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

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

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.