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A secreted Grignard peptide and its preparation method

A secretory and Grignard technology, applied in the biological field, can solve the problems of complex production process, difficult process, and high cultivation cost

Active Publication Date: 2021-09-10
CHONGQING ACAD OF ANIMAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The Grignard cyclic peptide secreted by wild-type Lactobacillus gasseri is affected by the culture method and the composition of the medium, and its secretion is very small, and the culture cost is high
Currently, Grignard cyclic peptide is heterologously expressed in Escherichia coli (Kawai et al.Animal Sci J 2003, 74:45–51), but the expression product is a linearized protein with poor activity and stability, which is easy to It is degraded by the bacteria's own protease and cannot achieve high-efficiency expression (the expression level is only 370-480 μg / L). At the same time, it is expressed in E. coli cells. Post-processing extraction requires cell rupture and removal of E. coli endotoxins. The actual production process is complicated , these factors have affected its industrialization
The expression product is a linearized protein with poor activity, and the expression product is located in the cell, which needs to be extracted by lysing the cell. The process is difficult and difficult to industrialize

Method used

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  • A secreted Grignard peptide and its preparation method
  • A secreted Grignard peptide and its preparation method
  • A secreted Grignard peptide and its preparation method

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Embodiment Construction

[0033] specific implementation plan

[0034] (1) Through the in-fusion cloning technique, first construct the Grignard loop peptide expression cassette ( figure 1 ). Primers are listed below.

[0035] Table 1 Primer sequences and amplified fragments

[0036]

[0037] Underlines indicate homologous sequences

[0038] First, the vector pP43MNK (BioVector NTCC Inc.) was double-digested with HindIII and PstI, and the digestion reaction system was as follows: 10 μL 10×M buffer, 5 μL vector (50 ng / μL), 5 μL HindIII and 5 μL PstI, mixed with 5 μL ddH2O in In a water bath at 37°C for 60 min, the enzyme digested product was used to recover the linearized vector. Using the Lactobacillus gasseri genome as a template, use P1 / P2, P3 / P4, and P5 / P6 as primers to amplify the gaaA gene fragment, gaaBC gene fragment, and gaaDI gene fragment, respectively, because primers P3 and P6 have homologous sequences with the vector , P2 and P5, P4 and P1 also have homologous sequences (such as ...

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Abstract

The invention provides a secreted Grignard loop peptide and a preparation method thereof. Through the in-fusion cloning technology, the related synthetic genes of gassericin A were recombined in a fixed tandem manner gaaABCDI After the expression cassette is connected with the carrier, it is transformed into genetically engineered bacteria such as Bacillus subtilis (preservation number is CGMCC NO.18446), and the obtained secreted Grignard cyclopeptide has good stability and activity, and stronger antibacterial activity. The expression product obtained by the preparation method has an expression amount of 2 g / L in the fermentation broth, has the advantages of high efficiency, high expression, simple medium, and easy fermentation for large-scale production, and provides a high-efficiency method for feed, food and medicine Inexpensive way of producing and preparing.

Description

technical field [0001] The invention relates to a cyclic peptide of lactic acid bacteria and a preparation method thereof, belonging to the field of biotechnology. [0002] technical background [0003] Antimicrobial peptides, also known as host defense peptides, are small molecule polypeptides produced by organisms that are related to host defense and innate immunity, generally consisting of 2-50 amino acids. Antimicrobial peptides have the functions of inhibiting and killing pathogens, promoting immunity, promoting wound healing and mucosal defense. Gassericin A is a cyclic bacteriocin antimicrobial peptide mainly secreted by Lactobacillus gasseri and other Lactobacillus microorganisms. It consists of 58 amino acid residues and has a molecular weight of 5652Da. Through the cyclization of amide bonds, it has strong inhibitory and killing effects on Listeria, Bacillus cereus, Staphylococcus, etc., and is a potential antibiotic substitute (Kawai et al.Biosci Biotechnol Bioche...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/335C12N15/31C12N15/75C12N15/62
CPCC07K14/335C07K2319/00C12N15/75
Inventor 杨飞云周晓容肖融苏国旗
Owner CHONGQING ACAD OF ANIMAL SCI