Andrias davidianus cathelicidin-ME2, and coded sequence and purpose thereof

A technology of antimicrobial peptides and giant salamanders, applied in the field of genetic engineering, can solve the problems of high cost of antimicrobial peptides, complex methods of antimicrobial peptides, and low yield, and achieve the effect of inhibiting bacterial growth

Inactive Publication Date: 2018-02-06
广州郡雅科技应用有限公司
View PDF0 Cites 2 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The method of directly extracting antimicrobial peptides from Chinese giant salamanders is complicated and the yield is very small;

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Andrias davidianus cathelicidin-ME2, and coded sequence and purpose thereof
  • Andrias davidianus cathelicidin-ME2, and coded sequence and purpose thereof
  • Andrias davidianus cathelicidin-ME2, and coded sequence and purpose thereof

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0040] Example 1: Obtaining cDNA of Cathelicidin-ME2 antimicrobial peptide from Chinese giant salamander

[0041] The inventor used TaKaRa Smart TM The cDNA library construction kit was used to construct a cDNA library of Chinese giant salamander skin tissues. Through random selection of clones and sequencing, the measured sequence was compared with the sequence in the NCBI gene bank (GenBank), and the Cathelicidin-ME2 antimicrobial peptide was obtained. cDNA.

[0042] 1. Extraction of total RNA from mucosal epithelium of Chinese giant salamander

[0043] Take the Chinese giant salamander mucosal epithelial tissue in a cryopreservation tube, then release the Chinese giant salamander, put the cryopreservation tube in liquid nitrogen and transport it back to the laboratory, and store it in a liquid nitrogen tank for later use.

[0044] Take 0.1 g of mucosal epithelial tissue and low-temperature ground into powder with liquid nitrogen. Add 2 mL RNAiso Reagen and let it melt at room tem...

Example Embodiment

[0083] Example 2: Recombinant expression of Chinese giant salamander Cathelicidin antibacterial peptide in Bacillus subtilis

[0084] 1. According to the SQ ID NO: 2 sequence, the primers are calculated as follows:

[0085] P1: 5’-GCAATGACAGCCGTGGGTACA -3’;

[0086] P2: 5’-GC AAGCTT AGGGCGAACTCCATCTTGGT-3'(Introduction of Hind Ⅲ restriction site).

[0087] Amplify Cathelicidin-ME2 gene with pET30a-cathAM plasmid as template, the PCR system is as follows:

[0088]

[0089] Reaction procedure: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 s, annealing at 57°C for 30 s, extension at 72°C for 1 min, 35 cycles; extension at 72°C for 10 min. 1% agarose gel electrophoresis to detect PCR products.

[0090] 2. Using Bacillus subtilis genomic DNA as template, set primers to amplify P according to the known sequence in Genbank 43 Promoter sequence, the EcoRI restriction site is introduced into primer P3, and the designed primers are as follows:

[0091] P3: 5’-CG GAATTC CTTGTA...

Example Embodiment

[0128] Example 3: Separation and purification of recombinant Chinese giant salamander Cathelicidin-ME2 antibacterial peptide

[0129] The obtained fermentation supernatant containing the recombinant Chinese giant salamander Cathelicidin-ME2 antimicrobial peptide was replaced with equilibration buffer (25mM PBS, pH6.0) on a Sephadex G-25 column, and then applied to a CM sepharose FF cation exchange column. Collect the flow peaks, wash the column with the aforementioned equilibration buffer to plateau. Then, it was eluted with a linear gradient of the mixed solution of solution A (25 mM PBS, pH 8.0) and solution B (25 mM PBS containing 1 M NaCl, pH 8.0), collected each elution peak, and used Tricine / Tris SDS- PAGE electrophoresis (silver stain) analysis (the optimized elution scheme is elution with 100, 200, 500 mM NaCl in PBS). The target peptide was eluted in 25 mM PBS containing 200 mM NaCl, pH 8.0. Finally, the recombinant Chinese giant salamander Cathelicidin-ME2 antibacteri...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention provides a Chinese giant salamander Cathelicidin-ME2 antimicrobial peptide, a gene encoding the antimicrobial peptide and uses of the antimicrobial peptide. The antimicrobial peptide contains the amino acid sequence shown in SEQ ID NO:1. The gene encoding the antimicrobial peptide contains the base sequence encoding the amino acid sequence shown in SEQ ID NO:1. The antibacterial peptide has obvious inhibitory effect on a variety of bacteria, and the minimum inhibitory concentration (MIC) of Escherichia coli (CMCC44102), Escherichia coli (ATCC25922) and Salmonella typhimurium (CMCC50115) reached 62.5 μg/mL. The antimicrobial peptide can be used to prepare, for example, feed additives, preservatives, agricultural biological control agents, drugs for controlling microbial infections, and the like.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a Chinese giant salamander Cathelicidin-ME2 antibacterial peptide, a gene sequence encoding the antibacterial peptide and an application of the antibacterial peptide. Background technique [0002] Due to the indiscriminate use and abuse of antibiotics, many bacteria have produced drug-resistant strains, and the development of new antibiotics is not fast enough, so the contradiction between the development of new antibiotics and the demand for antibacterial is becoming increasingly prominent. In addition, the problem of antibiotic residues in animal products has attracted more and more attention, which directly endangers human health. The unique antibacterial mechanism of antimicrobial peptides provides the possibility to solve this problem, so it is very promising to be developed into a new class of highly effective antibacterial drugs. [0003] Antimicro...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
IPC IPC(8): C07K14/46C12N15/12A23K20/147
CPCC07K14/463A23K20/147
Inventor 不公告发明人
Owner 广州郡雅科技应用有限公司
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap