31A61K39/108A61P31/04C12R1/19
Inventor 石虎王振华马兴树陈淑芳杨广平
Owner HEBEI KEXING PHARMA
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