食品工业科技2013，Vol.34Issue(2)：198-201,4.
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
摘要
Abstract
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-ma/A 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 5×105 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Key words
Enterobacter sakazakii/α-glucosidase/cloning /prokaryotic expression/polyclonal antibody分类
生物科学引用本文复制引用
徐晓可,吴清平,张淑红,张菊梅,李程思,郭伟鹏..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[J].食品工业科技,2013,34(2):198-201,4.基金项目
广东省中国科学院全面战略合作项目(2010B090301037) （2010B090301037）
广东省科技计划项目(No.2010B020316003) （No.2010B020316003）
