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Production process of human-alpha phylaxin-1 protein with colibacillus

A technology of Escherichia coli and defensin, which is applied in the field of producing human α-defensin 1 protein by Escherichia coli, which can solve the problems that have not been reported before, achieve good transcription and translation efficiency, prevent human immune response, and shorten the development cycle

Inactive Publication Date: 2008-12-24
甘肃亚盛盐化工业集团有限责任公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Li Jingpeng and others obtained the cDNA clone of human α-defensin through chemical synthesis (Journal of Northeast Agricultural University, 1995, 26: 383), and Liu Shuiping and others also cloned the cDNA fragment of human defensin gene (Journal of Hunan Medical University, 2002, 27: 17), however, the technology of producing human α-defensin using the Escherichia coli system as a tool has not been reported yet

Method used

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  • Production process of human-alpha phylaxin-1 protein with colibacillus
  • Production process of human-alpha phylaxin-1 protein with colibacillus
  • Production process of human-alpha phylaxin-1 protein with colibacillus

Examples

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Embodiment 1

[0051] Example 1: Chemical Synthesis and Cloning of α-Defensin 1 Mature Peptide Coding Sequence

[0052] 1. Optimal design and chemical synthesis of HNP1 mature peptide coding sequence

[0053] The HNP1 mRNA sequence was obtained from Genbank ( NM 004084 ) and its mature peptide amino acid sequence ( P59665 ), thus deducing its mature peptide coding sequence (90bp). On the basis of sequence sequence analysis, the original sequence is optimized and designed according to the differences between biologically preferred codons and their rare codons. That is, the rare codons in HNP1 were deleted and replaced with codons favored by E. coli. In order to achieve a good translation termination effect, two stop codons, TGA and TAA, were added at the end of the optimized coding sequence to facilitate the expression of the target protein in E. coli. The modified sequence is shown in Table 2.

[0054] The two complementary strands of the redesigned HNP1 mature peptide coding sequence ...

Embodiment 2

[0088] Example 2: Construction, induced expression and activity identification of mHNP1 T7 expression vector

[0089] 1. Construction of mHNP1 T7 expression vector

[0090] (1) Strains and plasmids:

[0091] Escherichia coli BL21(DE3)pLysS was preserved in our laboratory. pET32a plasmid (Novagen, see Figure 4 ) was a gift from Dr. Zeng Xiankun, School of Medicine, Southeast University.

[0092] (2) Tool enzymes and reagents:

[0093] Restriction enzyme, T 4 Ligase and Taq DNA polymerase were purchased from Dalian Bao Biological Engineering Co., Ltd., Shanghai Sangong Co., Ltd., and Beijing Dingguo Biotechnology Co., Ltd., respectively.

[0094] (3) Design and synthesis of primers:

[0095] The PCR primers were optimized and designed according to the present invention to design the coding sequence of mHNP1 mature peptide, all of which were synthesized by Dalian Bao Biology Co., Ltd. The primer sequences are as follows:

[0096] P1 5'GCG GGA TCC GCC TGC TAT TGC CGT ...

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Abstract

The invention provides a method for producing biologically active substance human alpha defensin 1 protein by using Escherichia coli and its applicable optimized design DNA sequence and recombinant expression plasmid. Including: use the sequence SEQ ID NO: 1 obtained after the optimized design of the human α-defensin 1 mature peptide coding sequence to construct the prokaryotic expression vector plasmid pET32a-mHNP1, introduce the plasmid into Escherichia coli BL21(DE3)pLysS, and chemically induce the expression of human Alpha-defensin 1. This method can overcome the low expression or even no expression caused by the toxic effect of α-defensin 1 protein on the host bacteria itself, and mass-produce active human α-defensin 1 protein, which meets the requirements of relevant structure, biochemical properties research and antibody preparation. It can also be applied to the field of medicine and health.

Description

technical field [0001] The present invention relates to a new method for producing human α-defensin 1 using transgenic engineering bacteria, that is, to optimize the coding sequence of human α-defensin 1 (Human neutrophil peptide, HNP1) mature peptide coding sequence according to the principle of biological codon usage preference, Construct a Trx fusion prokaryotic expression vector that can promote the formation of disulfide bonds, and transform the E. coli strain BL21(DE3)pLysS, which can reduce the low expression level of toxic protein groups, to form a high-efficiency E. coli expression system. The present invention also provides a method for using the genetic engineering strain constructed in the present invention. Background technique [0002] Defensins are a kind of polypeptide antibiotics ubiquitous in higher organisms. They have a broad-spectrum poisonous effect on pathogenic microorganisms and are an important part of the body's immune defense system (Ganc T et al,...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/12C12N15/70C12P21/02C07K14/435C12N15/63
Inventor 周长生陈其新李春丽陈正华
Owner 甘肃亚盛盐化工业集团有限责任公司
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