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Recombinant bacillus subtilis for producing antibacterial peptide Cecropin B, construction method and application of recombinant bacillus subtilis

A technology for the construction of Bacillus subtilis, which is applied in the field of recombinant Bacillus subtilis, can solve the problems of poor stress resistance of probiotics, single probiotic effect, and restrictions on wide application, and achieve intestinal damage repair, short fermentation time, and production strong effect

Active Publication Date: 2019-09-20
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, most probiotics used as feed additives have defects such as poor stress resistance, single probiotic effect, and slow effect, which limits their wide application in animal husbandry production.

Method used

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  • Recombinant bacillus subtilis for producing antibacterial peptide Cecropin B, construction method and application of recombinant bacillus subtilis
  • Recombinant bacillus subtilis for producing antibacterial peptide Cecropin B, construction method and application of recombinant bacillus subtilis
  • Recombinant bacillus subtilis for producing antibacterial peptide Cecropin B, construction method and application of recombinant bacillus subtilis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1 Construction of recombinant Bacillus subtilis producing antimicrobial peptide Cecropin B

[0045] In this example, the starting plasmid PDG1730 and wild-type Bacillus subtilis WB800 were donated by Professor Ming Sun of the School of Life Science and Technology of Huazhong Agricultural University, and Escherichia coli DH5α was purchased from Beijing Quanshijin Biotechnology Co., Ltd.

[0046] S1: Design primers for P43 gene and SacB gene respectively, and amplify to obtain corresponding gene fragments.

[0047] According to the fragment sequence of the P43 gene (promoter) in NCBI (accession number NCBI-Gene ID: EF473728.1) and the sequence of the signal peptide SacB gene fragment (accession number NCBI-Gene ID: MH614588.1), the primers were designed as follows:

[0048] Forward primer P43-F: CGggatccgagctcagctttatgagtgg, see SEQ ID NO: 1;

[0049] Reverse primer P43-R: GCAAACTTTTTGATGTTCATgtgtacattcctctcttacc, see SEQ ID NO: 2;

[0050] Forward primer SacB-F...

Embodiment 2

[0080] Example 2 Using Gene Recombinant Bacillus subtilis to secrete and express cecropin B

[0081] The correct recombinant B. subtilis B.s-CB strain will be verified for fermentation. The specific steps are as follows: pick a single clone and inoculate it in 5 mL of LB liquid medium containing Spc resistance, activate at 37° C. and 200 r / min for 14 hours, and when the OD value reaches 0.6-0.8. Inoculate in 50mL LB medium with an inoculum size of 1% by volume, culture at 37°C and 200r / min for 24h, take the fermentation broth of recombinant Bacillus subtilis and the fermentation broth of control bacteria (wild-type Bacillus subtilis WB800) for Western -blot detection.

[0082] Western blot steps:

[0083] 1. Use the small molecule protein glue kit (Solabu) to prepare separation gel, interlayer gel, and stacking gel according to the instructions;

[0084] 2. Protein loading: Add the prepared recombinant Bacillus subtilis fermentation broth and control bacteria (wild type Bac...

Embodiment 3

[0091] Example 3 Determination of antibacterial activity of recombinant genetically engineered strains

[0092] Bacillus subtilis B.s-CB was inoculated in the fermentation medium (the formula is the same as before) in proportion and fermented for 24 hours, with Escherichia coli O139, Escherichia coli O157, Escherichia coli K88, Escherichia coli K99, Staphylococcus aureus and Salmonella as Indicator bacteria (10 7 CFU / mL), respectively pipette 0.1mL and spread evenly on LB solid plate (90mm×20mm), (6 plates are Escherichia coli O139, Escherichia coli O157, Escherichia coli K88, Escherichia coli K99, Staphylococcus aureus and Salmonella. On the plate, A is the supernatant of the fermentation broth of recombinant bacteria, B is the supernatant of the fermentation broth of wild-type Bacillus subtilis WB800, and C is the commercial standard of cecropin B); place the Oxford cup (diameter Φmm×10mm) carefully Place 3 Oxford cups on each plate coated with indicator bacteria, adjust th...

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Abstract

The invention belongs to the technical field of agricultural microorganism genetic engineering, and relates to recombinant bacillus subtilis for producing antibacterial peptide Cecropin B, a construction method and application of the recombinant bacillus subtilis. A silkworm-derived antimicrobial peptide Cecropin B gene is integrated into a bacillus subtilis WB800 genome by homologous recombination to construct recombinant bacillus subtilis B. s-CB which can stably express Cecropin B. The recombinant bacillus subtilis B. s-CB prepared by the invention is applied to experimental offspring mice, and the results show that the recombinant bacillus subtilis B. s-CB can improve intestinal functions of the offspring mice, improve feed conversion rate, enhance organism immunity, finally improve production performance of the offspring mice, and provide technical support for research and production of feed additives.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering of agricultural microorganisms, and in particular relates to a recombinant Bacillus subtilis producing antibacterial peptide Cecropin B, a construction method and an application thereof. Background technique [0002] The antibacterial peptide Cecropin B (Cecropin B, CB) is the first natural insect antibacterial peptide (Anti-bacterial peptides, ABP) (Dan H et al 1980) obtained from C. The size is around 4kD. CB is easy to form α-helix under hydrophobic conditions, and its charge is between 8.2-9.6. It has many advantages such as strong thermal stability and good water solubility. Studies have shown that among the many known cecropin families, CB has obvious antibacterial activity against bacteria, viruses, fungi, cancer cells, etc. (Suttmann H et al 2008, Tajui Chia et al 2010). In addition, the antimicrobial peptide CB has great application value in animal husbandry. Wang Tiedong an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/75C07K14/435A23K10/18C12R1/125
CPCA23K10/18C07K14/43586C12N15/75
Inventor 王喜亮李毕平周玉岩李越石德时金秀娥金卉
Owner HUAZHONG AGRI UNIV
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