Expression method of earthworm antibacterial peptide

Abstract

The invention discloses an expression method of an earthworm antibacterial peptide. According to the expression method, an improved maltose manipulator promoter Pglv-M1 is constructed, a bacillus subtilis expression vector consisting of an anamylase signal peptide sequence SP amyQ, an SUMO (small ubiquitin-related modifier) sequence and an antibacterial peptide sequence is constructed, and maltoseis adopted as an inducer to express an earthworm antibacterial peptide; an obtained target protein is cut by using SUMO protease, and affinity chromatography purification is implemented to obtain theearthworm antibacterial peptide LumbricinI (6-34). The purification product has antibacterial functions on all of escherichia coli, staphylococcus aureus, saccharomyces cerevisiae and the like. According to the method, bacillus subtilis is adopted as an expression host, so that endotoxin is avoided, and convenience in purification can be achieved; the promoter Pglv-M1 is used, the maltose is adopted as the inducer, and the inhibition function of the maltose upon the promoter Pglv-M1 can be alleviated; sequences of different sections are optimized, and the expression yield can be increased; and a fusion protein can be secreted out of cells, so that purification steps can be reduced.

Description

technical field [0001] The invention belongs to the technical field of biotechnology and genetic engineering. Specifically, the invention relates to a method for expressing earthworm antimicrobial peptides by using an improved maltose-operated bacillus subtilis expression system. Background technique [0002] With the abuse of antimicrobial drugs, antimicrobial drug residues in the environment and in animals and plants will lead to the emergence of drug-resistant bacteria. Both of these will directly or indirectly affect the life, health and safety of the human body, so the research and development of new antibacterial drugs is imminent. As an important soil animal, earthworms live in a soil environment containing a large number of microorganisms. However, after receiving partial damage, earthworms can heal themselves and rarely suffer from infection. Scientific research shows that earthworms contain a variety of antibacterial peptides, and earthworm antibacterial peptides ...

AI Technical Summary

Problems solved by technology

However, among the inducible promoters of Bacillus subtilis, the promoters with higher expression efficiency are more expensive, toxic or easily inhibited. For example, Li Fuwei et al. expressed ew T1 using the promoter Pgrac carried in the pHT43 plasmid, but the promoter P grac The inducer IPTG is expensive and toxic, and because the sequence of the protein tag is not added before the antimicrobial peptide gene sequence, the directly expressed antimicrobial peptide will also cause harm to the expression host Bacillus subtilis, so the expression yield is not high, and the purification is relatively difficult. Difficult; In addition, the stronger inducible promoter of Bacillus subtilis has P xyl etc. Promoter P xyl The inducer is xylose, which is safer but more expensive and inhibited by glucose

Therefore, the utilization of the Bacillus subtilis expression system is limited

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