文献类型：期刊文献

中文题名：B淋巴细胞刺激因子基因的克隆及在大肠杆菌中的高效表达

英文题名：Cloning and High-Level Expression of B Lymphocyte Stimulator (BLys) in E.coli

作者：王锦之[1];周卿[1];周宇荀[1];马昱澍[1];魏东芝[1]

机构：[1]华东理工大学生物反应器工程国家重点实验室,上海200237

年份：2005

卷号：31

期号：2

起止页码：249

中文期刊名：华东理工大学学报（自然科学版）

外文期刊名：Journal of East China University of Science and Technology

收录：CSTPCD;;EI(收录号：2005209110104);Scopus;北大核心:【北大核心2004】；CSCD:【CSCD2011_2012】；

基金：上海市重点学科基金

语种：中文

中文关键词：B淋巴细胞刺激因子;大肠杆菌;克隆和表达;丝裂霉素;协同作用

外文关键词：B lymphocyte stimulator; E.coli; cloning and expression; mitomycin; synergistic effect

摘要：聚合酶链式反应(PCR)扩增获得了编码BLys(134-285AA)的cDNA片段,该片段以限制性内切酶Bgl 和Hind 双酶切插入pET32a载体。测序表明除(ATA263→ATG263)突变并导致了氨基酸(I263M)突变外,其余序列与Genbank报道的序列一致。蛋白表达用异丙基-β-D-硫代半乳糖苷(IPTG)诱导,SDS-PAGE表明:在33ku处有明显的融合蛋白表达,其表达水平高达总菌体蛋白的50%。点印迹证实融合蛋白能与anti-His6Tag抗体反应。生物学活性研究表明BLys协同丝裂霉素能明显地抑制HeLa细胞的生长。
cDNA fragment encoding BLys amino acid 134-285 amplified by polymerize chain reaction (PCR) was digested by restriction enzyme Bgl II and Hind III and inserted into pET32a plasmid in frame. Results of recombiant plasmid sequencing indicated that, except one base mutation occurred at the site of 263 amino acid (ATA263->ATG263) and led to amino acid (I263M) mutation, other sequences were the same as Genbank reported. Protein expression was induced by IPTG. SDS-PAGE analysis showed a well-marked fusion protein expressive band at the site of 33 ku. The expression revealed that its level constituted as high as 50% of the total protein. Fast dot blot assay confirmed that the fusion protein was immunoactive to six-histidine tag monoclonal antibody. Bioactivity analysis showed that BLys combined with mitomycin could significantly inhibit the growth of HeLa cells.