固有免疫调控蛋白PKR串联亲和纯化系统的建立及应用  

Establishment and application of a tandem affinity purification system of innate immune regulatory protein PKR

作　　者：李玉叶[1] 梁兆端[1] 吴思宇[1] 谢炯[1] 何军芳[1] 吴敏昊[1] 黄曦[1] 张萍[1] 

机构地区：[1]中山大学中山医学院免疫学教研室,热带病教育部重点实验室,广州510080

出　　处：《中华微生物学和免疫学杂志》2011年第6期487-491,共5页Chinese Journal of Microbiology and Immunology

基　　金：国家自然科学基金（30901345）;广东省自然科学基金（9151008901000083）

摘　　要：目的构建固有免疫调控蛋白——双链RNA（dsRNA）调控的蛋白激酶（PKR）的串联亲和纯化系统（TAP），初步研究PKR蛋白功能，以用于与PKR相互作用的新蛋白的鉴定与功能分析。方法通过PCR扩增PKR目的基因，克隆至真核表达载体pcTAP—A中。将重组质粒pcTAP-PKR通过脂质体法转染PKR稳定沉默（PKRkd）的HeLa细胞，并检测PKR对固有免疫信号通路之一，即促分裂素原活化蛋白激酶（MAPK）激活的调控；最后利用串联亲和纯化试剂盒纯化分离与PKR相互作用的蛋白，验证PKR蛋白复合物的纯化情况。结果将重组质粒pcTAP-PKR转染PKRkd HeLa细胞后，24h后即可检测到PKR融合蛋白，相对分子质量约为75×10^3，其表达量随着转染时间的增加而减少；PKR的过表达可发生自我磷酸化，并引起了MAPK信号通路的激活；Westernblot结果显示，小规模TAP纯化试验成功分离纯化了PKR蛋白。结论成功建立了一个PKR表达和串联亲和纯化系统，并证明PKR具有调控MAPK信号通路活性，为今后鉴定与PKR相互作用的蛋白研究奠定了基础。Objective To establish a tandem affinity purification（TAP） system of innate immuneregulatory protein PKR and analyze PKR function, for the future screen and identification of novel PKR-interaction proteins. Methods PKR gene was amplified by PCR, and then cloned into a mammalian expression vector pcTAP-A. Recombinant pcTAP-PKR was transfected into PKR knock-down（ PKRka） HeLa cells by LipofectAMINE 2000, and the PKR overexpressed HeLa cells were harvested for mitogen-activated protein kinases（MAPK） activation analysis. Cell extracts of PKR overexpressed cells were purified using TAP kit and examined by Western blot. Results Cal modulin resin（CBP） and streptavidin resin（SBP） tagged PKR was detected in PKRkd HeLa ceils as early as 24 h upon transfection with pcTAP-PKR, and its expression de- creased at later time points. The overexpression of PKR was autophosphorylated, and thus involved in the regulation of MAPK actviation. After small-scale TAP kit purification, PKR protein was detectable by West- ern blot. Conclusion We have successfully established a TAP system that over-expresses functional PKR, providing a useful tool for the future study on the identification of PKR interacting proteins.

关 键 词：双链RNA 蛋白激酶PKR 亲和 纯化 

分 类 号：R392[医药卫生—免疫学]