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Preparation method of high-purity cryptogein

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

Pending Publication Date: 2019-04-19
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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  • Preparation method of high-purity cryptogein
  • Preparation method of high-purity cryptogein
  • Preparation method of high-purity cryptogein

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preparation example Construction

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

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

[0040] S2, the encoding gene of described Cryptogein-TEV-Hoc fusion recombinant protein is transferred into plasmid;

[0041] S3, the plasmid obtained in step S2 is transferred into the E...

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 Accession: CAA84224.1). The Cryptogein gene can be obtained by using techniques known 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 Cryptogein amino acid sequence for synthesis (Suzhou Jinweizhi Biotechnology Co., Ltd., China).

Embodiment 2

[0052] Example 2. Construction of pVB-Cryptogein-TEV-Hoc plasmid

[0053] The backbone of the plasmid was derived from the pUC18 plasmid. Using the pUC18 plasmid as a template, the ampicillin resistance gene and plasmid replication region were amplified by PCR using KOD high-fidelity DNA polymerase. The PCR forward primer was 18 bases in the upstream sequence of the ampicillin resistance gene of pUC18, and the reverse primer was the 18 bases reverse complementary sequence in the downstream sequence of the pUC18 plasmid replication region. BglII restriction sites were added to the 5' ends of the forward and reverse primers. At the same time, we used T4 phage DNA as a template and used specific primers to amplify the Hoc protein gene fragment by PCR. The 5' end of the forward primer introduced the gene sequence of TEV protease. Then, the synthesized Cryptogein protein gene fragment and the TEV-Hoc protein gene fragment were fused into a DNA molecule by bridge PCR, wherein the ...

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Abstract

The invention provides a preparation method of a high-purity cryptogein, which comprises the following steps: (1) sequentially connecting a coding gene of a target protein with TEV protease and T4 bacteriophage Hoc protein gene fragments to prepare a coding gene of the fusion recombinant protein; (2) transferring the coding gene of the fusion recombinant protein into a plasmid; (3) transferring the plasmid into an escherichia coli host; (4) infecting the escherichia coli host with Hoc-T4 bacteriophage deleted by Hoc protein, and culturing the host to express T4 bacteriophage displaying the fusion recombinant protein; (5) separating T4 bacteriophage displaying fusion recombinant protein; (6) separating the fusion recombinant protein from the T4 bacteriophage, and obtaining the target protein after TEV protease cleavage. The preparation method can simply and rapidly obtain high-purity cryptogein, and the obtained cryptogein solution can be directly used as an agricultural antibacterial bacteriostatic agent to achieve the effect of obviously promoting plant growth, thereby having good application prospect.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for preparing high-purity cryptin. Background technique [0002] Fertilizers and pesticides are the main products for improving crop yields and preventing pests and diseases, but at the same time they cause environmental, energy and cost problems. sexual development. And biological products (including biological fertilizers and biological pesticides) prepared from proteins derived from microorganisms or fungi are the main measures for the high-quality, high-efficiency and pollution-free production of contemporary crops. In recent years, a large number of scientific researches have been carried out in this field at home and abroad. The early microbial proteins were mainly insecticidal crystal proteins (ICP) from Bacillus thuringiensis, and the new microbial proteins were mainly protein elicitors. Cryptogein and Activator, these proteins do not directly kill pes...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/62C12N15/70C07K14/37A01N61/00A01P1/00A01P3/00
CPCA01N61/00C12N15/70C07K14/37
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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