人组蛋白H1基因的原核表达、纯化及其活性检测  

Prokaryotic expression and purification of human histone-1 and its activity detection

作　　者：徐小洁[1] 范忠义[2] 王凌雪[1] 张浩[1] 丁丽华[1] 杜楠[2] 叶棋浓[1] 

机构地区：[1]军事医学科学院生物工程研究所,北京100850 [2]解放军总医院第一附属医院,北京100048

出　　处：《细胞与分子免疫学杂志》2011年第8期843-845,共3页Chinese Journal of Cellular and Molecular Immunology

基　　金：全军医药卫生科研基金资助(06J021);国家重大科学研究计划(2007CB914603);国家自然科学基金资助项目(30800205;31071174)

摘　　要：目的:克隆人组蛋白H1全长编码区基因,获得其原核表达产物,并对融合蛋白进行纯化以及活性检测。方法:采用PCR技术从人乳腺文库中扩增出人组蛋白H1全长编码区基因,将其克隆到pGEX-KG载体中,在大肠杆菌Rossate中表达后,利用GST-Sepharose 4B亲和珠对原核表达产物进行纯化,以SDS-PAGE和Western blot鉴定表达与纯化产物。结果:从人乳腺文库中扩增获得约650 bp的DNA片段,并成功克隆至pGEX-KG载体上,经测序与目的序列完全一致;在Rossate菌中诱导表达出相对分子质量(Mr)约为52 000的目的蛋白,经纯化后获得了纯度较高的重组蛋白GST-H1,激酶实验证明该蛋白活性良好。结论:成功获得了活性良好的重组蛋白GST-H1,为后续研究细胞周期蛋白调控奠定了实验基础。AIM： To construct the prokaryotic expression vector of human histone-1,obtain the purified GST-H1 protein,and detect its activity.METHODS： Human histone-1 coding region was amplified from human mammary cDNA library,and was inserted into prokaryotic expression vector pGEX-KG.The recombinant plasmid pGEX-KG-H1 was transformed into E.coli Rossate.The expressed product was purified by GST-Sepharose 4B beads and identified by SDS-PAGE and Western blot analysis.RESULTS： The DNA fragment of about 650 bp was successfully amplified by PCR,cloned into pGEX-KG,and identified by sequencing.The recombinant protein of about Mr 52 000 was successfully induced,purified and tested well by Kinase assay.CONCLUSION： The recombinant protein of GST-H1 is obtained successfully,which lay the foundation for further research on cell cycle control.

关 键 词：人组蛋白H1 原核表达 纯化 

分 类 号：R392.12[医药卫生—免疫学]