关键词: 菌丝霉素, 串联表达, 菌內抑菌活性

Abstract: : The study was conducted to construct the tandom expression strains of the plectasin，and to analyze the bacteriostatic activity of the tandom expression products. According to the codon preference in E. coli expression system, the gene of plectasin was synthesized and inserted into plasmid pGEX4T1 to construct the recombinant plasmid pGEXPT/pe. The coadhesive end restriction and ligation strategy was used to add the repeat unit one by one. The recombinant vectors pGEXPT/pes2 and pGEXPT/pes3 with two and three repeat units of plectasin were constructed respectively. The expression vectors of tandom repeat plectasin gene were respectively transformed into E. coli DH5α and induced with IPTG. The expressed protein was detected by SDSPAGE and Western Blot analyses. Then the bacteriostatic activity was determined. The results showed that the fusion proteins were correctly expressed. DNA sequencing indicated that 135, 261 and 387 bp of gene fragments were obtained after amplifying by PCR. SDSPAGE results showed that the expressed fusion proteins had molecular weight of 30, 35 and 40 kDa bands and accounted for 64.5%, 26.4% and 25.3% of the total bacterium proteins, respectively. The Western Blot analysis demonstrated that all of the target proteins had immunological activity. The results of bacteriostatic activity in vivo indicated that all of the expressed fusion proteins were definite inhibitors of E. coli DH5α. However, bacteriostatic efficacy of the fusion expression products significantly increased and antibacterial time extended with the increasingly number of plectasin gene. The successful construction of the tandom expression strains and the inhibition of the expression products to host bacteria laid a foundation for product engineering of efficient bacteriostatic effect of plectasin.

Key words: plectasin, tandom expression, bacteriostatic activity in vivo