重组人活性蛋白C的基因克隆、表达载体构建以及在哺乳动物细胞中的高效表达

Cloning of recombinant human activated protein C(rhAPC) gene, constructing expression vector and high expression of rhAPC in mammalian cells

目的研究重组人活性蛋白C的基因克隆、表达载体构建以及在哺乳动物细胞中的表达。方法用RT-PCR法从人胎肝中克隆出人蛋白C轻重链基因,用PCR突变法获得人活性蛋白C的全基因,将所得全基因连接入哺乳动物细胞表达载体,并转染293细胞研究其表达和活性。结果成功得到重组人活性蛋白C基因,并成功构建哺乳动物细胞表达载体和转染293细胞获高效表达以及相关活性数据。结论通过以上方法可以获得重组人活性蛋白C基因,并实现了在哺乳动物细胞中的高效表达。

Objective To study gene cloning of rhAPC, expression vector construction and expression of rhAPC in mammalian cells. Methods The light and heavy chain genes of human protein C were cloned from human fetus liver by reverse transcription polymerase chain reaction（RT-PCR）. The whole gene of rhAPC by PCR mutation was acquired, which was connected into mammalian cell expression vector and transfected into 293 cells for studying its expression and activity. Results The rhAPC gene has been cloned successfully. Its mammalian cell expression vector has been constructed and transfected into 293 cells successfully, Moreover, high expression and relevant activity data of rhAPC have been gotten. Conclusion The rhAPC gene is acquired by the above methods. In addition, high expression of rhAPC is implemented in mammalian cells.