北京分离株Gassericin T基因的克隆、表达及活性检测

目的克隆格氏菌素T基因,在原核细胞中表达,并检测其抑菌活性。方法通过PCR技术,从3株L.gasseri北京分离株中特异性扩增格氏菌素T的成熟肽DNA编码片段(V11/gatA和V441/gatA),经TA克隆和序列测定后,进行同源比较分析。双酶切后的目的片段与表达载体pGEX6P1连接,得到高效融合表达质粒pGEXgatA/V11和pGEXgatA/V441,转化大肠杆菌BL21(DE3)PlysS,IPTG诱导表达,并检测表达产物的抑菌活性。结果目的蛋白以包涵体形式表达,表达量约29%。重组格氏菌素对于金黄色葡萄球菌、铜绿假单胞菌、痢疾志贺氏菌和伤寒沙门氏菌具有明显的抑制生长作用。结论所获得的重组格氏乳杆菌素具有明显的抑菌活性,有望成为新型抗菌素。

Cloning and Expression of Gassericin T Gene of L. gasseri Strain Isolated in Beijing and Microbiostatic Activity of Expressed Product

Objective To clone gassericin T gene,express in prokaryotic cells and identify the microbiostatic activity of expressed product.Methods Amplify the DNA fragments encoding the mature peptide of gassericin T from three L.gasseri strains isolated in Beijing by PCR.Analyze the homology of the amplified gene fragments V1-1/gatA and V44-1/gatA to the gassericin T gene of L.gasseri strain isolated in Japan.Insert the two amplified gene fragments into expression vector pGEX-6P-1 to construct recombinant plasmids pGEX-gatA/V1-1 and pGEX-gatA/V44-1 respectively.Transform the constructed recombinant plasmids to E.coli BL21(DE3) PlysS for expression under induction of IPTG.Results The homologies of V1-1/gatA and V44-1/gatA to the gassericin T gene of L.gasseri strain isolated in Japan were 98% and 99% respectively.The expressed product,existing in a form of inclusion body,contained about 29% of total somatic protein.Recombinant gassericin T showed significant inhibiting activity to the growth of Staphylococcus aureaus,Pseudomonas aeruginosa,Shigella dysenteriae and Tyuphoidal salmonellosis.Conclusion The obtained recombinant gassericin T might be a novel antibiotic.