Unlock instant, AI-driven research and patent intelligence for your innovation.

Hybrid antimicrobial peptide as well as preparation method and application thereof

A hybrid antibacterial peptide and gene technology, applied in the field of genetic engineering, can solve the problems of narrow antibacterial spectrum of human c-type lysozyme and achieve good antibacterial effect

Inactive Publication Date: 2017-11-03
SHANGHAI OCEAN UNIV
View PDF3 Cites 9 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, the only research on the tandem expression of small molecule antimicrobial peptides and lysozyme in Pichia pastoris is the expression of channel catfish parasin Ⅰ and human lysozyme (Hua Zhao, Jiayong Tang, Lei Cao, Gang Jia, DingbiaoLong, Guangmang Liu, Xiaoling Chen, Jingyi Cai, Haiying Shang. Characterization of bioactive recombinant antimicrobial peptide parasin I fused with humanlysozyme expressed in the yeast Pichia pastoris system. Enzyme Microb. Tech. 2015, 77:61-67.), but the lysozyme gene used is derived from humans, The human c-type lysozyme has a narrow antibacterial spectrum and is mainly active against Gram-positive bacteria

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
  • Hybrid antimicrobial peptide as well as preparation method and application thereof
  • Hybrid antimicrobial peptide as well as preparation method and application thereof
  • Hybrid antimicrobial peptide as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Construction of Hybrid Antimicrobial Peptide LD Genetic Engineering Bacteria

[0069] 1.1 Obtain tandem gene LD by SOE-PCR

[0070] Using the pPICZαA-defbl3 vector (constructed and saved in the previous stage of this experiment) as a template, according to the principle of nested PCR, LD-p3, LD-p3, LD-p4 and LD-p5 primers (Table 1) were used for PCR amplification to obtain the gene fragment defbl3l containing the optimized zebrafish β-defensin cDNA sequence (SEQ ID NO:2). According to the channel catfish lyc gene sequence (source GenBank: NM_001200789), using the website software ( http: / / www.jcat.de / Start.jsp ) to optimize it. The optimized gene sequence (SEQ ID NO:4) was designed with a 6×His tag sequence and a restriction endonuclease XhoI cutting site at the 5′ end, and 4 glycine flexible linkers and enterokinase were added at the 3′ end The nucleotide sequence of the cutting site was designed to obtain the gene fragment lycl containing the optimized cDNA sequen...

Embodiment 2

[0091] Expression, Purification, Antibacterial Activity and Stability of Recombinant Hybrid Antimicrobial Peptide LD

[0092] 2.1 Methanol-induced expression of recombinant hybrid antimicrobial peptide LD

[0093] Positive yeast transformants were inoculated into 100mL BMG medium (0.1mol / L sorbitol, 0.1mol / L potassium phosphate buffer pH6.0, 1.34%YNB, 4×10 -5 Biotin, 1% glycerol), 8 layers of gauze sealing, 29 ° C, 250r / min shaker shaking culture to OD 600 4.0 or so; transfer the bacteria from the BMG medium to the same volume of BMM medium (0.1mol / L potassium phosphate buffer pH6.0, 1.34%YNB, 4×10 -5 biotin, 0.75% methanol), 29°C, 250r / min to induce the expression of the target protein; add methanol every 24h to 0.75% of the final volume. 0.75% methanol induced expression of X-33 / pPICZαA yeast transformants and X-33 / pPICZαA-LD 4 After 120 hours of yeast transformants, the supernatant after centrifugation was concentrated with a 3kDa ultrafiltration centrifuge tube, and the...

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

PropertyMeasurementUnit
PCR efficiencyaaaaaaaaaa
Login to View More

Abstract

The invention relates to a hybrid antimicrobial peptide as well as a preparation method and application thereof. An amino acid sequence of the hybrid antimicrobial peptide is shown in SEQ ID NO:6, and a nucleotide sequence for encoding a gene of the hybrid antimicrobial peptide is shown in SEQ ID NO:5. Through in-vitro construction of multiple copy expression cassette expression vectors, a recombinant vector containing different copy numbers of expression cassettes for encoding the gene of the hybrid antimicrobial peptide is obtained, and a yeast transformant of the recombinant vector containing different copy numbers of expression cassettes is then obtained; through methanol induction, the gene is efficiently expressed in a pichia pastoris host cell, thereby obtaining the hybrid antimicrobial peptide. The preparation method can efficiently prepare the hybrid antimicrobial peptide, a used culture condition is simple, the yield is high, the cost is low, and the obtained hybrid antimicrobial peptide has a good antimicrobial effect and is suitable to be used as an antimicrobial agent, a feed additive and the like.

Description

technical field [0001] The invention relates to the field of genetic engineering, and more specifically relates to a novel hybrid antibacterial peptide and its preparation method and application. Background technique [0002] At present, a large number of antibiotics and preservatives are used in the field of aquatic product processing and farming. Although it can play a good effect, it will cause bacteria to develop drug resistance and affect the quality of aquatic products. Although natural antibacterial protein is a good substitute for antibiotics and preservatives, its content in organisms is low, and it is difficult to directly isolate, extract and purify from organisms; and its effect against exogenous microorganisms is limited. In particular, there is currently no commercial antibacterial protein from aquatic sources. "GB2760-2014" stipulates that egg white lysozyme cannot be used in aquatic products. There are only 3 types of feed antibiotics that can be used in a...

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): C07K19/00C12N9/36C12N15/62C12N15/81C12N1/19A23K20/195A61K38/47A61K38/17A61P31/04C12R1/84
CPCA61K38/00A23K20/195C07K14/461C07K2319/00C12N9/2462C12N15/62C12N15/815C12Y302/01017
Inventor 陶妍李雯宋长丰冯亚东
Owner SHANGHAI OCEAN UNIV