Escherichia coli ghost capable of realizing dual-protein expression and preparation method of escherichia coli ghost capable of realizing dual-protein expression

A technology of Escherichia coli and double protein, which is applied in the fields of botany equipment and methods, microorganism-based methods, biochemical equipment and methods, etc., can solve the problems of the expression efficiency and structural influence of the two proteins, and solve biological safety hazards, High safety and high cracking efficiency

Inactive Publication Date: 2020-04-03
JIANGSU AGRI ANIMAL HUSBANDRY VOCATIONAL COLLEGE
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Problems solved by technology

However, there is a problem that the expression effi...

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  • Escherichia coli ghost capable of realizing dual-protein expression and preparation method of escherichia coli ghost capable of realizing dual-protein expression
  • Escherichia coli ghost capable of realizing dual-protein expression and preparation method of escherichia coli ghost capable of realizing dual-protein expression
  • Escherichia coli ghost capable of realizing dual-protein expression and preparation method of escherichia coli ghost capable of realizing dual-protein expression

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Embodiment 1

[0034] A kind of preparation of the Escherichia coli slough of double protein expression, the steps are:

[0035] (1) Preparation of double-expression lysogenic plasmid pETDuet1-E-SNA: First, the E gene (276bp) was synthesized according to the cleavage gene E sequence of bacteriophage PhiX174 published by NCBI and connected to the pUC57 vector, named pUC57-E. Design forward / reverse PCR primers for the E gene fragment according to the first MCS sequence of the vector pETDuet1, and use pUC57-E as a template to amplify, so that the 5' and 3' ends of the PCR product are respectively consistent with the two ends of the linearized vector. sequences and restriction sites. according to IIOne Step cloning kit instructions, complete the construction of recombinant plasmid pETDuet1-E. According to the SNA sequence published by NCBI, synthesize its 453bp extramembrane region sequence without signal peptide and connect it to the pUC57 vector, named pUC57-SNA, according to the second MCS...

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Abstract

The invention discloses an escherichia coli ghost capable of realizing dual-protein expression. The ghost is prepared from dual-expression bacteriolysis plasmids, the dual-expression bacteriolysis plasmids use pETDuet1 as a carrier, besides, an E gene and staphylococcus aureus nuclease A are inserted, and bacteriolysis plasmids capable of realizing dual-protein expression of the E gene and the staphylococcus aureus nuclease A are realized. According to the ghost disclosed by the invention, gene fragments are respectively inserted in two independent multiple clone sites of the carrier, so thatthe problem that two protein structures in expression mutually influence can be effectively solved, besides, degradation of bacterium DNA is realized, the obtained ghost products are high in safety, dual-protein expression is realized, and the dual-protein expression efficiency is high. The escherichia coli ghost prepared from the pETDuet1-E-SNA bacteriolysis plasmids disclosed by the invention can degrade heredity substances (including drug resistance genes and toxin genes) of the escherichia coli, the use safety of the ghost can be effectively increased, and biological potential safety hazard existing in use of the ghost products can be avoided.

Description

technical field [0001] The invention relates to the technical field of preparation of Escherichia coli slough, more specifically, relates to a double-protein expressed Escherichia coli slough and a preparation method thereof. Background technique [0002] In 1985, Lubitz et al. found that the expression of the cleavage gene E of phage PhiX174 in Gram-negative bacteria could cause the bacteria to form transmembrane channels, and the cytoplasm and organelles escaped from the channels to form bacterial empty shells. Called sloughs or spores. Compared with formalin inactivated bacterial vaccines, the preparation process of sloughs does not damage the antigenic structure of bacterial cells, and its immunogenicity is better preserved, so it can be used as a new type of inactivated vaccine, vaccine carrier or Adjuvant use. At present, most of the relevant reports at home and abroad adopt the expression of E gene alone to prepare slough. However, this method cannot achieve the de...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/70C12N15/33C12N15/55C12R1/19
CPCC07K14/005C12N9/22C12N15/70C12N2795/00022
Inventor 郭长明袁橙孟婷张步彩管远红刘剑华李东旭高伟
Owner JIANGSU AGRI ANIMAL HUSBANDRY VOCATIONAL COLLEGE
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