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Method for constructing expression system of exogenous genes in campylobacter jejuni and shuttle plasmids thereof

A technology of Campylobacter jejuni and a construction method, which is applied to the construction method of an exogenous gene expression system and the field of its shuttle plasmid, and can solve the problems such as no test report.

Inactive Publication Date: 2010-06-09
NORTHWEST A & F UNIV
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Problems solved by technology

[0016] At present, biotransformants using the Lux gene as a reporter gene have been reported, but there is no experimental report on the successful transfer and expression of the Lux gene using Campylobacter jejuni as a recipient cell

Method used

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  • Method for constructing expression system of exogenous genes in campylobacter jejuni and shuttle plasmids thereof
  • Method for constructing expression system of exogenous genes in campylobacter jejuni and shuttle plasmids thereof
  • Method for constructing expression system of exogenous genes in campylobacter jejuni and shuttle plasmids thereof

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Embodiment Construction

[0068] A method for constructing an exogenous gene expression system in Campylobacter jejuni, the construction method comprising the following steps:

[0069] The implementation conditions of the following steps are all in an anaerobic environment, and the temperature is controlled at 37°C;

[0070] 1) Prepare the promoter

[0071] a. Use the kit to isolate and purify Campylobacter jejuni DNA;

[0072] b. For the obtained Campylobacter jejuni DNA, the promoter σ28 NC_002163 of the Campylobacter jejuni flaA gene was cloned by PCR; the specific primers and PCR process were as follows:

[0073] Upstream 5'-ACGCGAATTCAATATTTACCAAAAACTTTAACAACC-3'

[0074] Downstream 5'-ACGCGTCGACTCCTTTAAATAATTTCAAACTCATCCAT-3'

[0075] 94℃ 4min

[0076] Denaturation at 94°C for 1 min

[0077] Annealing reaction at 49°C for 1min

[0078] 72°C extension reaction for 1min

[0079] After 4 cycles

[0080] Denaturation at 94°C for 1 min

[0081] Annealing reaction at 59℃ for 1min

[0082] 72°...

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Abstract

The invention relates to a method for constructing an expression system of exogenous genes in a campylobacter jejuni. Under the condition of anaerobic environment and temperature controlled at 37 DEG C, the method comprises the steps of preparing promoters, constructing the shuttle plasmids, and establishing a bioluminescent transgene model of the campylobacter jejuni. By using electrotransformation to introduce the campylobacter jejuni ATCC33291 and ATCC35921 which are expressed under the selection pressure of kanamycin antibiotics, the method can observe bioluminescence without adding any substrate, visually expresses bioinformation, and has a wide research prospect.

Description

technical field [0001] The invention relates to a construction method of an exogenous gene expression system and a shuttle plasmid thereof, in particular to a construction method of an exogenous gene (Lux) expression system in Campylobacter jejuni and a shuttle plasmid thereof. Background technique [0002] In recent years, gene recombination technology has developed rapidly. People have been able to link genes with certain functions to appropriate vectors and transform them into bacteria, plants, and animal cells by chemical or physical methods. This technology has been applied It has broad application prospects in the manufacture of exogenous protein products, organ transplantation, animal and plant transgenic breeding, gene therapy, and biosensors. [0003] Nowadays, people use exogenous gene expression technology to produce biosensors. Different from traditional detection methods, the new biosensors are to directly inject a foreign aid gene (DNA or RNA) encoding a certai...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/74C12N15/65C12N15/63
Inventor 丁武寇莉萍刘变芳张静宋社果
Owner NORTHWEST A & F UNIV
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