Bikunin的分子克隆及真核分泌型表达载体的构建和表达  

Molecular cloning of Bikunin and construction of its eukaryotic secretory vector and expression in eukaryotic cells

作　　者：刘毕欧[1] 孙国勋[2] 赵明明[2] 施鹜丹[2] 康鹏云[2] 洪珞珈[2] 张辰宇[3] 

机构地区：[1]哈尔滨医科大学附属第一医院 [2]哈尔滨医科大学附属第四医院血液科,黑龙江哈尔滨150001 [3]南京大学生命科学院,江苏南京210093

出　　处：《哈尔滨医科大学学报》2011年第3期210-213,共4页Journal of Harbin Medical University

基　　金：黑龙江省教育厅海外学人科研资助项目(1055HQ017)

摘　　要：目的构建含有Bikunin的哺乳类细胞表达载体pSecTag2B-Bikunin,应用哺乳类细胞表达系统,表达分泌型Bikunin融合蛋白。方法用RT-PCR方法,从人肝细胞系L02扩增Bikunin编码区序列,将其克隆于pMD18-T Simple Vector中构建pMD18-T-Bikunin,经酶切、PCR及测序鉴定后,将Bikunin亚克隆至哺乳类细胞表达载体pSecTag2B中。用磷酸钙法转染NIH3T3细胞,24 h后,Western blot检测浓缩25倍的细胞培养上清中融合蛋白的表达。结果成功构建含有Bikunin的哺乳类细胞表达载体pSecTag2B-Bikunin。用磷酸钙法转染NIH3T3细胞,24 h后,Western blot检测浓缩25倍的细胞培养上清,有融合蛋白的表达。结论本实验成功克隆并在真核表达系统表达了分泌型Bikunin。Objective To construct mammalian vector pSecTag2B-Bikunin and to express secreted Bikunin fusion protein in mammalian cell expression system.Methods Amplified Bikunin CDS from human hepatocyte line L02 using RT-PCR was cloned into pMD18-T Simple Vector to construct the pMD18-T-Bikunin.After identification of enzyme cut,PCR and sequencing Bikunin was subcloned into mammalian vector pSecTag2B to construct successfully the pSecTag2B-Bikunin confirmed with enzyme cut and PCR.Fusion protein was detected by Western blot in culture medium concentrated 25 times after 24 h NIH3T3 cell transferred with calcium phosphate cell transfection.Results The pSecTag2B-Bikunin was constructed successfully by subcloning Bikunin into mammalian vector pSecTag2B.Fusion protein was detected by Western blot in culture medium concentrated 25 times after 24 h NIH3T3 cell transferred with calcium phosphate cell transfection.Conclusion We can successfully clone and express the secretory Bikunin in eukaryotic expression system.

关 键 词：BIKUNIN 分子克隆 表达载体 构建 

分 类 号：R78[医药卫生—口腔医学]