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Bacillus subtillis expressing metallothionein and method for producing metallothionein

A technology of Bacillus subtilis and metallothionein, which is applied in the field of producing metallothionein by recombinant Bacillus subtilis, to achieve the effects of increasing protein production, optimizing fermentation conditions, and avoiding bacterial endotoxin contamination

Inactive Publication Date: 2017-06-13
THIRD INST OF OCEANOGRAPHY MINIST OF NATURAL RESOURCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But so far there is no report on the use of probiotics to express metallothionein

Method used

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  • Bacillus subtillis expressing metallothionein and method for producing metallothionein
  • Bacillus subtillis expressing metallothionein and method for producing metallothionein
  • Bacillus subtillis expressing metallothionein and method for producing metallothionein

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Embodiment 1

[0040] Primers are designed according to the gene sequence Op-MT of the pleated oyster metallothionein of the present invention, and the sequence of the Op-MT gene is amplified by PCR. The PCR product was recovered and connected to the cloning vector pMD-19, and the plasmid pMD-19-Op-MT and PHT43-SUMO expression vector were digested with restriction endonuclease BamHI / SmaI, and then connected with T4 DNA ligase to construct the recombinant plasmid PHT43 -SUMO-Op-MT. Using Bacillus subtilis B.subtilis WB800N as the expression host, the secreted expression vector PHT43-SUMO-Op-MT was transformed by electroporation, and screened on LB plates containing 10 μg / mL chloramphenicol and 50 μg / mL neomycin to obtain Recombinant Bacillus subtilis engineering strain B.subtilis WB800N / PHT43-SUMO-Op-MT highly expressing metallothionein was stored at -80°C. The preserved recombinant Bacillus subtilis was transferred to the plate medium, cultured at 37°C for 24h, and set aside. Take a circle...

Embodiment 2

[0042] Primers are designed according to the gene sequence Op-MT of the pleated oyster metallothionein of the present invention, and the sequence of the Op-MT gene is amplified by PCR. The PCR product was recovered and connected to the cloning vector pMD-19, and the plasmid pMD-19-Op-MT and PHT43-SUMO expression vector were digested with restriction endonuclease BamHI / SmaI, and then connected with T4 DNA ligase to construct the recombinant plasmid PHT43 -SUMO-Op-MT. Using Bacillus subtilis B.subtilis WB800N as the expression host, the secreted expression vector PHT43-SUMO-Op-MT was transformed by electroporation, and screened on LB plates containing 10 μg / mL chloramphenicol and 50 μg / mL neomycin to obtain Recombinant Bacillus subtilis engineering strain B.subtilis WB800N / PHT43-SUMO-Op-MT highly expressing metallothionein was stored at -80°C. The preserved recombinant Bacillus subtilis was transferred to the plate medium, cultured at 37°C for 24h, and set aside. The activated...

Embodiment 3

[0044] Primers are designed according to the gene sequence Op-MT of the pleated oyster metallothionein of the present invention, and the sequence of the Op-MT gene is amplified by PCR. The PCR product was recovered and connected to the cloning vector pMD-19, and the plasmid pMD-19-Op-MT and PHT43-SUMO expression vector were digested with restriction endonuclease BamHI / SmaI, and then connected with T4 DNA ligase to construct the recombinant plasmid PHT43 -SUMO-Op-MT. Using Bacillus subtilis B.subtilis WB800N as the expression host, the secreted expression vector PHT43-SUMO-Op-MT was transformed by electroporation, and screened on LB plates containing 10 μg / mL chloramphenicol and 50 μg / mL neomycin to obtain Recombinant Bacillus subtilis engineering strain B.subtilis WB800N / PHT43-SUMO-Op-MT highly expressing metallothionein was stored at -80°C. The preserved recombinant Bacillus subtilis was transferred to the plate medium, cultured at 37°C for 24h, and set aside. Take a circle...

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Abstract

The invention provides a bacillus subtillis expressing metallothionein. The bacillus subtillis contains a fusion expression vector containing the metallothionein. The fusion expression vector carries encoding nucleic acid molecules of the metallothionein and can express the metallothionein inductively. The invention further provides a method for producing the metallothionein. The method comprises the steps of constructing a fusion expression vector of the metallothionein; adopting the fusion expression vector for converting the bacillus subtillis; culturing the bacillus subtillis and adding an inductive agent to obtain a culture containing the expressed metallothionein; purifying the culture optionally to obtain the purified metallothionein. According to the method, the production cost is lowered, the protein yield is increased, the defects that a traditional metallothionein protein method are overcome, and the ocean source metallothionein has the great practical production potential in the aspects of industrial production for developing natural and efficient ocean source heavy metal biological antidotes.

Description

technical field [0001] The present invention relates to a recombinant Bacillus subtilis expressing metallothionein, and also relates to a method for producing metallothionein by using the recombinant Bacillus subtilis. Background technique [0002] Metallothionein (MT) is a kind of highly inducible endogenous metal-binding protein ubiquitous in organisms, which can maintain the dynamic balance of necessary metal content in organisms, detoxify heavy metals, scavenge free radicals, and improve the body's immunity. , enhancing the body's anti-stress ability and other functions, and has a close relationship with the growth and development of animal bodies and the pathogenesis of some diseases. Therefore, metallothionein has been widely used in medicine, cosmetics, health food additives, and environmental protection. [0003] At present, most of the common metallothionein in the domestic market is purified from rabbit liver. Due to the special demand for metallothionein raw mate...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/75C12R1/125
Inventor 孙继鹏阎光宇邹俊杰何建林黄文文洪碧红
Owner THIRD INST OF OCEANOGRAPHY MINIST OF NATURAL RESOURCES
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