蓖麻毒素A链基因的克隆表达、纯化及其活性

Expression,Purification and Characterization of Ricin A from E. coli

DOI：

中文关键词: 蓖麻毒素 RTA RTB 融合表达 细胞杀伤

英文关键词: ricin RTA RTB fusion expression cell killing

基金项目:国家“十一五”科技支撑计划重大项目(2006BAD09A01);淮海工学院重点学科建设项目(HYK200605)

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中文摘要:

为了实现蓖麻毒素A链基因(rta)的克隆表达,制备有高生物活性的重组蓖麻毒素A链蛋白(RTA),借助重组腺病毒介导表达的RTB进入细胞,发挥RTA的细胞毒作用,检测其活性。重组质粒pET32a-His-RTA能正确表达RTA融合蛋白,相对分子质量约47 000,每升细菌培养物回收约50 mg的纯化蛋白质,在有RTB表达的系列中,细胞死亡率明显上升,最高可达50%~60%。说明利用pET32a表达系统可以快速获得大量有高生物活性的可溶性RTA融合蛋白质。以腺病毒为载体表达RTB可以帮助RTA进入细胞,对细胞发挥毒性作用。

英文摘要:

The target of this study is to achieve the cloning and expression of ricin A chain gene and produce the recombined ricin A chain protein(RTA) with high activity ane then assay it activity by RTB mediated by recombined adenovirus the RTA could enter cells and exert cytotoxicity.For this,the ricin A chain gene was amplified by PCR and cloned it into the fusion expression vector pET32a to construct plasmid(pET32a-His-RTA),the plasmid was transferred into E.coli BL21 and induced with low concentration of IPTG(1 mmol/L) at low temperature(20 ℃).Purified RTA by Ni-NTA column and the purified protein was identified by SDS-PAGE electrophoresis and Western-blot.It was found that the constructed recombinant plasmid(pET32a-His-RTA) was efficient expressed RTA fusion protein and about 50mg purified protein(Mr 47 000) was obtained from 1 liter culture broth.The cell death rate significantly increased and the highest being about 50-60 percentage point in the system containing RTB.

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