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

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.