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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 high cost, complicated operation, and many steps.

Pending Publication Date: 2020-03-13
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 harpinEa

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

Embodiment 2

[0030] Example 2. Construction of pVB-harpinEa-TEV-Hoc plasmid

[0031] The backbone of the plasmid is derived from the pUC18 plasmid. Using the pUC18 plasmid as a template, KOD high-fidelity DNA polymerase was used to amplify the ampicillin resistance gene and plasmid replication region by PCR. The PCR forward primer is the 18 bases in the upstream sequence of the pUC18 ampicillin resistance gene, and the reverse primer is the reverse complement of the 18 bases in the downstream sequence of the pUC18 plasmid replication region. Both the forward and reverse primers have a BglII restriction site added to the 5'ends. At the same time, we used T4 phage DNA as a template and used specific primers to amplify Hoc protein gene fragments by PCR. The 5'end of the forward primer introduced the gene sequence of TEV protease. Then, by bridge PCR, the synthesized harpinEa protein gene fragment and the TEV-Hoc protein gene fragment are fused into a DNA molecule, where the coding region of th...

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-Hoc was transformed into competent cells of Escherichia coli P301 strain by chemical transformation method, and cultured to bacteria under the pressure of ampicillin antibiotics in a shaker at 37°C at 200 rpm Density reaches 3×10 8 Pcs / ml. Hoc deleted with Hoc protein - T4 bacteriophage (given by the laboratory of Professor Rao, Department of Biology, Catholic University of America) infects the cultured E. coli cells at MOI (multiplicity of infection)=1, and continues to incubate in a shaker at 37°C at 200 rpm for 30 min. During this process, the harpinEa-TEV-Hoc recombinant protein is expressed under the control of the T4 phage particle assembly pathway and displayed on the capsid.

[0035] After the display is completed, the culture solution is immediately aliquoted in pre-cooled centrifuge tubes, and centrifuged at 43...

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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 Hoc protein gene segment to prepare a fusion recombinant protein encoding gene, wherein the encoding region of the hypersensitive protein is located at the upstream of a TEV-Hoc fusion protein gene and having consistent expression cassettes; (2) transferring a 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 Hoc-T4 bacteriophage deleted by Hoc protein, and culturing the host to express and display the T4 bacteriophage of the fusion recombinant protein; (5) separating the T4 bacteriophage showing the fusion recombinant protein; and (6) separating the fusion recombinant protein from the T4 bacteriophage, and carrying out TEV protease digestion to obtain the hypersensitive protein.

Description

Technical field [0001] The present invention belongs to the field of biotechnology, and specifically relates to a method for efficiently expressing hypersensitive proteins using T4 phage display technology. Background technique [0002] Fertilizers and pesticides are the main products for increasing crop yields and preventing pests and diseases, but at the same time they have caused environmental, energy, and cost problems. They have become major economic and social developments that are now plagued all countries in the world, and seriously restrict the sustainability of agriculture. development of. Biological products (including biological fertilizers and biological pesticides) prepared by using protein from microorganisms or fungi are the main measures for high-quality, high-efficiency 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 mainly...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N15/62C12N7/00C12P21/06C07K14/27A01N63/20A01P21/00A01P7/04
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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