迁移侵袭抑制蛋白MIIP的GST融合蛋白表达及生物学活性鉴定  

Expression and Biological Activity Determination of Migration and Invasion Inhibitor Protein-GST Fusion Protein

作　　者：高玉婧[1] 吕叶[2] 裴秀英[1] 孙玉宁[1] 杨怡[1] 姚青[1] 张茜[1] 李建宁[1] 

机构地区：[1]宁夏医科大学生育力保持教育部重点实验室,宁夏回族自治区生殖与遗传重点实验室生物化学与分子生物学系,银川750004 [2]宁夏医科大学总医院肿瘤医院肿瘤内科,银川750004

出　　处：《宁夏医科大学学报》2013年第12期1314-1317,1321,共5页Journal of Ningxia Medical University

基　　金：国家自然科学基金(81101601);宁夏自然科学基金(NZ11125);宁夏医科大学"博士学位建设学科"开放课题(KF2010-25)

摘　　要：目的构建人MIIP蛋白与谷胱甘肽转移酶(GST)重组的融合蛋白原核表达载体并进行表达及生物学活性鉴定。方法 RT-PCR扩增得到人MIIP基因全长编码序列,采用重组DNA技术将其克隆至原核表达载体pGEX-4T-1,构建获得重组原核表达载体pGEX-4T-1-MIIP,经酶切鉴定及测序验证完全正确后,将其转化至大肠杆菌BL21细胞中,用异丙基硫代-β-D半乳糖苷(IPTG)诱导表达,经纯化获得目的蛋白,用Western blot鉴定所得融合蛋白。结果构建了pGEX-4T-1-MIIP原核表达载体,在大肠杆菌中表达获得了GST-MIIP融合蛋白,经Glutathione Agarose纯化和Western blot检测,证实所得GST-MIIP融合蛋白具有生物学活性。结论成功获得了有生物学活性的GST-MIIP融合蛋白。Objective To construct the prokaryotic expression vector of human MIIP protein and glutathione-S- transferase( GST) for protein expression and purification. Methods Whole MIIP coding sequence was obtained by RT- PCR,and then,it was cloned into prokaryotic expression vector pGEX- 4T- 1 to generate novel recombinant vector pGEX- 4T- 1- MIIP. After identification of pGEX- 4T- 1- MIIP by restriction enzyme digestion and sequencing,the recombinant vector was transformed into E. coli BL21 and induced by isopropyl- β- D- thiogalactoside( IPTG),eventually,the target protein GST- MIIP was obtained after purification. GST- MIIP was then identified by Western blot. Results Prokaryotic expression vector of pGEX-4T- 1- MIIP was successfully constructed. GST- MIIP fusion protein with bioactivity was successfully obtained after the recombinant plasmid expressed in E. Coli,which was determined by purification of Glutathione Agarose and Western blot analysis. Conclusion The fusion protein of GST- MIIP with bioactivity has been successfully obtained.

关 键 词：pGEX-4T-1-MIIP GST-MIIP 原核表达 融合蛋白 

分 类 号：Q78[生物学—分子生物学]