抗菌肽SMAP-29基因的设计、合成、表达纯化及抗菌活性的检测-病原生物学专业毕业论文.docxVIP

- - PAGE 10 - Design, Synthesis and Expression of SMAP-29 Gene and Assay the Antibacterial Activity of SMAP-29 ABSTRACT Objectives: The novel gene of the SMAP-29 was designed and synthesized, the recombinant prokaryotic expression plasmid pGEX-4T-1/SMAP-29 was constructed. After the SMAP-29 gene was overexpressed in E. coli and expression productions were purified, antibacterial activity peptides SMAP-29 were obtained. Methods： The design of SMAP-29 gene based on the SMAP-29 protein sequence and the bias code of amino acid of E. coli and mRNA secondary structure free energy. The new gene was synthesized via gene splicing by overlap extension and cloned in the prokaryotic expression vector pGEX-4T-1. The reconstructed vector was identified by PCR, restriction enzyme analysis and sequencing. GST-SMAP-29 fusion protein was expressed in E.coli BL21 by IPTG induction. Using affinity chromatography, thrombin splicing and HPLC purification, the high purified SMAP-29 was obtained. The antibacterial activity of the SMAP-29 against Escherichia coli JM109, Enterococcus faecalis ATCC 29212 ， Enterococcus faecalis （ Resistant fluoroquinolones ） and Staphylococcus albus (Clinic strain) were assayed by Microbroth dilution. Results： The novel gene SMAP-29 was synthesized and the recombinant prokaryotic expression vector pGEX-4T-1/SMAP-29 was constructed. The new fusion protein band(GST-SMAP-29) was identifyed by SDS, its molecular weight is about 29 kDa, after splicing by thrombin，the antibacterial peptides SMAP-29 are about 3.2KDa. Using Microbroth dilution assay, the results indicated that the SMAP-29 has the comparatively strong bactericidal bioactivity. Conclusions： The expressing SMAP-29 in E.coli has antibacterial activity against Escherichia coli JM109, Enterococcus faecalis ATCC 2921，Enterococcus faecalis（Resistance fluoroquinolones）and Staphylococcus albus (Clinic strain). Postgraduate: Han Fulang (Major in Pathogenic Biology ) Directed by: Prof