Cotesia vestalis Teratocyte Defensin CvT-Def gene and encoded antibacterial polypeptide

A technology of Plutella xylostella and antibacterial polypeptide, which is applied in the field of molecular biology

Inactive Publication Date: 2015-12-09
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are few literature reports on the antimicrobial peptides of the teratocytes of Plutella xylostella

Method used

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  • Cotesia vestalis Teratocyte Defensin CvT-Def gene and encoded antibacterial polypeptide
  • Cotesia vestalis Teratocyte Defensin CvT-Def gene and encoded antibacterial polypeptide
  • Cotesia vestalis Teratocyte Defensin CvT-Def gene and encoded antibacterial polypeptide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment example 1

[0031] Implementation case 1: Screening of transcriptome data of teratogenic cells of Braconis plutella xylostella

[0032] 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 partial 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. The whole data filtering strategy is as follows figure 1 shown.

Embodiment example 2

[0033] Implementation Case 2: cDNA Cloning of Antibacterial Polypeptide Defensin (CvT-Def) in Teratocytes of Plutella xylostella

[0034] 1.1 Collection of abnormal cells

[0035]In a small test tube with a length of 5 cm and a diameter of 1.5 cm, one female of Plutella xylostella xylostella that had mated for 2 days after emergence was introduced, and then a 3rd instar mid-stage Plutella xylostella larva was picked with a brush and put into the tube, and the production was observed. After the egg behavior occurred twice, the larvae were removed with a brush, and the operation was repeated with another unparasitic larvae to control about 30 female bees per parasite. The parasitic Plutella xylostella larvae were fed for 3, 4, 5, 6, and 7 days under the conditions of 25±1° C. and 75% relative humidity, respectively, and then dissected. During dissection, the diamondback moth was removed from the cabbage leaf, and the surface of the insect body was sterilized with 75% ethanol, a...

Embodiment 3

[0058] Example 3: Synthesis of the defensin CvT-Def polypeptide and assay of its antibacterial function

[0059] 3.1 Synthesis and purification of the defensin CvT-Def polypeptide in the teratogenic cells of Braconis xylostella xylostella (completed by Shanghai Sangon Bioengineering Co., Ltd.)

[0060] a. Instrument

[0061] Singapore Glass Instrument Factory BZ24 / 29D=2.5CM Beijing Innovation Tongheng LC3000 preparative liquid chromatograph; Shimadzu's LC10Avp high performance liquid chromatograph Waters' ZQ-2000 mass spectrometer

[0062] b. Peptide synthesis process

[0063] The peptide chain was synthesized by the solid-phase method of standard Fmoc chemistry. The specific synthesis consists of the following cycles:

[0064] 1. Deprotection: Fmoc-protected columns and monomers must use a basic solvent (piperidine) to remove the protective group of the amino group.

[0065] 2. Activation and cross-linking: The carboxyl group of the next amino acid is activated by an acti...

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Abstract

The invention relates to the field of molecular biology, and aims at providing a Cotesia vestalis Teratocyte Defensin CvT-Def gene and an encoded antibacterial polypeptide. A nucleotide sequence of the gene is as shown in SEQ ID NO. 1, and an amino acid sequence of the antibacterial polypeptide is as shown in SEQ ID NO. 2. The Cotesia vestalis Teratocyte Defensin CvT-Def gene and an amino acid sequence encoded thereby are of a novel hymenopteran-derived antibacterial protein, can be developed into food additives and medicaments having application value and are applied to multiple fields of agriculture, food sanitation, industry and the like.

Description

technical field [0001] The present invention belongs to the field of molecular biology. The invention relates to a CvT-Def gene of the teratogenic cell defensin of Braconis xylostella xylostella and its coded antibacterial polypeptide, in particular to the application of the gene in preparing artificially synthesized polypeptides with antibacterial activity. Background technique [0002] In recent years, due to the widespread use of antibiotics, many pathogenic bacteria have developed resistance to them, and the development of new green antibiotics has become increasingly important. Antimicrobial peptides have the characteristics of strong antibacterial ability, wide antibacterial spectrum, and not easy to produce drug resistance. They have broad application prospects in medicine and natural food preservatives, so they have become a research hotspot in the study of antibacterial drugs. The content of natural antimicrobial peptides is very small, and the separation is diffic...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C07K14/435A61K38/00A61P31/04A23L3/3463
Inventor 陈学新谷启娟时敏
Owner ZHEJIANG UNIV
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