GST-hS100A9融合蛋白的原核表达、纯化及鉴定  

Prokaryotic expression,purification and identification of GST-human S100A9 fusion protein

作　　者：游莉[1] 徐兰兰[1] 郭元元[1] 邹正渝[1] 黎玉叶[1] 孙双双[1] 罗进勇[1] 周兰[1] 

机构地区：[1]重庆医科大学医学检验系临床检验诊断学教育部重点实验室,重庆400016

出　　处：《中国生化药物杂志》2011年第4期253-256,共4页Chinese Journal of Biochemical Pharmaceutics

基　　金：国家自然科学基金(30772548)资助

摘　　要：目的制备GST-hS100A9融合蛋白。方法从pHAHA-hS100A9中PCR扩增获取hS100A9基因,亚克隆至原核表达载体pGST-moluc,构建pGST-hS100A9;后者转化BL21菌后,异丙基-β-D-硫代半乳糖苷(IPTG)诱导重组菌表达融合蛋白GST-hS100A9,经谷胱甘肽-琼脂糖4B球珠(GS4BB)分离纯化、SDS-PAGE及Western blot鉴定,BCA法蛋白定量,CCK-8测定GST-hS100A9对乳腺癌细胞MCF-7增殖的作用。结果经酶切、测序分析证实pGST-hS100A9质粒构建正确;重组菌株经IPTG诱导后,表达相对分子质量约36 000的蛋白;纯化后纯度达94%;该蛋白可被S100A9的抗体特异识别;该蛋白产量约为20 mg/L菌液;且其抑制MCF-7的增殖。结论成功构建GST-hS100A9融合蛋白的原核表达质粒pGST-hS100A9;该质粒可在BL21中诱导表达GST-hS100A9,产率高,且对MCF-7增殖有抑制作用。Purpose To prepare the fusion protein GST-human S100A9.Methods hS100A9 gene was amplified by PCR and then subcloned to vector pGST-moluc.E.coli BL21 was transformed by the recombinant pGST-hS100A9 and induced by IPTG.The fusion protein GST-hS100A9 was expressed in E.coli BL21 and was purified with Glutathion-Sepharose 4B beads,then identified by SDS-PAGE and Western Blot.Quantitative analysis of GST-hS100A9 was made by BCA.CCK-8 was used to detect the effect of GST-hS100A9 on mammary carcinoma cell MCF-7.Results Digestion and sequence analysis confirmed that the plasmid GST-hS100A9 was correct.A 36 kD protein,as expected,was obtained eventually（about 20 mg protein/L bacterial culture） and its purity was about 94%.At the same time,it was specifically recognized by antibody of hS100A9.It inhibited the proliferation of mammary carcinoma cell MCF-7.Conclusion GST-hS100A9 fusion protein expression plasmid pGST-hS100A9 was successfully constructed.High yield of GST-hS100A9 was induced by BL21 and it inhibited the proliferation of MCF-7.It will facilitate the further investigation of hS100A9.

关 键 词：GST-hS100A9融合蛋白 诱导表达 蛋白纯化 蛋白鉴定 

分 类 号：Q786[生物学—分子生物学]