产单核细胞李斯特菌plcB基因的克隆与原核表达  

Cloning and prokaryotic expression of the plcB gene from Listeria monocytogenes

作　　者：田德斌[1] 袁舟[1] 殷月兰[1] 黄金林[1] 董慧[1] 焦新安[1] 

机构地区：[1]扬州大学江苏省人兽共患病学重点实验室,扬州225009

出　　处：《中国人兽共患病学报》2007年第7期652-655,共4页Chinese Journal of Zoonoses

基　　金：国家自然科学基金(30425031);FANEDD专项资金(200358)资助

摘　　要：目的克隆并原核表达产单核细胞李斯特菌plcB基因。方法利用PCR技术从产单核细胞李斯特菌基因组中扩增不含编码信号肽的plcB基因。采用T-A克隆构建pGEM-T-plcB重组质粒,BamHI、XhoI双酶切pGEM-T-plcB获得plcB片段后再插入pGEX-6p-1原核表达载体,获得pGEX-6p-1-plcB重组表达质粒并在表达宿主菌BL21中诱导表达。对表达产物进行纯化和SDS-PAGE、Western-blotting、磷脂酶活性实验分析。结果克隆的plcB基因长834bp,与GenBank上公布的序列完全相同;表达的与GST标签蛋白融合的广谱磷脂酶C蛋白(GST-PC-PLC)具有磷脂酶生物活性和免疫原性。结论成功构建产单核细胞李斯特菌plcB基因的原核表达质粒,并在大肠杆菌中得到有效表达,这为进一步研究PC-PLC蛋白的致病与免疫机理奠定了基础。The plcB gene without signal peptide sequence was amplified by PCR from Listeria monocytogenes and the recombinant plasmid pGEM-T-plcB was constructed by using T-A cloning. After double digestion with BamHⅠ and XhoⅠ, the plcB gene was then subcloned into prokaryotic expression vector pGEX-6p-1 to construct expression plasmid pGEX-6p-1-plcB and this plasmid was expressed in expression host E. coli BL21 cells with induction. The expressed products were purified and analysed by SDS-PAGE, Western blotting and the phospholipase activity experiment. It was demonstrated that the cloned plcB gene appeared to be 834 bp in length, entirely identical with that published in GenBank The fusion protein GST-tagged broadrange phospholipase C （GST-PC-PLC） showed bioactivity and immunogenicity in lecithinase test and Western blotting assay respectively. It is concluded that the prokaryotic expression plasmid for L. rnonocytogenes plcB gene was successfully constructed and effectively expressed in E. coil These results would provide basis for the further studies on the pathogenic and immune mechanisms of the PC-PLC protein.

关 键 词：产单核细胞李斯特菌 plcB基因 广谱磷脂酶C 原核表达 

分 类 号：R378.99[医药卫生—病原生物学]