Fusion Expression, Purification And Antimierobial Activity Analysis Of Cecropin P1 In Escherichia Coli

This study is a basic work for the molecular improvement and product development of antimicrobial peptides("863 "Program).Such problems about drug residue and antibiotic-resistant strains were increasingly serious with the wide and misused application of traditional antibiotics. The great concern to develop effective but human, animals and environmental compatible antibiotic alternatives is one of the focus researches. Antimicrobial peptide, as one of the most important constituent components of host defense system, possesses antimicrobial activities against a wide variety of microorganisms including G+, G-, fungi, protozoon, enveloped virus and cancer cell etc, and exhibits unique mechanism against bacteria. These properties make it as great potential and can be applied in many fields as one of alternative antibiotics. However, the acquisitionpath is very limited, which restricts its development.Cecropin P1 is a novel cationic protein originally purified from porcine small intestine. It has broad antimicrobial activity against both gram-negative and most gram-positive bacteria. The functional cecropin P1 was successfully expressed in E.coli by gene engineering method in this experiment which has insignificance in the development of basis of theory and practice to expressing cecropin P1 and other small peptide, and also in the exploration of universal method and technology of gene engineering of peptides and antimicrobial peptides research.In this study, according to the biased coden used in E.coli, the gene sequence encoding cecropin P1 was designed. The gene fragment of cecropin P1 was first obtained through gene SOEing; The DNA sequence was inserted into pET-32a(+) and pET-41b(+)vector, constructed the fusion expression vector of cecropin P1;The first constructed vector was better to express soluble fusion protein, then it was transformed into E.coli BL21(DE3) as expression host. After IPTG induction, the result of SDS-PAGE showed that cecropin P1 fusion protein was successfully expressed; The optimized induction condition was established that IPTG were added to the final concentration of 0.03 mM when the bacterial cells density reached OD=0.6, then continuously culture the cells for 1h at 37℃. The expression level of soluble cecropin P1 fusion protein exceeded 25% of the total soluble proteins under the induction of optimized condition; Cecropin P1 fusion protein was higher than 85% purity by Ni2+chromatography purification as judged SDS-PAGE, the production is 200mg fusion protein was obtained per liter culture of induced bacteria; After desalination and concentration, The cecropin P1 fusion protein was cleaved by recombinant enterokinase, then the activedetection showed that the recombinant cecropin P1(0.17mg/mL) has inhibition activity to the growth of E.coli ATCC25922, which suggested the functional cecropin P1 has been achieved by gene engineering method; In order to increase the yield, tandem repeats have been constructed, although they were not successfully expressed, this work laid the foundation for the design and filtration for the future researches.