Plectasin expressed through bacillus subtilis and expressing method thereof

A technology of Bacillus subtilis and mycelia, applied in chemical instruments and methods, antibacterial drugs, pharmaceutical formulations, etc., can solve problems such as difficulty, high cost of chemical synthesis, and inability to form large-scale production

Inactive Publication Date: 2017-04-05
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, it is more difficult to directly extract from organisms, because the content of antimicrobial peptides in organisms is small, and the cost of chemical synthesis is too high to form large-scale production

Method used

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  • Plectasin expressed through bacillus subtilis and expressing method thereof
  • Plectasin expressed through bacillus subtilis and expressing method thereof
  • Plectasin expressed through bacillus subtilis and expressing method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1 Gene Synthesis

[0024] Whole-gene synthesis of the plectasin gene with signal peptide SacB and protein tag 6×His-SUMO, and two enzyme cutting sites EcoRI and BamHI were designed at both ends of the gene. The gene sequence is as follows: figure 1 shown. Gene synthesis was completed by Shanghai Sangon Bioengineering Co., Ltd.

Embodiment 2

[0025] Example 2 Construction of pGJ148-Plectasin expression plasmid

[0026] The gene synthesized in Example 1 and the shuttle vector pGJ148 were double-digested with EcoRI and BamHI respectively, and after recovery from the gel, they were treated with T4 ligase overnight at 4°C. The ligation product was transformed into Escherichia coli competent cell DH5α and the plasmid was extracted. The recombinant plasmid structure is as figure 2 shown. The correct recombinant plasmid pGJ148-Plectasin verified by PCR, double enzyme digestion and sequencing was electrotransformed into the host expression bacteria Bacillus subtilis WB800N.

Embodiment 3

[0027] Example 3 Recombinant strain vial-induced expression

[0028] Pick the above-mentioned recombinant strain WB800N-pGJ148-Plectasin in 10mL LB medium, add neomycin and chloramphenicol so that the final concentration is 10μg / mL, culture overnight at 37°C, 220r / m, transfer to 150mL LB culture After culturing for 4 hours, add maltose for induction, so that the final concentration of maltose is 1%, 3%, 5%, 7%, 9%. After 48 hours of culture, the supernatant of the bacterial liquid was collected for Western-blot analysis. Maltose at various concentrations could induce the expression of the fusion protein. As the concentration of maltose increased, the expression of the fusion protein also increased. When , the expression of the fusion protein reached the highest level. But when the concentration of maltose reached 9%, the expression of fusion protein decreased.

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Abstract

The invention provides plectasin expressed through bacillus subtilis and an expressing method thereof. The sequence of the plectasin is shown as a sequence table SEQ ID No.1. The expressing method includes the steps that a plectasin gene with the signal peptide SacB and protein tag 6*His-SUMO is connected onto an expression vector pGJ148 and then the connected product is electrically transferred into competent escherichia coli cells DH5alpha; correctly-authenticated recombinant plasmid is converted into the bacillus subtilis WB800N; and under the action of an inductive agent maltose, fusion protein can be expressed, and plectasin protein can be obtained by means of purification. The protein has an efficient killing effect on gram positive bacteria and does not have hemolysis to red blood cells of human bodies, production cost is reduced, and application of the plectasin as an anti-infection drug is further promoted; and meanwhile a theoretical basis is provided for application of the plectasin.

Description

technical field [0001] The invention specifically relates to a plectasin expressed by Bacillus subtilis and an expression method. Background technique [0002] With the long-term and widespread use of antibiotics, their negative effects have become increasingly prominent, and bacterial resistance has seriously threatened human health. Antimicrobial peptides are considered to be ideal drugs to replace antibiotics because of their high antibacterial activity, spectrum sterilization, and difficulty in producing drug resistance. Defensins are a class of endogenous antimicrobial peptides that widely exist in organisms and are an important part of the body's natural immune system. Plectasin is the first fungal defensin isolated from the saprophytic ascomycete Pseudolectania nigrella by Mygind et al. in 2005. Mature Plectasin consists of 40 amino acid residues, and its higher order structure contains an α-β model consisting of an α-helix and two antiparallel β-sheets, stabilized ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/37C12N15/75C12N15/66A61K38/16A61P31/04
CPCA61K38/00C07K14/37C12N15/66C12N15/75Y02A50/30
Inventor 单安山张丽聪
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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