四种SSB蛋白的表达纯化及其对HCVRNA的绑定分析

Expression and purification of four single-stranded DNA-binding proteins and their binding on HCV RNA

目的表达和纯化四种单链DNA结合（SSB）蛋白，并探讨其对HCVRNA的绑定。方法构建四种SSB蛋白的表达质粒，各简写为TTH，SSOB，KOD和BL21。TTH转化BL21（DE3）感受态细胞，SSOB，KOD和BL21分别转化Transetta（DE3），IPTG诱导表达。镍柱亲和层析纯化，经SDS—PAGE检测表达纯化后的SSB蛋白。并通过TAE电泳检测其对HCVRNA的绑定情况。结果四种SSB蛋白表达质粒转入合适的感受态细胞，IPTG诱导，纯化后经SDS—PAGE电泳，分别发现单一预期大小的蛋白条带，说明我们成功表达纯化出较高纯度的SSB蛋白。并与HCVRNA孵育后，经TAE电泳后发现四种SSB蛋白均能绑定HCVRNA。结论我们表达和纯化了四种SSB蛋白，并首次发现SSB蛋白绑定HCVRNA，为未来研究SSB蛋白在RNA上的应用提供一个重要基础。

Objective Express and purify four single-stranded DNA-bindiug （SSB） proteins, and evaluate the binding of SSB proteins on HCV RNA. Methods The expression plasmids of four SSB proteins were conducted, termed TTH, SSOB, KOD and BL21, respectively. The BL21 （DE3） was transformed by the expression plasmid of TTH, Transetta （ DE3 ） were transformed by the expression plasmid of SSOB, KOD and BL21, then protein expression was induced with IPTG, the expression products were analysised by SDS PAGE. To evaluate the binding of SSB on HCV RNA, RNA-SSB protein complexes were applied to a 1.2% TAE agarose gel. Results Suitable competent cells were transformed with the expression plasmids, induced by IPTG. SSB proteins were purified by affinity chromatography, to visualize their purity all SSB proteins were applied to SDS-PAGE analysis. All four proteins showed single clear bands. We have successfully obtained the SSB protein expression plasmid, expressed and purified SSB protein. TAE agarose gel electrophoresis was used to confirm SSB protein-RNA binding activity. The each of SSB-RNA complex migrated more slowly than the sole RNA, which suggested SSB protein could specifically bind to RNA. Conclusions We have expressed and purified four SSB proteins, and for the first time found that SSB protein can bind HCV RNA. Our results may provide a basis for future studies of the novel functions of SSB proteins on RNA.