阪崎肠杆菌α-葡萄糖苷酶的原核表达及其多克隆抗体制备  

Prokaryotic expression of Enterobacter sakazakii α-Glucosidase and preparation of its polyclonal antibody

作　　者：徐晓可[1] 吴清平[1] 张淑红[1] 张菊梅[1] 李程思[1] 郭伟鹏[1] 

机构地区：[1]广东省菌种保藏与应用重点实验室,广东省微生物应用新技术公共实验室,广东省华南应用微生物重点实验室-省部共建国家重点实验室培育基地,广东省微生物研究所,广东广州510070

出　　处：《食品工业科技》2013年第2期198-201,共4页Science and Technology of Food Industry

基　　金：广东省中国科学院全面战略合作项目(2010B090301037);广东省科技计划项目(No.2010B020316003)

摘　　要：以阪崎肠杆菌(ATCC29544)的基因组DNA为模板通过PCR扩增出α-葡萄糖苷酶基因malA,将其克隆入表达载体PET22b(+),构建重组表达质粒pET22b-malA。将重组表达质粒转化大肠杆菌BL21(DE3),进行优化表达。采用尿素洗涤包涵体并切胶回收纯化融合蛋白,然后再免疫新西兰大白兔制备多克隆抗体。通过ELISA测定效价,并通过免疫荧光法鉴定与菌体表面结合能力。结果表明克隆的目的基因全长1677bp,与Genebank的malA比较具有100%的同源性。带His标签的融合蛋白以包涵体形式表达,其最优化的表达条件是37℃下用0.8mmol/L IPTG诱导5h。ELISA测定效价为5×105;免疫荧光法鉴定表明多克隆抗体能与阪崎肠杆菌表面结合。本实验为阪崎肠杆菌的免疫磁珠检测奠定了基础。The α -glucosidase gene malA was amplified by PCR from Enterobacter sakazakii （ATCC29544） genomic DNA. Then it was cloned into the expression vector pET22b（＋） to acquire the recombinant expression plasmid pET22b-malA. It was transformed into Escherichia coli BL21（DE3） to optimize expression. The fusion protein was purified by urea and separated by SDS -PAGE and recovered by gel extraction. White rabbits originate in New Zealand were immunized with the separated protein. The titer of polyclonal antibody was detected by ELISA and the binding capacity was identified by immunofluorescence assay. The result showed that the target gene was 1677bp and 100% identical to the corresponding gene in Genebank. SDS -PAGE analysis indicated that His -fusion protein was mostly lied in inclusion bodies,and the optimal expression condition was induced for 5h with 0.8mmol/L IPTG at the temprature of 37℃. The titer of polyclonal antibody was about 5 ×10 5 by ELISA. Immunofluorescence assay showed that the polyclonal antibody could bind E. sakazakii. These results would lay a foundation to prepare immunomagnetic beads for detection of E. sakazakii.

关 键 词：阪崎肠杆菌 Α-葡萄糖苷酶 克隆 原核表达 多克隆抗体 

分 类 号：Q78[生物学—分子生物学]