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Method of producing three kinds of human alpha alexins by gene engineering bacteria mixed culture

A mixed culture and defensin technology, applied in the direction of microorganism-based methods, genetic engineering, biochemical equipment and methods, etc., can solve the problems of increased cooling costs, high dissolved oxygen levels, and slow bacterial growth, achieving short development cycles, High expression efficiency and the effect of inhibiting toxic effects

Inactive Publication Date: 2010-09-01
甘肃亚盛盐化工业集团有限责任公司
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

25-30°C is the commonly used culture temperature. At this temperature, the bacteria grow slowly and the dissolved oxygen level is high. If the temperature is too low, the cost of cooling will increase.
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 production of human α-defensins using the Escherichia coli system as a tool, especially the technology for synergistic production of three defensins has not been reported yet

Method used

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  • Method of producing three kinds of human alpha alexins by gene engineering bacteria mixed culture
  • Method of producing three kinds of human alpha alexins by gene engineering bacteria mixed culture
  • Method of producing three kinds of human alpha alexins by gene engineering bacteria mixed culture

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Embodiment 1

[0070] Embodiment 1: Preparation of human alpha defensin genetically engineered bacteria

[0071] 1. Optimal design and chemical synthesis of α-defensin (HNP) mature peptide coding sequence

[0072] HNP1 and HNP2 mRNA sequences (NM_004084), HNP3 mRNA sequences ( NM_005217 ) and the amino acid sequence of HNP1 and HNP2 mature peptide (P59665), the amino acid sequence of HNP3 mature peptide ( P59666 ), HNP1, HNP2, and HNP3 mature peptide coding sequences have only individual base sequence differences (see Table 1, italics), that is, the first three bases of HNP1 are GCC encoding glycine, while HNP2 lacks this amino acid, and the first three bases of HNP3 sequence One codon is GAC encoding aspartic acid. Therefore, the coding sequence of one peptide was first synthesized, and the other sequences were amplified by PCR method.

[0073] Table 1 Coding sequences of mature peptides of HNP1, HNP2 and HNP3 in GENBANK

[0074]

[0075] Through sequence analysis, the original sequ...

Embodiment 2

[0150] Example 2: Mixed expression and activity detection of three human α-defensins

[0151] 1. Chemically induced expression of human α-defensin mixture in mixed culture system

[0152] The correctly identified human α-defensin genetically engineered bacteria PHS1, PHS2, and PHS3 were respectively prepared into glycerol bacteria and streaked on LB solid medium containing ampicillin for preservation. Pick fresh single colonies from the three bacterial plates and add them to 3ml of LB medium containing ampicillin for shaking overnight. Take the overnight culture of the three bacteria the next day and mix them in equal amounts. After mixing, inoculate into new LB medium according to 1% volume, shake vigorously at 37°C for about 3h to OD 600 ≈0.6. Add 1M IPTG to the culture solution to make the final concentration reach 1mmol / L, low temperature induction (30°C) shaking culture, take small samples to detect protein expression at 2h, 4h, and 6h after induction, and finally collec...

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Abstract

The present invention relates to a new method for producing human alpha phylaxin protein by utilizing transgenic codibacillus mixed culture. Said method includes the following steps: using human alpha phylaxin mature peptide to optimize coding sequence, constructing said sequence into proper tac type expression vector pGEx4T1, then transferring said sequence into the screened cytotoxicity-resisting colibacillus BL21(DE3) pLys S so as to construct three kinds of engineering strains of PHS1, PHS2 and PHS3 containing exogenous gene. They are characterized by that they can resist toxicity of expression protein to cell so as to make the expression quantity be obviously raised. The above-mentioned three kinds of engineering strains are undergone the processes of mixed culture and chemical induction, and can be used for expressing human alpha phylaxin, and can simultaneously produce mixed protein of three kinds of human alpha phylaxin proteins.

Description

technical field [0001] The invention relates to a method for producing human α-defensin by using genetically engineered bacteria mixed culture, that is, inserting the genetically modified human α-defensin (Human neutrophil peptides, HNP) 1, 2, 3 mature peptide coding sequence into a fusion prokaryotic expression vector , transform Escherichia coli, obtain three kinds of genetically modified engineering bacteria, and produce three kinds of human α-defensin protein mixtures in a mixed culture system. The invention provides the method and applicable engineering bacteria used in the invention. Background technique [0002] Defensin is a kind of microbial antibiotic peptide commonly found in higher organisms. It has a broad-spectrum poisonous effect on pathogenic microorganisms and is an important part of the body's immune defense system (Ganc T et al, Eur J Haematol, 1990, 44 : 1; Zhang Manchao, Molecular Biology of Foreign Medicine, 1994, 68: 70). Defensins are mainly produce...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/12C12N15/70C12P21/02A61K38/17C12R1/19
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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