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Method for efficiently expressing hypersensitive protein by using T4 phage display technology

A phage display and hypersensitive protein technology, applied in the field of high-efficiency expression of hypersensitive proteins, can solve the problems of lengthy process, reduced yield of microbial proteins and polypeptides, and many steps.

Pending Publication Date: 2020-03-17
湖北微生元生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These processes are complicated to operate, expensive, and due to many steps and lengthy processes, the yield of microbial proteins and peptides will be reduced, and their biological activity and stability will be reduced

Method used

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  • Method for efficiently expressing hypersensitive protein by using T4 phage display technology
  • Method for efficiently expressing hypersensitive protein by using T4 phage display technology
  • Method for efficiently expressing hypersensitive protein by using T4 phage display technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1. Sequence acquisition of hypersensitive protein (harpinEa)

[0029] The sequence of gene hrpN encoding harpinEa protein derived from strain Erwinia amylovora has been published (GeneBank number: M92994.3). The hypersensitive protein (harpinEa) gene can be obtained using well-known techniques in the industry such as gene synthesis, PCR amplification, extraction of chromosomal DNA and establishment of a library. The present invention adopts the gene synthesis method to send the hypersensitive protein (hrapinEa) amino acid sequence to be synthesized (Suzhou Jinweizhi Biotechnology Co., Ltd., China).

Embodiment 2

[0030] Embodiment 2, construct pVB-harpinEa-TEV-Soc plasmid

[0031] The backbone of the plasmid was from the pUC18 plasmid. Using the pUC18 plasmid as a template, the ampicillin resistance gene and the plasmid replication region were amplified by PCR using KOD high-fidelity DNA polymerase. The forward primer of PCR is 18 bases in the upstream sequence of pUC18 ampicillin resistance gene, and the reverse primer is the reverse complementary sequence of 18 bases in the downstream sequence of pUC18 plasmid replication region. A BglII restriction enzyme site was added to the 5' ends of the forward and reverse primers. At the same time, we used T4 phage DNA as a template and obtained the Soc protein gene fragment by PCR amplification using specific primers, wherein the 5' end of the forward primer introduced the gene sequence of TEV protease. The synthetic harpinEa protein gene fragment and the TEV-Soc protein gene fragment were fused into a DNA molecule by bridging PCR, wherein ...

Embodiment 3

[0033] Example 3. In vivo expression and phage display of fusion recombinant protein

[0034] The T4 phage synchronous display expression plasmid pVB-harpinEa-TEV-Soc was transformed into competent cells of E. coli P301 strain by chemical transformation method, and under the pressure of ampicillin antibiotics, it was cultured in a shaker at 37°C at a speed of 200rpm until the bacteria Density up to 3×10 8 pieces / ml. Soc with Soc protein deletion - T4 phage (gifted by the laboratory of Professor Rao, Department of Biology, Catholic University of America) infected the cultured Escherichia coli cells at MOI (multiplicity of infection) = 1, and continued to culture in a shaker at 37° C. at 200 rpm for 30 min. During this process, the harpinEa-TEV-Soc recombinant protein was expressed under the regulation of the T4 phage particle assembly pathway and displayed on the capsid.

[0035] After the display is completed, the culture solution is immediately divided into pre-cooled centri...

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Abstract

The invention relates to a method for efficiently expressing hypersensitive protein by using a T4 phage display technology. The method comprises the following steps: (1) sequentially connecting an encoding gene of the hypersensitive protein with TEV protease and a T4 bacteriophage Soc protein gene segment to prepare a fusion recombinant protein encoding gene, wherein a encoding region of the hypersensitive protein is located at the upstream of a TEV-Soc fusion protein gene and having consistent expression cassettes; (2) transferring the coding gene of the fusion recombinant protein into a pUC18 plasmid; (3) transferring the plasmid into an escherichia coli host; (4) infecting the escherichia coli host by using Soc-T4 bacteriophage without Soc protein, and culturing the T4 bacteriophage ofwhich the host expresses and displays the fusion recombinant protein; (5) separating the T4 bacteriophage showing the fusion recombinant protein; and (6) separating the fusion recombinant protein fromthe T4 bacteriophage, and carrying out TEV protease digestion to obtain the hypersensitive protein.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for efficiently expressing hypersensitive proteins using T4 phage display technology. Background technique [0002] Chemical fertilizers and pesticides are the main products to increase crop yields and prevent pests and diseases, but at the same time, they have caused problems in the environment, energy and cost, and have become a major issue that plagues the economic and social development of countries around the world and seriously restricts the sustainability of agriculture. develop. The use of biological products (including biological fertilizers and biological pesticides) prepared from proteins from microorganisms or fungi is the main measure for high-quality, efficient and pollution-free production of contemporary crops. In recent years, a lot of scientific research has been carried out in this area at home and abroad. The early microbial proteins were ma...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N15/62C12N7/00C12P21/06C07K14/27A01N63/50A01P21/00A01P3/00A01P7/04C12R1/92
CPCC12N15/70C12N15/62C12N7/00C12P21/06C07K14/27C07K2319/00
Inventor 刘胜蒙亮刘欣刘军鹏李维石聿勇胡艳红张会慧
Owner 湖北微生元生物科技有限公司
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