Eureka AIR delivers breakthrough ideas for toughest innovation challenges, trusted by R&D personnel around the world.

Bacteriostatic protein and encoding gene and application thereof and strain method

A technology of encoding genes and proteins, applied in the field of antibacterial proteins and their encoding genes, uses and strains, can solve the problems of poor antibacterial effect and limited application, and achieve the effect of improving antibacterial effect

Active Publication Date: 2020-12-18
深圳市康佑生物技术有限公司
View PDF4 Cites 1 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

With the deepening of research and application, it has been found that chicken lysozyme has poor antibacterial effect on some Gram-positive bacteria (such as Staphylococcus aureus) and many Gram-negative bacteria (such as Escherichia coli), which limits its use in many field application

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
  • Bacteriostatic protein and encoding gene and application thereof and strain method
  • Bacteriostatic protein and encoding gene and application thereof and strain method
  • Bacteriostatic protein and encoding gene and application thereof and strain method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Embodiment 1, lysozyme mutant design

[0034] Studies have shown that superimposing antimicrobial peptides on the basis of lysozyme amino acids can improve the antibacterial effect of lysozyme. In our previous research, we obtained a scorpion-derived antimicrobial peptide IsCT (CN104263749A) with good antibacterial effect through experiments. This patent hopes to pass The amino acid sequence of the antimicrobial peptide IsCT was superimposed on chicken lysozyme protein to improve the antibacterial properties of chicken lysozyme. The design process of the chicken lysozyme mutant LyIc containing the antimicrobial peptide IsCT is as follows: (1) In order to prevent the amino acid sequence of the antimicrobial peptide IsCT from wrapping inside the mutant LyIc protein and affecting its antibacterial activity, the amino acid sequence of the antimicrobial peptide IsCT was The sequence is superimposed on the N-terminus of the chicken lysozyme protein; (2) Due to the strong anti...

Embodiment 2

[0035] Embodiment 2, chicken lysozyme mutant LyIc gene synthesis and expression vector construction

[0036] According to the codon preference of Pichia pastoris, the nucleotide sequence encoding the mutant LyIc was optimized by online codon optimization software to obtain the gene lys-Is, the nucleotide sequence of which is shown in SEQ ID NO.1. The gene lys-Is is 486bp in full length (without signal peptide), encoding 161 amino acids, including antioxidant peptide (DEDTQAMP), antimicrobial peptide IsCT (ILGKILKGIKKLF), amino acid linker (GGGSSSSGGGG) and chicken lysozyme (129 amino acids). The optimized gene lys-Is was obtained by General Biosystems (Anhui) Co., Ltd. through the whole gene synthesis method. A pair of primers were designed according to the sequence of lys-Is (primer sequences were fw: 5'-agtc gaattcGATGAAGACACTCAAGCTATG-3' and rev: 5'-ttctctaga TTACAATCTACAAC CTCTGAT-3') for the amplification of gene lys-Is, agarose electrophoresis Detect the PCR amplificat...

Embodiment 3

[0037] Embodiment 3, construction and screening of recombinant Pichia pastoris

[0038]The expression vector pPICZαA-lys-Is was linearized with SacI and then transformed into Pichia pastoris X33, and the recombinant transformants were spread on Zeocin resistance plates with different concentrations (100 μg / ml-500 μg / ml). Use a toothpick to pick the yeast recombinant transformants grown on the plate one by one into a 24-well plate containing 2mL of BMGY medium in each well, culture at 30°C, 220rpm for about 36h, and then add 1% (v / v) methanol for induction culture . After culturing at 30°C and 220rpm for 48 hours, the supernatant was collected by centrifugation for antibacterial assay. The specific steps of the antibacterial test are as follows: (1) Cultivate Micrococcus luteus (CICC10680) to the logarithmic growth phase (OD600 is about 1), and add the cultured bacterial solution to the sterile medium at 1 μL / mL to prepare flat. (2) Use a hole puncher with a radius of 3 mm t...

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 present invention discloses a bacteriostatic protein and an encoding gene and an application thereof and a strain method. Chicken lysozyme is used as a starting template, an amino acid sequence ofscorpion-derived antimicrobial peptide IsCT is superposed to an N terminal of the chicken lysozyme protein through rational design, and meanwhile, an antioxidant peptide joint and an amino acid jointare directionally introduced to obtain a mutant LyIc; then according to a codon preference of pichia pastoris, a whole-gene synthesis technology is adopted to obtain a gene lysIs corresponding to themutant LyIc and a nucleotide sequence of the lysIs is shown as SEQ ID NO.1; then the lysozyme mutant gene lysIs is linked into an expression vector pPICZ alpha A, the vector is transferred into pichia pastoris X33, the recombinant microorganisms are subjected to fermentation culture and the lysozyme mutant LyIc is separated and purified; and finally, bacteriostatic effects of the lysozyme mutantLyIc and original lysozyme on common pathogenic bacteria are compared and analyzed. The lysozyme mutant LyIc effectively improves the bacteriostatic effect on common pathogenic bacteria (escherichia coli, salmonella and staphylococcus aureus) and lays foundation for wide application of the chicken lysozyme.

Description

technical field [0001] The invention belongs to the technical field of biotechnology, and in particular relates to an antibacterial protein, its encoding gene, application and bacterial strain. Background technique [0002] In recent years, the abuse of antibiotics has caused many problems, such as: the emergence of super-resistant bacteria; antibiotic residues and so on. From the perspective of sustainable development, humans must effectively control the usage and dosage of antibiotics. Since the 1990s, many researchers at home and abroad have begun to look for alternatives to antibiotics. After many studies, it has been found that lysozyme, as a natural protein, can effectively kill many microorganisms, so it is expected to become a substitute for antibiotics in many fields. As a glycoside hydrolase, lysozyme can decompose the cell wall of microorganisms, causing the bacteria to lose the protection of the cell wall and rupture and die under the action of intracellular hig...

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): C12N9/36C12N15/56C12N1/19A61P31/04C12R1/84
CPCC12N9/2462C12Y302/01017A61P31/04Y02A50/30
Inventor 王建荣王鹏王玲玲詹仕贤
Owner 深圳市康佑生物技术有限公司
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
Eureka Blog
Learn More
PatSnap group products

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

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com