Panton-Valentine杀白细胞素原核表达、纯化及生物学活性鉴定  

Prokaryotic expression and biological activity identification of the Panton-Valentine leukocidin of Staphylococcus aureus

作　　者：常文娇[1,2] 马筱玲[1] 沈继龙[2] 高朋[1] 戴媛媛[1] 张翠萍[1] 周馨[1] 周文静[1] 

机构地区：[1]安徽医科大学附属省立医院检验科,合肥230001 [2]教育部重要遗传病基因资源利用重点实验室,合肥230032

出　　处：《安徽医科大学学报》2011年第1期6-10,共5页Acta Universitatis Medicinalis Anhui

基　　金：安徽省2007年度科技攻关计划(编号:07010302192)

摘　　要：目的原核表达金黄色葡萄球菌Panton-Valentine杀白细胞素(PVL)蛋白,并进行生物学活性鉴定。方法提取PVL阳性金黄色葡萄球菌DNA,利用PCR扩增LukF-PV和LukS-PV编码基因,亚克隆入表达载体pET28 a,转化入大肠埃希菌(E.coli)BL21株,经异丙基-β-D硫代半乳糖苷(IPTG)诱导表达,纯化后获得可溶性的蛋白,Western blot对其鉴定。并以人多形核粒细胞(PMNs)为靶细胞检测其生物学活性。结果成功构建了LukF-PV和LukS-PV基因的表达载体,经IPTG诱导和亲和层析纯化法获得重组蛋白,Western blot鉴定证实其为重组PVL蛋白。实验表明该蛋白具有诱导人多形核粒细胞(PMNs)凋亡的作用。结论成功地表达获得了具有生物学活性的PVL重组蛋白,为PVL快速检测方法的建立和致病机制研究奠定基础。Objective To clone and express Panton-Valentine leukocidin gene from Staphylococcus aureus strain and evaluate its biological activity in vitro. Methods LukF-PV and LukS-PV encoding gene were obtained by PCR amplification from genomic of Staphylococcus aureus. The PCR products were ligated to pMD18-T. The reeombinants were confirmed by BamH I and Xho I digestion, PCR, and DNA sequencing, and then were cloned into expression vector pET28a. The reeombinants were transformed into E. coli BL 21 （ DE3 ） which followed by induction of IPTG. Fusion expression was purified through affinity chromatography with NiSO4 and identified the fusion expression with SDS-PAGE and Western blot. The cytolytic activity was tested by incubation of rPVL with human polymorphonuelear neutrophils （PMNs） in vitro. Results LukF-PV and LukS-PV encoded gene, which was high homologous to the sequence previously reported, was obtained. The expression was confirmed by SDS-PAGE and Western blot. And the biological activity identification showed that the expressed proteins obviously promoted the apoptosis rate of the PMNs in vitro. Conclusion The genes of LukS-PV and LukF-PV are successfully cloned into plasmid pET28a and expressed respectively, which provide a basis for analyzing the toxicity related to the diseases and further studies about the pathogenesis of PVL.

关 键 词：杀白细胞素类/遗传学 基因表达 细胞凋亡 葡萄球菌 金黄色 

分 类 号：R341[医药卫生—基础医学] R378.11