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Pig breed brucella bax inhibitor-1 (BI-1) gene deletion strain and construction method and application thereof

A gene deletion strain, Brucella suis technology, applied in the biological field, can solve problems such as unclear functions of prokaryotic organisms, and achieve the effects of enhanced polymerization, increased permeability, and slowed growth

Active Publication Date: 2020-01-17
NORTHWEST A & F UNIV
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Problems solved by technology

[0004] Bax inhibitor-1 (BI-1) is a highly conserved transmembrane protein in eukaryotes and prokaryotes. It mainly plays the role of inhibiting BAX-induced apoptosis in eukaryotes, but its function in prokaryotes is not known yet. clear

Method used

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  • Pig breed brucella bax inhibitor-1 (BI-1) gene deletion strain and construction method and application thereof
  • Pig breed brucella bax inhibitor-1 (BI-1) gene deletion strain and construction method and application thereof
  • Pig breed brucella bax inhibitor-1 (BI-1) gene deletion strain and construction method and application thereof

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Experimental program
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Embodiment

[0052] Construction of homologous recombination deletion plasmid

[0053] 1. Primer Design

[0054] According to the genome sequence of Brucella suis published by NCBI (accession number: NC_017251.1), the PCR primers BI1-UF / BI1-UR for the upper and lower homology arms of the BI-1 gene were designed (containing Sph I and Speech I restriction site) and BI1-DF / BI1-DR (containing Sal I and Eco R I restriction site), and primers BI1-TF / BI1-TR for identifying the deletion strain inside the BI-1 gene sequence; according to the pEGFP-C1 plasmid gene sequence published by NCBI (Accession No.: U55763.1), design Kana Mycin resistance gene PCR primers KanR-F / KanR-R (containing Sal I and Speech I restriction site). The primer sequences are as follows:

[0055] ;

[0056] 2. Acquisition of target fragments

[0057] (1) PCR of the upper and lower homology arms BI1-UP and BI1-DW of the BI-1 gene

[0058] With the Brucella suis live vaccine S2 strain genome as template, ...

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Abstract

The invention discloses a pig breed brucella bax inhibitor-1 (BI-1) gene deletion strain and a construction method and application thereof. The strain is obtained by replacing a pig breed brucella BI-1 gene with a kanamycin resistance gene, and the method includes the main steps that a pig breed brucella S2 vaccine strain genome is taken as a template, PCR amplification is conducted to obtain an upstream homologous arm and a downstream homologous arm of the BI-1 gene, the upstream homologous arm and the downstream homologous arm of the BI-1 gene and a kanamycin resistance gene fragment also obtained through PCR amplification are together cloned into a pUC18 plasmid, and a homologous recombination deletion plasmid pUC18-BI1-KanR is obtained. The plasmid is used for electrotransformation ofa pig breed brucella competent cell, and through PCR identification, an obtained positive transformant is the pig breed brucella BI-1 gene deletion strain. Compared with a wild strain, the deletion strain shows the characteristics of decreasing the growth rate, reducing colonies, increasing cell membrane permeability, enhancing cell polymerization and the like, and it is indicated that the deletion strain has the potential of serving as a brucella attenuated live vaccine.

Description

technical field [0001] The invention relates to a method for constructing a Brucella suis BI-1 gene deletion strain, which belongs to the field of biotechnology. Background technique [0002] Brucellosis is caused by Brucella ( Brucella ) is a zoonotic infectious disease, which mainly causes abortion and infertility in sick animals. It mainly manifests as undulating fever, osteoarthritis, and reproductive system inflammation in humans, which seriously threatens human life and health and the safety of animal husbandry. Healthy and sustainable development. Vaccination is the most effective way to prevent brucellosis. However, there are still many deficiencies in the immune protection and the distinction between wild virus infection and vaccination in my country's commercially available brucellosis vaccines, and there is no such vaccine in the world. Effective vaccine against brucellosis in humans. Therefore, the development of brucellosis vaccine and related research need to...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/74G01N33/569A61K39/10A61P31/04C12R1/01
CPCC07K14/23C12N15/74G01N33/56911A61K39/098A61P31/04A61K2039/522A61K2039/552Y02A50/30C12Q1/6876
Inventor 王爱华张广冻靳亚平周栋钟方粒陈蕾秦佩佩支飞杰李俊玫田路路
Owner NORTHWEST A & F UNIV
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