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Hermetia illucens L antibacterial peptide, preparation method and application thereof

A technology of soldier flies and antibacterial peptides, which is applied in the antibacterial function of soldier flies and antibacterial peptides, and can solve the severe impact of treatment effects. and other problems, to achieve good application prospects, simple method, and broad-spectrum antibacterial activity

Inactive Publication Date: 2012-12-12
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the drug resistance of pathogenic microorganisms is constantly increasing, and the drug resistance spectrum is also expanding, so the impact on clinical disease resistance, anti-infection, agricultural animal and plant diseases, etc. is relatively serious.

Method used

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  • Hermetia illucens L antibacterial peptide, preparation method and application thereof
  • Hermetia illucens L antibacterial peptide, preparation method and application thereof
  • Hermetia illucens L antibacterial peptide, preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] (1) Extraction of total RNA from soldier fly

[0050] Soak spare equipment (anatomical scissors, centrifuge tubes, various types of Tips, etc.) in 1% diethylpyrocarbonate (DEPC, purchased from Shanghai Sangong) water for 12 hours;

[0051] Each equipment treated with DEPC was sterilized by autoclave for 30 minutes;

[0052] Soak the original material, the light-spotted soldier fly with 75% alcohol for 30 minutes;

[0053] Wash the treated material 2-3 times with ultrapure water;

[0054] The tissue samples were cut with dissecting scissors, placed in liquid nitrogen, and immediately put into an RNase-free mortar for grinding, and the total RNA was extracted with an ultra-pure total RNA rapid extraction kit (former Pinghao Biotechnology Co., Ltd.) under sterile conditions. For RNA, see the instruction manual of the RNA Rapid Extraction Kit for detailed steps.

[0055] Store RNA at -70°C for later use.

[0056] (2) Synthesis of the first-strand cDNA of the light-spott...

Embodiment 2

[0066] (1) Design of degenerate primers

[0067] Consult the relevant database of antimicrobial peptides, analyze the conserved regions of various antimicrobial peptides according to the antimicrobial peptides that Diptera insects may produce, use Primer5.0 to design degenerate primers, and finally design 6 pairs of primers, the results are shown in Table 1 .

[0068] Table 1 Primers required for antifungal peptide gene amplification

[0069]

[0070] (2) PCR amplification of antimicrobial peptide gene

[0071] Using the cDNA of the reverse-transcribed soldier fly as a template and the designed 6 pairs of annex primers as primers, temperature gradient PCR was carried out. The system and conditions of PCR amplification are as follows:

[0072] The PCR amplification system is:

[0073]

[0074] The reaction was carried out in the form of temperature gradient PCR, and the reaction parameters were: pre-denaturation at 94°C for 5 minutes; denaturation at 94°C for 30 sec, T...

Embodiment 3

[0096] According to the Stomoxyn ZH1 gene sequence amplified in Example 2, combined with the desired expression vector pPICZαA, design primers with restriction sites, upstream primer P1: CCG GAATTC AGAGGATTTCGTAAGCA (the underline is the restriction site of EcoR Ⅰ, and the protection base is in the box); downstream primer P2: AAT GCGGCCGC The primers of AGTAGCAGCAACAACAGCA (underlined is the restriction site of Not Ⅰ, and the box is protected base) were synthesized by Shanghai Yingjun Company.

[0097] Using the cDNA of the soldier fly cDNA as a template, using the designed primers P1 and P2 with restriction sites, the corresponding Stomoxyn ZH1 gene was amplified by PCR (the amplification conditions were the same as in Example 2), and the amplified The product was detected by 2.5% agarose gel electrophoresis.

[0098] Example 4 Construction of recombinant plasmid pPICZαA-Stomoxyn ZH1

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Abstract

The present invention discloses a new Hermetia illucens L antibacterial peptide StomoxynZH1, a preparation method and an application thereof. Preparation steps of the antibacterial peptide comprise: extracting the total RNA of insect Hermetia illucens L as a template, performing RT-PCR to obtain a first strand of cDNA as a template, and performing PCR amplification to obtain a PCR product; carrying out digestion to obtain a PCR product and a eukaryotic expression vector to construct a recombinant expression vector; transforming the resulting recombinant expression vector into yeast host cellsto obtain a recombinant strain expressing the antibacterial peptide StomoxynZH1; and carrying out inducing culture on the resulting recombinant strain to obtain a culture, and carrying out centrifugal separation on the culture to obtain the expressed antibacterial peptide. The antibacterial peptide of the present invention has broad spectrum antimicrobial activities and provides good application prospects in medicine, agriculture disease prevention and control, feed additives and other fields, wherein the broad spectrum antimicrobial activities comprise gram-negative bacteria escherichia coliresistance, gram-positive bacteria staphylococcus aureus resistance, and fungi rice blast pathogenic bacteria resistance.

Description

technical field [0001] The invention relates to the technical field of genetic bioengineering. It specifically relates to a light-spotted soldier fly (Hermetia illucens L.) antimicrobial peptide gene, a preparation method of a light-spotted soldier fly antibacterial peptide, and a light-spotted soldier fly antibacterial peptide in antibacterial functional applications. Background technique [0002] Insect antimicrobial peptides are a kind of antibacterial active substances produced by insects when they are accidentally injured or infected by pathogenic bacteria. It is an immune response product in the non-specific immune defense system in the organism. Advantages in many aspects, such as small molecular weight, good thermal stability, broad-spectrum antibacterial effect, and pathogenic bacteria are not easy to develop resistance. [0003] At present, the drug resistance of pathogenic microorganisms is constantly increasing, and the drug resistance spectrum is also expandin...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C12N15/81C07K14/435A61K38/17A61P31/04A61P31/10A23K1/17A01N47/44A01P1/00A01P3/00A23K20/195
Inventor 张吉斌周定中王茂淋左怀雨喻子牛
Owner HUAZHONG AGRI UNIV
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