Check patentability & draft patents in minutes with Patsnap Eureka AI!

Expressing method of antimicrobial peptide CC31 in bacillus subtilis

A Bacillus subtilis, expression method technology, applied in the direction of microorganism-based methods, biochemical equipment and methods, using vectors to introduce foreign genetic material, etc., can solve the problem that Subtilis has no obvious codon preference, etc., and achieve easy amplification and easy Good operation and stability

Inactive Publication Date: 2016-01-13
HEILONGJIANG BAYI AGRICULTURAL UNIVERSITY
View PDF5 Cites 8 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

(3) Long-term studies have shown that B. Subtilis has no obvious codon preference

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
  • Expressing method of antimicrobial peptide CC31 in bacillus subtilis
  • Expressing method of antimicrobial peptide CC31 in bacillus subtilis
  • Expressing method of antimicrobial peptide CC31 in bacillus subtilis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Construct expression vector

[0042] Synthesis of a Antimicrobial Peptide Gene and Construction of Cloning Vector

[0043] Using SOEingPCR technology to complete the gene synthesis of hybrid antimicrobial peptide CC31 in vitro. The antimicrobial peptide was designed with two pairs of four primers as follows, each pair has complementary sequences, and the antimicrobial peptide gene was synthesized by two-step PCR, and connected with the pMD18-T cloning vector to form the cloning recombinant plasmid pMD18-T / CC31.

[0044] CC31 amino acid sequence: GWLKKIGKKIERVGQHTRIIPPLGYFAKKT

[0045] CC31: 113bp

[0046] GC TCTAGA GGTTGGCTGAAAAAAATCGGCAAGAAGATTGAACGTGTTGGCCAACATACCCGTATTATTCCGCTGCCGCTGGGTTATTTTGCGAAAAAAACC CCCGGG GGA

[0047] F1: 5′-GC TCTAGA GGTTGGCTGAAAAAAATCGGCAAGAAGATTGA-3′

[0048] R1: 5′-ACGGGTATGTTGGCCAACACGTTCAATCTTOTTGCCGATTTTTTTC-3′

[0049] F2: 5′-ACGTGTTGGCCAACATACCCGTATTATTCCGCTGCCGCTGGGTTATTTTGCGAA-3′

[0050] R2: 5′-TCC CCCGGG TTAGGTTTTTTTC...

Embodiment 2

[0075] The purified product CC31 obtained in Example 1 was tested for activity.

[0076] The minimum bactericidal concentration was determined by the 96-well plate method. Pick a single colony of Escherichia coli, Streptococcus dysgalactiae, Staphylococcus aureus, Salmonella, Lactobacillus brevis, Lactobacillus crispatus, Lactobacillus delbrueckii, Bifidobacterium, Pichia SMD1168 and Pichia GS115 in a suitable liquid In the culture medium, four kinds of pathogenic bacteria were cultured overnight at 37°C with 200rpm air shaking, four kinds of lactic acid bacteria were cultured overnight at 37°C with 200rpm shaking, and two kinds of Pichia pastoris were cultured at 30°C and 200rpm air shaking for 48 hours. The antimicrobial peptides were serially diluted with 1×PBS: 2000, 1500, 750, 500, 375, 250, 187.5, 125, 93.75, 62.5, 46.875, 31.25, 23.475 and 15.625 μM, a total of 16 gradients. Dilute the cultured bacterial solution to 2×10 5 ~7×10 5CFU / mL, drop the diluted test bacteri...

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 discloses an expressing method of antimicrobial peptide CC31 in bacillus subtilis. Through the molecular biological technique, an expression vector pHT43 is selected to establish a recombinant expression vector, the recombinant expression vector pHT43 / CC31 is successfully established and is converted into bacillus subtilis WB800N, and accordingly expression engineering bacteria pHT43 / CC31 / WB800N are obtained. In LB culture media, by means of IPTG inducible expression, an expression product is purified through the high performance liquid chromatography (HPLC) so that CC31 protein can be obtained, and the correctness is detected through the mass-spectrography (MS). According to the method, the antimicrobial peptide CC31 is expressed in the bacillus subtilis WB800N, separation and purification are easy, and operating is also easy. The expression product has antimicrobial activity to both gram positive bacteria and gram negative bacteria and does not have bacteriostatic activity to beneficial bacteria, the heat stability is good, and the method is suitable for large-scale industrial production and can be applied to the fields of medicine, food, feed additives and cultivation.

Description

technical field [0001] The invention relates to the field of biology, in particular to a method for expressing antibacterial peptide CC31 in Bacillus subtilis. Background technique [0002] Antimicrobial peptides are part of the innate immune mechanism of animals, plants and humans, and are induced when the body is attacked by viruses, bacteria, etc. Antimicrobial peptides have a broad-spectrum antibacterial effect, especially for killing some pathogenic bacteria that produce antibiotic resistance, which has aroused people's interest in it. Antimicrobial peptides have great application potential in agriculture, medical treatment, food and other fields. So far, thousands of natural antimicrobial peptides have been discovered, but there are relatively few species with good medical selection index. Obtain ideal new artificial antimicrobial peptides from natural antimicrobial peptide resources. [0003] The design feature of the hybrid antimicrobial peptide is to combine the ...

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): C12N15/75C12R1/125
Inventor 姜宁张爱忠张晨雪朱双祁丽全佳慧黄福佳张伟庆
Owner HEILONGJIANG BAYI AGRICULTURAL UNIVERSITY
Features
  • R&D
  • Intellectual Property
  • Life Sciences
  • Materials
  • Tech Scout
Why Patsnap Eureka
  • Unparalleled Data Quality
  • Higher Quality Content
  • 60% Fewer Hallucinations
Social media
Patsnap Eureka Blog
Learn More

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

© 2025 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com