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 alpha defensin 1 protein by using Escherichia coli, can solve problems such as unreported, achieve the effects of good transcription and translation efficiency, short development period, and easy processing and purification

Inactive Publication Date: 2006-08-02
甘肃亚盛盐化工业集团有限责任公司
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  • 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.

[0054] The modified sequence is shown in Table 2.

[0055] The two complementary strands of the redesigned HNP1 mature peptide coding ...

Embodiment 2

[0087] Example 2: Construction, induced expression and activity identification of mHNP1 T7 expression vector

[0088] 1. Construction of mHNP1 T7 expression vector

[0089] (1) Strains and plasmids:

[0090] 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.

[0091] (2) Tool enzymes and reagents:

[0092] 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.

[0093] (3) Design and synthesis of primers:

[0094] 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:

[0095] P1 5'GCG GGA TCC ATG GCC TGC TAT TGC C...

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Abstract

The present invention provides production process of human alpha phylaxin-1 protein with colibacillus and the optimized designed DNA sequence and recombinant expression plasmid. The production process includes: optimizing design with alpha phylaxin-1 mature peptide code sequence to obtain sequence SEQ ID No. 1; constituting prokaryotic expression vector plasmid pET32a-mHNP1, introducing the plasmid into colibacillus BL21(DE3)pLysS, and chemically inducing expression of human alpha phylaxin-1. The process can overcome the toxin of human alpha phylaxin-1 protein on host bacillus causing low or even no expression, and produce active human alpha phylaxin-1 protein in great amount for meeting the need of relevant structure and biochemical research and antibody preparation as well as for application in medical field.

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 Applications(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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