Method for displaying trehalose synthase on bacillus subtilis spore capsid protein Cot surfaces

A technology of Bacillus subtilis and trehalose synthase, applied in the field of molecular biology, can solve the problems of lack of research reports, affecting the surface display of target proteins, etc., and achieve high safety effects

Inactive Publication Date: 2015-12-09
QILU UNIV OF TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, because the enzyme is greatly affected by the spatial structure, whether these Cot spore capsid proteins will affect the surface display of the target protein after binding to the target protein has not been reported yet.

Method used

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  • Method for displaying trehalose synthase on bacillus subtilis spore capsid protein Cot surfaces
  • Method for displaying trehalose synthase on bacillus subtilis spore capsid protein Cot surfaces

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Preparation of recombinant spores displaying trehalose synthase TreS on the surface of Bacillus subtilis spores using CotB as molecular carrier.

[0067] Acquisition of Bacillus subtilis capsid protein CotB gene

[0068] Bacillus subtilis 168 genomic DNA was extracted from the cultivated Bacillus subtilis bacterial liquid according to the instructions of the Ezup column bacterial genome DNA extraction kit. In addition, primers were designed according to the coding sequence of the CotB gene in the NCBI database, and BamHI and XbaI restriction sites were introduced into the upstream and downstream primers, respectively. The primers were provided by Sangon Bioengineering (Shanghai) Co., Ltd. CotH was amplified using the extracted Bacillus subtilis genomic DNA as a template by using 2×HiFi-PCRMaster polymerase from Treasure Bioengineering Co., Ltd. The nucleotide sequences of the primers are as follows, where the underline represents the restriction site.

[0069] CotB-F:...

Embodiment 2

[0108] Preparation of recombinant spores displaying trehalose synthase TreS on the surface of Bacillus subtilis spores using CotC as molecular carrier.

[0109] Acquisition of CotC Gene of Bacillus subtilis Bacillus Capsid Protein

[0110] Bacillus subtilis 168 genomic DNA was extracted from the cultivated Bacillus subtilis bacterial liquid according to the instructions of the Ezup column bacterial genome DNA extraction kit. In addition, primers were designed according to the coding sequence of the CotH gene in the NCBI database, and BamHI and XbaI restriction sites were introduced into the upstream and downstream primers respectively. The primers were provided by Sangon Bioengineering (Shanghai) Co., Ltd. CotC was amplified using the extracted Bacillus subtilis genomic DNA as a template using 2×HiFi-PCRMaster polymerase from Treasure Bioengineering Co., Ltd. The nucleotide sequences of the primers are as follows, where the underline represents the restriction site.

[0111]...

Embodiment 3

[0148] Preparation of recombinant spores displaying trehalose synthase TreS on the surface of Bacillus subtilis spores using CotD as molecular carrier.

[0149]Acquisition of Bacillus subtilis capsid protein cotD gene

[0150] Bacillus subtilis 168 genomic DNA was extracted from the cultivated Bacillus subtilis bacterial liquid according to the instructions of the Ezup column bacterial genome DNA extraction kit. In addition, primers were designed according to the coding sequence of the CotD gene in the NCBI database, and BamHI and XbaI restriction sites were introduced into the upstream and downstream primers respectively. The primers were provided by Sangon Bioengineering (Shanghai) Co., Ltd. CotD was amplified using the extracted Bacillus subtilis genomic DNA as a template using 2×HiFi-PCRMaster polymerase from Treasure Bioengineering Co., Ltd. The nucleotide sequences of the primers are as follows, where the underline represents the restriction site.

[0151] CotD-F: GG...

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Abstract

The invention relates to a method for displaying trehalose synthase on bacillus subtilis spore capsid protein Cot surfaces. The method is characterized in that Cot series spore capsid protein is used as the molecular vehicle for displaying heterologous protein on spore surfaces, series primers of the Cot molecular vehicle are designed, the molecular vehicle can be replaced and target heterologous protein can be changed through double enzyme digestion, the gene technology is used to display the trehalose synthase on bacillus subtilis spore surfaces, and the spore capsid protein which can efficiently display the trehalose synthase is screened. The method has the advantages that the trehalose synthase efficiently displayed on the spore surfaces has no antibacterial activity, meets the requirements of enzyme preparation applied to food and facilitates next-step production of trehalose.

Description

technical field [0001] The present invention relates to a method for displaying trehalose synthase on the surface of the Bacillus subtilis spore coat protein Cot. The invention particularly relates to several novel spore coat proteins as molecular carriers to integrate foreign protein genes with them, thereby screening out A molecular carrier capable of high-throughput displaying the amount of target protein belongs to the technical field of molecular biology. Background technique [0002] Trehalose is a non-reducing sugar composed of two glucose molecules with α, α, 1,1-glycosidic bonds. It can stabilize the structure of cell membranes and proteins under harsh environmental conditions such as high temperature, high cold, high osmotic pressure, and dehydration. , effectively protect cell membranes and protein molecules from denaturation and inactivation, thereby maintaining the life process and biological characteristics of living organisms. In the industrial production of ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/75C12N1/21C12N15/62
Inventor 王腾飞王瑞明赵一瑾陆奉勇
Owner QILU UNIV OF TECH
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