Antimicrobial peptide MP1106, preparation method and application thereof

A technology of MP1106 and antimicrobial peptide, which is applied in the field of genetic engineering and molecular biology, can solve the problems of high cytotoxicity, high production cost, and weak bactericidal activity, and achieve the effect of wide application prospects and high-efficiency expression

Inactive Publication Date: 2014-12-31
FEED RESEARCH INSTITUTE CHINESE ACADEMY OF AGRICULTURAL SCIENCES
View PDF3 Cites 17 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Some natural antimicrobial peptides are not easy to achieve large-scale production due to their weak bactericidal activity, low stability, high cytotoxicity, and high production costs, which have restricted the further utilization of antimicrobial peptides (Findlay, Zhanel et al.2010, Nicole K .Brogden and Brogden 2011)

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
  • Antimicrobial peptide MP1106, preparation method and application thereof
  • Antimicrobial peptide MP1106, preparation method and application thereof
  • Antimicrobial peptide MP1106, preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] The acquisition of embodiment 1 antimicrobial peptide MP1106 gene fragment

[0048] 1. Optimization of the gene sequence of the antimicrobial peptide MP1106

[0049] According to the yeast codon table: Pichia pastoris[gbpln]: 137 CDS's (81301 codons), from: www.kazusa.or.jp / codon / .

[0050]

[0051]

[0052] The amino acid sequence of MP1106 according to the design: GFGCNGPWSEKDMHCHNHCKSIKGYKGGYCAKGGFICKCY

[0053] After codon optimization, the gene sequence is as follows:

[0054] GGT TTT GGT TGT AAC GGT CCA TGG TCT GAA AAG GAT ATG CAT TGT CAT AAC CAT TGT AAG TCT ATT AAG GGT TAC AAG GGT GGT TAC TGT GCT AAG GGT GGT TTT ATT TGT AAG TGT TAC

[0055] 2. Gene expression cassette design

[0056] protected base XhoⅠ Kex 2 MP1106 kill password Xba I protected base

[0057] When designing the gene expression cassette, in order to connect the gene into the pPICZαA vector, two restriction enzyme sites Xho Ⅰ and Xba Ⅰ in the multiple cloning sit...

Embodiment 2

[0064] Embodiment 2 Preparation of recombinant yeast containing the gene of MP1106

[0065] 1. Acquisition and subcloning of genes and expression cassettes

[0066] The plasmid mini-extraction kit extracts the pUC57 plasmid containing MP1106 and the expression cassette (operate according to the kit instructions), and then performs double digestion with Xho I and Xba I endonucleases, and the vector pPICZαA is also carried out with the same enzyme digestion system, the system is as follows :

[0067]

[0068] 37°C, water bath for 4h.

[0069] Then the two obtained double-digested fragments were ligated with T4 DNA ligase, and the ligation system was as follows:

[0070]

[0071] 2. The recombinant plasmid pPICMP1106 was linearized with enzyme Pme Ⅰ and then used for yeast transformation. The linearization system is as follows:

[0072]

[0073]

[0074] Reaction conditions: 37°C, 4h.

[0075] 3. Preparation of competent Pichia pastoris X-33

[0076] Pick a sing...

Embodiment 3

[0086] Example 3 Shake flask horizontal screening of positive transformants and high-density fermentation

[0087] 1. Screening of positive transformants

[0088] Pick the identified positive transformant in 10ml YPD liquid medium, culture at 30°C, 250rpm for 16-18h, inoculate the overnight bacterial solution in 10ml BMGY medium with 1% inoculum, cultivate to OD at 30°C, 250rpm 600nm About 5.0, collect the bacterial liquid, 4°C, 4000rpm, centrifuge for 5 minutes, remove the supernatant, collect the bacterial cells, resuspend the cells with 50ml BMMY medium until the OD600nm is about 1.0, transfer the above bacterial liquid to a 250ml shake flask, bottle Cover the mouth with 4 layers of sterilized gauze to start the induction, which is counted as the initial induction 0h, and then add anhydrous methanol to the final concentration of 0.5% every 24h, induce 120h, and respectively at 0h, 24h, 48h, 72h, 96h, 120h Take 500 μl, centrifuge at 12,000 rpm for 10 minutes, collect the su...

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 novel antimicrobial peptide MP1106 (Seq ID No.1) designed based on Plectasin, and provides a high-efficiency expression of the novel antimicrobial peptide MP1106 (Seq ID No.1) in pichia pastoris. Through the preference of yeast codon for optimization of antibacterial peptide MP1106 encoding gene and construction of recombinant plasmid, the recombinant plasmid is transformed into the pichia pastoris, secretory expression of high density fermentation protein in a 5 L fermentation tank can be achieved, the total protein level reaches 2.134g / L, and purified recombinant MP1106 yield reaches 831mg / L. In addition, recombinant MP1106 cation-exchange chromatography one-step purification method is established, and novel antimicrobial peptide MP1106 in-vitro properties show that the novel antimicrobial peptide MP1106 has strong inhibitory activity against staphylococcus aureus, is free of cell toxicity and stable in high temperature, alkaline environment, serum incubation conditions, and is resistant to papain, pepsase and the like. The novel antimicrobial peptide MP1106 is superior in properties, can realize large-scale industrial production, and is potential to be developed into a novel anti staphylococcus aureus preparation.

Description

technical field [0001] The invention relates to the fields of genetic engineering and molecular biology, in particular to a novel antimicrobial peptide MP1106, its preparation method and application. Background technique [0002] Staphylococcus aureus is one of the most common pathogens in human and livestock infections. It can infect skin and soft tissues, cause local infections such as pneumonia and infective endocarditis, and cause systemic infections such as bacteremia and sepsis, which can cause serious clinical diseases (Chambers and Deleo 2009, Nickerson, West et al. 2009), and Staphylococcus aureus is prone to drug resistance, and even multi-drug resistant strains mutate, making it more and more difficult to control (Lowy 1998). Methicillin-resistant Staphylococcus aureus (MRSA) emerged two years after methicillin was introduced into clinical use in 1960 (Graveland, Duim et al. 2011). In recent years, MRSA has been found not only in hospitals (hospital-acquired MRS...

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
Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/37C12N15/31C12N15/81C12N1/19C12P21/02A61K38/16A61P31/04
CPCC07K14/37C12N15/81
Inventor 王建华曹鑫涛张勇滕达王秀敏毛若雨
Owner FEED RESEARCH INSTITUTE CHINESE ACADEMY OF AGRICULTURAL SCIENCES
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