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Gene engineering producing method of antibacterial peptide IB-367

A technology of IB-367 and production method, applied in the field of genetic engineering expression of antimicrobial peptides, to achieve the effect of reducing the probability of mismatch

Inactive Publication Date: 2007-02-28
TIANJIN TASLY PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0008] The present invention aims to solve the problem of high-efficiency expression of antibacterial peptide IB367 by means of genetic engineering

Method used

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  • Gene engineering producing method of antibacterial peptide IB-367

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Genetic engineering expression of IB-367

[0020] Construction of expression vectors and acquisition of transformants

[0021] The nucleic acids SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQID NO.5 were synthesized on the plasmid pUC18 vector according to conventional methods, and SmaI (ccc / ggg) was used according to the instructions +EcoRI(g / aattc) digested SEQ IDNO.1-pUC18, recovered small fragments by low melting point agarose method ("Molecular Cloning"), and used PhsAI(gacNN / NNgtc)+EcoRI(g / aattc) to vector pET42a( +) Carry out enzyme digestion, recover large fragments by low-melting point agarose method, and ligate the recovered product with T4 DNA ligase according to the instructions to obtain an expression vector containing the DNA sequence encoding IB-367, and transform the expression vector into newly prepared Escherichia coli The competent cells of DH5α were screened and identified as transformants, and the plasmid was extracted by conventional m...

Embodiment 2

[0027] Antibacterial effect detection

[0028] Method: Determination by inhibition zone and bactericidal concentration

[0029] Agarose diffusion antimicrobial assay (US Patent 6,337,314, January 8, 2002).

[0030] Obtain the IB-367 expressed by genetic engineering according to the method of Example 1, to 5 golden yellow strains of different sources (standard bacterial strain Staphylococcus aureus S.aureus, CMCC (B) 26003 and hospital clinical case isolation Staphylococcus aureus bacterial strain) The minimum bactericidal concentration (MIC) of Staphylococcus aureus is 0.1-3 μg / ml, which is effective against different sources (standard strain Pseudomonas aeruginosa Mignla, CMCC (B) 10104 and hospital clinical case isolates of Pseudomonas aeruginosa strains). The minimum bactericidal concentration (MIC) of five strains of Pseudomonas aeruginosa was 0.04-6 μg / mL.

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Abstract

The invention relates the antibiosis peptide IB-367 gene engineering expression method, comprising synthesized encoding IB-367 DNA sequence, which is connected with the carrier to express and purify. The encoding IB-367 DNA sequence adopts codons which is used high frequency, reducing the mismatch rate. The Tm value is moderate, and the IB-367 expressed in procaryotic cell fusion expression system is 48-52% of total protein.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a genetic engineering expression method of antimicrobial peptides. Background technique [0002] The discovery of antibiotics has made people no longer helpless against various diseases caused by pathogenic microorganism infection, and has made a great contribution to the protection of human health. However, with the widespread use of antibiotics, pathogenic strains continue to develop drug resistance, and some infectious diseases that were very effectively controlled by antibiotics in the past have revived in recent years. And due to the continuous enhancement and spread of drug resistance, existing antibiotics have become powerless. In addition, the widespread abuse of antibiotics and drug residues, allergic reactions to some antibiotics and many other problems have become increasingly serious and prominent, and a new cl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09C12N15/11C12P21/02C12N15/12
Inventor 杨旭吴全忠高峰姜燕
Owner TIANJIN TASLY PHARMA CO LTD
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