Insect antimicrobial peptide Thanatin and method for producing deletion mutant thereof

A technology for deletion mutants and antibacterial peptides is applied in the field of preparation of insect antibacterial peptides and their deletion mutants, which can solve the problems of difficult large-scale industrial production, short Thanatin sequences, and difficult expression, and achieve high antibacterial activity and environmental friendliness. Effect

Inactive Publication Date: 2008-05-07
沈子龙
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  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0011] In order to solve the problem that the Thanatin sequence is short and difficult to express, in the prior art, the Thanatin-encoded gene is often used to form a bivalent gene together with ot

Method used

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  • Insect antimicrobial peptide Thanatin and method for producing deletion mutant thereof
  • Insect antimicrobial peptide Thanatin and method for producing deletion mutant thereof
  • Insect antimicrobial peptide Thanatin and method for producing deletion mutant thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0038] Construction of expression vectors and acquisition of transformants

[0039] Synthesize primers SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3, (Table 1) respectively according to known conventional methods by those skilled in the art, use SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.1 and SEQ ID NO.3 respectively constitute 2 pairs of primers, which are amplified by PCR, separated by agarose electrophoresis, and recovered by gel to obtain the target fragment. The target fragment was connected to the pMD18T (Shanghai Sangong) vector, transformed into Escherichia coli DH5α (Novagen, USA), and positive clones were identified by blue-white screening, named pMD18T-TH and pMD18T-TH1. According to the instructions, the vectors pET32a(+) (Novagen, USA), pMD18T-TH and pMD18T-TH1 were respectively digested with BamH I and XhoI (Promega, USA) respectively, and the corresponding fragments were recovered by low-melting point agarose method ("Molecular Cloning" ), connect the recovered product...

Embodiment 2

[0051] Determination of Antibacterial Activity

[0052] A polypeptide sample solution with a concentration of 6 mg / ml was prepared with sterilized physiological saline. The bacteria used in the test were inoculated in the nutrient broth, cultured at 37°C for 24 hours, diluted 1:105 times with sterile saline before use, and 12 bacterial culture tubes were taken and numbered, and the nutrient meat was added to the first tube Add 1.8ml broth, and add 1.0ml broth to the remaining 11 tubes. Add 0.2ml of polypeptide solution to the first tube, mix well, take out 1.0ml and add it to the second tube, repeat this process, dilute to the 12th tube in turn, add 0.2ml of bacterial solution to each tube, shake gently, place at 37 Cultivate for 24 hours. The lowest concentration of peptides for sterile growth is the minimum sample concentration (MIC) that inhibits bacterial growth. Table 2 is Thanatin prepared in Example 1 and its deletion mutants to the minimum inhibitory concentrations ...

Embodiment 3

[0056] Determination of polypeptide hemolytic activity: Human red blood cells (where obtained) were suspended in phosphate (where purchased, produced by whom) buffer (pH 7.4) to obtain red blood cell suspension (5% v / v). Dissolve the polypeptide in phosphate buffer solution to make about 5mg / ml stock solution, take 14 1.5ml centrifuge tubes, add 1ml polypeptide stock solution to the first centrifuge tube, add 0.5ml phosphate buffer to the other tubes solution, take 0.5ml of peptide stock solution from the first tube and add it to the second tube, mix evenly with a micro mixer, then take 0.5ml solution from the second tube, add it to the third tube and mix evenly, and so on, That is, dilute to the 14th tube sequentially by the doubling dilution method, discard 0.5ml, add 0.5ml of prepared 5% red blood cell suspension to each tube to a final volume of 1.0ml, shake gently, and incubate in a 37°C incubator After 60 min, it was centrifuged at 4000 rpm for 10 minutes, and the supern...

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Abstract

The invention relates to a method for using the genetic engineering method to prepare insect Thanatin and fifteenth amino acid (T) deletion mutant thereof. The invention is characterized in that the method has low cost and simple post processing, thereby being commercially produced in the large scale.

Description

【Technical field】 [0001] The invention relates to the field of biotechnology, more specifically, the invention relates to a preparation method of an antibacterial insect antimicrobial peptide and a deletion mutant thereof. 【Background technique】 [0002] The discovery of antibiotics is of great significance in the history of human medicine. Its use has saved countless lives and increased human life expectancy by more than ten years. But with the widespread use of antibiotics, bacteria have gradually adapted and developed resistance to them. Although there are hundreds of antibiotics in clinical use at present, they are all small chemical molecules, and the newly developed antibiotics are generally structural modifications of them. It is difficult to solve the problem of bacterial resistance to antibiotics. With the emergence of drug-resistant bacteria, the development of new antibiotics against drug-resistant bacteria has become an international concern. [0003] Many orga...

Claims

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Application Information

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IPC IPC(8): C07K14/435C12N15/12C12N15/70
Inventor 沈子龙吴国球
Owner 沈子龙
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