Brucella three-gene recombinant plasmid, construction method thereof and expression and application thereof in escherichia coli

A Brucella, gene recombination technology, applied in the field of genetic engineering, can solve the problems of infringement of the reproductive system and lymphatic system, economic loss of animal husbandry, threat to human health, etc., and achieve the effect of high expression efficiency, low cost and enhanced activity

Active Publication Date: 2019-03-19
SHANDONG AGRICULTURAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

It is mainly infected through the digestive tract, respiratory tract, reproductive tract and damaged skin, muco...

Method used

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  • Brucella three-gene recombinant plasmid, construction method thereof and expression and application thereof in escherichia coli
  • Brucella three-gene recombinant plasmid, construction method thereof and expression and application thereof in escherichia coli
  • Brucella three-gene recombinant plasmid, construction method thereof and expression and application thereof in escherichia coli

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Experimental program
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Embodiment 1

[0047] Three kinds of gene recombination plasmids of embodiment 1 Brucella

[0048] The embodiment of the present invention provides a kind of brucella three gene recombination plasmids, and described recombination plasmid is to take pET-28a (+) plasmid as carrier, in pET-28a (+) restriction site BamHI and xhoI Insert the full-length synthetic Omp10-Omp28-L7 / L12 gene between them, and construct the pET-28a(+) recombinant plasmid containing the Omp10-Omp28-L7 / L12 gene fragment and the kanamycin selection tag, namely pET-28a(+ )-Omp10-Omp28-L7 / L12 recombinant plasmid, the gene amplification sequence of the Omp10-Omp28-L7 / L12 gene fragment is shown in SEQ ID NO:5.

[0049] SEQ ID NO: Gene amplification sequence of 5Omp10-Omp28-L7 / L12 gene fragment

[0050]

Embodiment 2

[0051] The construction method of three kinds of gene recombination plasmids of embodiment 2 Brucella

[0052] The embodiment of the present invention provides a kind of construction method of three gene recombinant plasmids of Brucella, comprising the following steps:

[0053] 1) search Brucella standard strain (28M strain) Omp10, Omp28 and L7 / L12 (SEQ ID NO:1,2,3) nucleotide sequence from NCBI, and pET-28a (+) plasmid spectrogram By comparing the information, it is determined that the double restriction sites that need to be introduced during primer design are BamHI and Xhol I;

[0054] 2) Construction of recombinant plasmid pET-28a(+)-Omp10-Omp28-L7 / L12 recombinant plasmid

[0055] ①PCR amplification of Omp10-linker, linker-Omp28-linker and L7 / L12-linker genes

[0056] According to Brucella Omp10, Omp28 and L7 / L12 genes, the following primers were designed:

[0057] Primer1:5′-CGC GGATCC ATGAAACGCTTCCGCATCGTT-3′

[0058] Wherein, the underlined part is the restriction...

Embodiment 3

[0128] Expression of three kinds of gene recombinant plasmids of embodiment 3 Brucella in escherichia coli

[0129] The embodiment of the present invention provides the expression of three kinds of gene recombinant plasmids of Brucella in Escherichia coli, comprising the following steps:

[0130] 1) Transform the pET-28a(+)-Omp10-Omp28-L7 / L12 recombinant plasmid and pET-28a(+) constructed in Example 2 into Escherichia coli BL21(DE3) plysS competent cells, and coat with kana Mycin LB agar plate, identified after 8-12 hours of culture;

[0131] Among them, the steps of BL21(DE3)plysS competent cells are as follows:

[0132] a. Take 50 μL of competent cells melted on an ice bath, add 10 μL of target DNA, mix gently, and place in an ice bath for 30 minutes.

[0133] b. Heat shock in a water bath at 42°C for 45 seconds, then quickly transfer the tube to an ice bath for 2 minutes, and do not shake the centrifuge tube during this process.

[0134]c. Add 500 μL of sterile LB broth ...

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Abstract

The invention discloses a brucella three-gene recombinant plasmid, a construction method thereof and expression and application thereof in escherichia coli, belonging to the technical field of geneticengineering. According to the technical scheme, a pET-28a(+) plasmid is taken as as a carrier, and a synthetic full-length gene Omp10-Omp28-L7/L12 is inserted between pET-28a(+) restriction enzyme cutting sites BamHI and XhoI so as to establish a pET-28a(+)recombinant plasmid containing an Omp10-Omp28-L7/L12 gene segment and a kanamycin screening label, namely pET-28a(+)-Omp10-Omp28-L7/L12 recombinant plasmid. The brucella three-gene recombinant plasmid has the beneficial effects that brucella Omp10-Omp28-L7/L12 is successfully cloned and is linked and converted to the recombinant plasmid, sothat the efficient soluble expression of the recombinant plasmid in an escherichia coli expression system is realized, an expressed protein is capable of inhibiting the infection, proliferation and transfer capabilities of brucella in the body fluid and cell levels, promoting the level of an antibody in a mouse and prolonging the immune time, and a novel treatment through is provided for the treatment of brucella diseases.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a three-gene recombination plasmid of Brucella, a construction method and its expression and application in Escherichia coli. Background technique [0002] Brucella (brucella) belongs to Brucella, is a Gram-negative coccus, a facultative intracellular parasitic Gram-negative small cocci, most of which are arranged in a single arrangement, and very few are 2 connected or in the form of short rods. Parasitic on the reticuloendothelial system. The bacteria have no flagella and do not form spores, but the virulent strains can have thin capsules. [0003] Animals can be infected with brucellosis, but sheep, pigs, and cattle are most susceptible. People can also be infected with Brucella, and Brucella sheep is the most infectious and the most harmful, followed by Brucella bovis. It is mainly infected through the digestive tract, respiratory tract, reproductive tract and ...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N15/65C12N15/66C12N15/62C07K19/00A61K39/10A61K39/116A61P31/04G01N33/569
CPCA61K39/098A61P31/04C07K14/23C07K2319/00C12N15/65C12N15/66C12N15/70G01N33/56911
Inventor 朱瑞良王秋菊胡莉萍魏凯黄河崔文平沙洲徐煜琳朱琳楚遵锋
Owner SHANDONG AGRICULTURAL UNIVERSITY
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