人黑色素瘤相关抗原Restin的克隆和表达  

Cloning and expression of Restin,a human melanoma associated antigen

作　　者：王卫华[1,2] 路凡[1] 沈琦[1] 

机构地区：[1]第四军医大学基础部生物化学与分子生物学教研室,陕西西安710032 [2]解放军总医院老年消化科,北京100853

出　　处：《现代生物医学进展》2009年第23期4407-4410,共4页Progress in Modern Biomedicine

基　　金：国家高技术研究发展计划(863计划)项目(No.2006AA02Z157)

摘　　要：目的:通过对原核表达载体、表达条件和宿主菌的优化,利用原核表达系统有效表达可溶性的人Restin融合蛋白。方法:应用PCR方法扩增获得Restin编码区,克隆入原核表达载体pET44a(+),分别转化E.coli BL21(DE3)和E.coli Rosetta-gamiTM2(DE3),不同的IPTG浓度诱导表达Restin重组融合蛋白,SDS-PAGE和Western Blot分析表达产物。结果:成功构建了重组载体pET44a(+)-Restin,并在E.coli Rosetta-gamiTM2(DE3)中获得了可溶性表达,表达量占菌体总蛋白的8.9%。结论:可溶性Restin融合蛋白的获得为后续的功能研究、蛋白制备及其抗体研制奠定了基础。Objective： To construct the recombinant plasmid of human Restin and express the protein in a prokaryotic system. Methods： Human Restin gene coding region was obtained by PCR method and was cloned into pET44a（＋） vector, a prokaryotic expression vector. The plasmid was transformed into E.coli BL21 （DE3） and E. coli Rosetta-gami^TM2 （DE3） to express Restin fusion protein in response to IPTG induction. The products were analyzed by SDS-PAGE and Western-Blot. Results： The recombinant plasmid pET44a（＋） Restin was constructed and the fusion protein was expressed in E. coli. And the molecular mass was about 97 kD and presence of soluble protein in E. coil Rosetta-gamiTM2（DE3）. Conclusion： The soluble fusion protein of Restin in E.coli were expressed successfully, which establish a basis for further study about biological functions of Restin.

关 键 词：RESTIN 原核表达 重组融合蛋白 

分 类 号：Q78[生物学—分子生物学]