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Method for preparing high purity cryptoprotein

A high-purity cryptid protein technology, which is applied in the field of preparation of high-purity cryptic protein, can solve the problems of reducing the yield of microbial proteins and polypeptides, reducing biological activity and stability, and lengthy processes, and achieves good practical application value, Easy to accept and simplify the effect of purification process

Inactive Publication Date: 2019-04-23
EZHOU INST OF IND TECH HUAZHONG UNIV OF SCI & TECH +1
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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 preparing high purity cryptoprotein
  • Method for preparing high purity cryptoprotein
  • Method for preparing high purity cryptoprotein

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preparation example Construction

[0038] The first aspect of the present invention provides a method for preparing a high-purity cryptogram, the steps comprising:

[0039] S1. Linking the coding gene of cryptogein with TEV protease and T4 phage Soc protein gene fragments sequentially to prepare the Cryptogein-TEV-Soc fusion recombinant protein coding gene, the coding region of the cryptogein protein is in the TEV-Soc fusion protein gene Upstream, and the expression frame is consistent; the upstream of the coding DNA sequence of the Cryptogein-TEV-Soc fusion recombinant protein is introduced into the Soc promoter sequence, and the downstream is introduced into the Soc terminator sequence. In one embodiment of the present invention, the cryptogein protein is derived from Chain A of the Cryptogein protein of the strain Phytophthora cryptogea whose GeneBank number is CAA84224.1.

[0040] S2. Transfer the gene encoding the Cryptogein-TEV-Soc fusion recombinant protein into a plasmid;

[0041] S3, transforming the ...

Embodiment 1

[0050] Example 1, Sequence Acquisition of Cryptogein

[0051] The sequence encoding the Cryptogein protein derived from the strain Phytophthora cryptogea has been published (GeneBank number: CAA84224.1). The Cryptogein gene can be obtained by 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 method of gene synthesis, and the amino acid sequence of Cryptogein is synthesized (Suzhou Jinweizhi Biotechnology Co., Ltd., China).

Embodiment 2

[0052] Embodiment 2, construction pVB-Cryptogein-TEV-Soc plasmid

[0053] 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 Cryptogein protein gene fragment and the TEV-Soc protein gene fragment were fused into a DNA molecule by bridging PCR, w...

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Abstract

The invention provides a method for preparing high-purity cryptoprotein. The method comprises the steps of (1) sequentially connecting a coding gene of a target protein with a TEV protease and a T4 phage Soc protein gene fragment to prepare a fusion recombinant protein coding gene; (2) transferring the gene encoding the fusion recombinant protein into a plasmid; (3) transferring the plasmid into abacterium coli host; (4) infecting the bacterium coli host with a Soc-deficient Soc-T4 phage, and cultivating the host to express the T4 phage displaying the fusion recombinant protein; (5) isolatingthe T4 phage displaying the fusion recombinant protein; (6) isolating the fusion recombinant protein from the T4 phage, and obtaining the target protein by TEV protease digestion. The method can obtain the high-purity cryptoprotein easily and quickly, an obtained cryptoprotein solution can be directly used as an agricultural antibacterial bacteriostatic agent to achieve the effect of promoting plant growth, and the method has a good application prospect.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a preparation method of high-purity cryptogram. Background technique [0002] Chemical fertilizers and pesticides are the main products to increase crop yield and prevent pests and diseases, but at the same time, they have caused problems in terms of environment, energy and cost, which have become major problems that plague the economic and social development of countries all over the world, seriously restricting the sustainable development of agriculture. sexual development. 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 mainly insecticidal cr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/62C12N15/70A01N37/46A01P1/00A01P3/00A01P21/00
CPCA01N37/46C12N9/50C12N15/70C07K14/37C07K2319/50
Inventor 刘胜蒙亮刘欣李维石聿勇李威胡艳红
Owner EZHOU INST OF IND TECH HUAZHONG UNIV OF SCI & TECH
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