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Recombined bacillus subtilis secretion type expression vector

A Bacillus subtilis, expression vector technology, applied in the field of general expression vector pBC38C, can solve the problem of no expression vector, etc., and achieve the effect of good biological activity and good genetic stability

Inactive Publication Date: 2010-07-21
MILITARY VETERINARY RES INST PLA MILITARY MEDICAL ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] A lot of work has been done on the development and use of Bacillus subtilis as a cloning and expression system at home and abroad, but so far, there is still no commercial expression vector, and the research and development of expression vectors for Bacillus subtilis has become the focus of people's research one

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  • Recombined bacillus subtilis secretion type expression vector
  • Recombined bacillus subtilis secretion type expression vector
  • Recombined bacillus subtilis secretion type expression vector

Examples

Experimental program
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Effect test

Embodiment 1

[0017] Construction of embodiment 1 plasmid pBC38C

[0018] The present invention clones a new promoter from Bacillus subtilis 1700 strain (purchased from Institute of Microbiology, Chinese Academy of Sciences), and its nucleotide sequence is shown in SEQ ID No.2. This example takes the promoter of Bacillus subtilis 1700 strain as an example to illustrate the construction method of plasmid pBC38C.

[0019] 1. Cloning of the promoter

[0020] Genomic DNA of Bacillus subtilis 1700 strain was extracted with primer pair:

[0021] Upstream primer: 5′-CgA CTCgAg TgTCgACgTgCATgCAggCCg-3';

[0022] Downstream primer: 5′-CgA ATGAT TATAATggTACCgCTATCAC-3', the upstream primer introduces the XhoI restriction site (underlined part), and the downstream primer introduces the ClaI restriction site (underlined part), to amplify the promoter of Bacillus subtilis.

[0023] The reaction system is: 10×Ex Taq Buffer (Mg 2+ plus) 5 μL, dNTP Mixture (2.5 mM each) 4 μL, upstream primer 1 μL, ...

Embodiment 2

[0034] Secreted expression of embodiment 2 green fluorescent protein

[0035] This example uses green fluorescent protein as the reporter gene to evaluate the expression level of the recombinant secreted expression vector

[0036] 1. Construction of recombinant expression vector containing green fluorescent protein gene

[0037] Nhe I / EcoR I double-digested the plasmid pEGFP-C1 containing green fluorescent protein (constructed and preserved by the Virology Laboratory of the Military Veterinary Research Institute of the Academy of Military Medical Sciences), recovered a small fragment (GFP gene), and double-digested the plasmid pET-28a (+) (purchased from Invitrogen) to reclaim large fragments, connect the two recovered fragments under the action of T4DNA ligase, construct the recombined intermediate vector pET-28a-GFP, extract a large number of intermediate plasmids, use Nhe I and Not I The intermediate vector pET-28a-GFP was double digested and the GFP gene fragment was reco...

Embodiment 3

[0052] Example 3 Genetic Stability Analysis of Recombinant Secreted Expression Vector pBC38C-GFP

[0053] Utilizing the characteristics of the recombinant secretory expression vector carrying the chloramphenicol resistance gene, it was continuously passaged to 120 generations in the liquid medium containing chloramphenicol and chloramphenicol-free, and the stability of the plasmid was measured every 5 generations . The stability rate of the plasmid is equal to the number of colonies grown from 100 single colonies selected from a plate without chloramphenicol and planted on a plate containing chloramphenicol. The specific operation is as follows:

[0054] Pick a single colony of Bacillus subtilis containing the recombinant plasmid pBC38C-GFP, inoculate one LB broth and one LB broth containing chloramphenicol, and culture at 37°C with shaking (180rpm / min). When cultured to the 5th generation, 10th generation, 15th generation, and 20th generation, the cultures of LB broth and L...

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Abstract

The invention provides a recombined bacillus subtilis secretion type expression vector, which is constructed by inserting bacillus subtilis plasmid pGJ103 as a starting plasmid into an expression cassette with a bacillus subtilis promoter and a signal peptide sequence. The expression cassette comprises the bacillus subtilis promoter, the signal peptide sequence, a multiple-cloning-site sequence and a termination sequence which can be connected in an operative way. The expression vector pBC38C constructed in the invention can realize secretory expression of homologous genes and heterologous genes in bacillus subtilis, the expressed protein has good biological activity, and the parasitifer used by the expression vector is safe for animals and the environment and has bio-security. In addition, the expression vector has good genetic stability, and the recombined expression vector can not be lost after multiple passages and can continue carry out the secretory expression of foreign genes in bacillus subtilis cells. The expression vector of the invention can play an important role in the protein expression and the preparation of subunit vaccines for major diseases.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a general expression vector pBC38C capable of secreting and expressing homologous and heterologous genes in Bacillus subtilis. Background technique [0002] Bacillus subtilis is a kind of Bacillus, a single cell is 0.7-0.8×2-3μm, without capsule, with flagella all over the body, and can move. It is a typical Gram-positive bacterium, and the spores are oval or columnar, located in the center of the bacterium or slightly biased. The surface of the colony is rough and opaque, stained white or yellowish, and often forms wrinkles when growing in liquid medium. The cell wall does not contain endotoxin and has only a single layer of outer membrane, which can directly secrete many proteins into the medium. It can form spores, has strong environmental adaptability and environmental friendliness, and is a production bacterium of some important industrial enzyme preparations. [...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/75C12N15/64C12R1/125
Inventor 金宁一陈晓月李昌李霄金扩世鲁会军田明尧金洪涛
Owner MILITARY VETERINARY RES INST PLA MILITARY MEDICAL ACAD OF SCI
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