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Cotesia plutellae antimicrobial peptide defensin gene, antimicrobial peptide and application

A technology of Braconis xylostella xylostella and antibacterial polypeptides, which is applied in the fields of molecular biology and genetic engineering, and can solve the problems of less research on the innate immune system

Active Publication Date: 2012-12-12
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are few studies on the innate immune system of Plutella xylostella at present, and there is no literature report on the isolation of antimicrobial peptides from Plutella xylostella

Method used

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  • Cotesia plutellae antimicrobial peptide defensin gene, antimicrobial peptide and application
  • Cotesia plutellae antimicrobial peptide defensin gene, antimicrobial peptide and application
  • Cotesia plutellae antimicrobial peptide defensin gene, antimicrobial peptide and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment example 1

[0034] Implementation Case 1: Screening of Transcriptome Data of Braconis xylostella xylostella

[0035]Firstly, from each website (APD: http: / / www.bicnirrh.res.in / antimicrobial / ; CAMP: http: / / aps.unmc.edu / AP / main.php; NCBI: http: / / www.ncbi. nlm.nih.gov) collect as many known antimicrobial peptide sequences as possible to establish an antimicrobial peptide database, and then perform local BlastX with the transcriptome sequencing data of Plutella xylostella, to obtain some antibacterial peptide sequences similar or homologous to known antimicrobial peptide sequences peptide sequence. Then use RACE technology to obtain the full length of the fragment, after sequencing verification, submit it to NCBI for sequence alignment, and according to the arrangement characteristics of the six cysteines in the sequence, it is speculated that the sequence belongs to the insect defensin family.

[0036] The whole data filtering strategy is as follows figure 1 shown.

Embodiment example 2

[0037] Implementation Case 2: cDNA Cloning of Antibacterial Polypeptide Defensin (Cv-def) of Braconis xylostella xylostella

[0038] 1.1, TRIzol method to extract total RNA

[0039] (1) Freeze a certain amount (about 5 Plutella xylostella) for 1 minute, put it into a 1.5ml centrifuge tube, add 1ml TRIzol reagent, thoroughly grind and homogenate, and let stand at room temperature for 5 minutes.

[0040] (2) Add 0.2ml chloroform to the centrifuge tube, shake for 15s, transfer the mixture into a TIANGEN centrifuge tube, and let stand for 2min.

[0041] (3) Centrifuge at 12000g for 15min at 4°C, take the supernatant, and transfer it to a new 1.5ml centrifuge tube.

[0042] (4) Add 0.5ml of isopropanol to the centrifuge tube, mix the liquid in the tube gently, and let stand at room temperature for 10 minutes.

[0043] (5) Centrifuge at 12000g for 10min at 4°C and discard the supernatant.

[0044] (6) Add 1ml of 75% ethanol to the centrifuge tube, gently wash the precipitate, cen...

Embodiment 3

[0060] Embodiment 3: Antibacterial polypeptide defensin (Cv-def) polypeptide synthesis, antibacterial function determination of Braconis plutella xylostella

[0061] 3.1 Synthesis and purification of the antibacterial polypeptide defensin Cv-def polypeptide of Braconis plutella xylostella (completed by Shanghai Sangon Bioengineering Co., Ltd.)

[0062] a. Instrument

[0063] Singapore Glass Instrument Factory BZ24 / 29 D=2.5CM Beijing Innovation Tongheng LC 3000 preparative liquid chromatograph; Shimadzu LC 10Avp high performance liquid chromatograph Waters ZQ-2000 mass spectrometer

[0064] b. Peptide synthesis process

[0065] The peptide chain was synthesized by the solid-phase method of standard Fmoc chemistry. The specific synthesis consists of the following cycles:

[0066] 1. Deprotection: Fmoc-protected columns and monomers must use a basic solvent (piperidine) to remove the amino protection group.

[0067] 2. Activation and crosslinking: The carboxyl group of the ne...

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Abstract

The invention relates to the technical field of genetic engineering and aims to provide cotesia plutellae antimicrobial peptide defensin gene, antimicrobial peptide and application. The nucleotide sequence of the cotesia plutellae antimicrobial peptide defensin gene is shown as the SEQ ID NO.1. The amino acid sequence of the antimicrobial peptide of the cotesia plutellae antimicrobial peptide defensin gene coding is shown as the SEQ ID NO.2. Another antimicrobial peptide shown as the SEQ ID NO.3 is obtained by removing signal peptide precursor of the antimicrobial peptide. The invention further provides an antimicrobial peptide synthesis method and application in food additives or drugs for curing gram-positive bacterium or gram-negative bacterium infection. The cotesia plutellae antimicrobial peptide defensin (Cv-def) gene and the nucleotide sequence of the cotesia plutellae antimicrobial peptide defensin (Cv-def) gene coding can be applied in cotesia plutellae to express new antifungal protein and coding sequence, can be developed into food additives and drugs having application value and can be applied in various fields of agriculture, industry and food sanitation and the like.

Description

technical field [0001] The invention belongs to the technical fields of molecular biology and genetic engineering. The present invention relates to an antimicrobial peptide gene of Plutella xylostella and its encoded antibacterial peptide and its application, in particular to an antibacterial peptide gene of Plutella xylostella antimicrobial peptide defensin (Cv-def) and its encoding The antibacterial polypeptide and the application of the gene in the preparation of artificially synthesized polypeptides with antibacterial activity. Background technique [0002] Due to the abuse of traditional antibiotics and the continuous emergence of drug-resistant strains, the clinical treatment of infectious diseases has been seriously affected. Antimicrobial peptides have the advantages of strong sterilization and not easy to cause drug resistance, and have become a new research hotspot of antibacterial drugs. At present, the production of antimicrobial peptides is low, and the separa...

Claims

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Application Information

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IPC IPC(8): C12N15/12C07K14/435A61K38/17A61P31/04A23L1/305
Inventor 陈学新王知知时敏赵伟
Owner ZHEJIANG UNIV