分枝杆菌噬菌体D29裂解酶gp10的原核表达及抗脓肿分枝杆菌活性  

Prokaryotic Expression and Antibacterial Activity to Mycobacterium abscessus of Mycobacteriophage D29 Lyase gp10

作　　者：熊玉霞[1] 杨致邦[2] 于拽拽[1] 戴维[3] 蒋任举[1] 蒋英[1] 

机构地区：[1]重庆医科大学神经科学研究中心,重庆400016 [2]重庆医科大学基础医学实验教学中心病原生物学与免疫学实验室,重庆400016 [3]重庆医科大学

出　　处：《中国生物制品学杂志》2011年第11期1269-1272,共4页Chinese Journal of Biologicals

摘　　要：目的克隆并原核表达分枝杆菌噬菌体D29裂解酶gp10基因,观察重组蛋白对脓肿分枝杆菌的抑菌作用。方法以噬菌体D29基因组DNA为模板,PCR扩增gp10基因片段,克隆至pET-32a(+)载体上,构建重组原核表达质粒pET-32a-gp10,转化E.Coli BL21(DE3),IPTG诱导表达。表达的重组蛋白经SDS-PAGE分析,亲和层析纯化后,采用杯碟法检测其抑菌活性。结果 PCR扩增获得长1 454 bp的gp10基因片段,重组表达质粒pET-32a-gp10经PCR、双酶切及测序证明构建正确;表达的重组蛋白相对分子质量约为54 800,表达量占菌体总蛋白的97.4%,破菌上清及破菌沉淀中均可见目的蛋白的表达,纯化后纯度可达90%,浓度为0.5 mg/ml;重组蛋白对脓肿分枝杆菌有抑菌作用。结论已成功表达重组分枝杆菌噬菌体D29裂解酶gp10,其对脓肿分枝杆菌具有抑菌作用,为抗脓肿分枝杆菌及其他非结核分枝杆菌感染新药物的研制提供了依据。Objective To clone mycobacteriophage D29 lyase gpl0 gene, express in prokaryotic cells and observed its antibacterial activity to Mycobacterium abscess. Methods The gpl0 gene fragment was amplified by PCR using the genomic DNA of mycobacteriophage D29 as a template and cloned into vector pET-32a （＋）. The constructed recombinant plasmid pET-32a-gp10 was transformed to E. coli BL21 （DE3） for expression under induction of IPTG. The expressed recombinant protein was analyzed by SDS- PAGE, purified by affinity chromatography, and determined for antibacterial activity by cylinder plate method. Results The gpl0 gene fragment at a length of t 454 bp was amplified by PCR. PCR, restr[ctiot analysis and sequencing proved that recombinant piasmid pET-32a-gp10 was constructed correctly. The expressed recombinant protein, with a relative molecular mass of about 54 800, contained about 97.4% of total somatic protein. Target protein was expressed in either ultrasonic supernatant or precipitate of recombinant E. coli, at a concentration of 0. 5 mg/ml and reached a purity of 90% after purification. The recombinant protein showed an- tibacterial activity to M. abscessus. Conclusion Recombinant mycobacteriophage D29 lyase gpl0 was successfully expressed and showed antibacterial activity to M. abscessus, which provided a basis for developing novel drugs for M. abscessus and other nontuberculosis mvcobacteria infections.

关 键 词：分枝杆菌 非典型性 细菌噬菌体 裂解酶 基因表达 抗菌活性 

分 类 号：Q786[生物学—分子生物学] R392-33[医药卫生—免疫学]