在真核细胞表达制备重组人前蛋白转化酶枯草溶菌素9  

Preparation of Human PCSK9 in Eukaryotic Cells

作　　者：贺梦影 张长江[1] 魏红[1] 段小波[1] 谭文[1] 

机构地区：[1]华南理工大学生物科学与工程学院广东省发酵与酶工程重点实验室,中国广东广州510006

出　　处：《生命科学研究》2015年第6期471-478,520,共9页Life Science Research

基　　金：深圳华大基因研究院与华南理工大学"创新基金项目"(SW20130804)

摘　　要：分别优化、合成和构建了在毕赤酵母和哺乳动物细胞表达人前蛋白转化酶枯草溶菌素9(proprotein convertase subtilisin/kexin type 9,PCSK9)的PCSK9-p PICZαA和PCSK9-pcDNA4.0重组质粒,将重组质粒转化至GS115细胞和转染至中国仓鼠卵巢(chinese hamster ovary,CHO)细胞,诱导细胞表达包含his标签的PCSK9重组蛋白。通过SDS-PAGE电泳和Western-blot分析两种系统表达重组蛋白的能力,发现GS115表达的重组蛋白相对分子质量大于人天然PCSK9蛋白且容易发生降解,而CHO细胞能够高表达与人天然PCSK9蛋白相对分子质量相同的、稳定的、易于纯化的特异重组蛋白。采用His/Ni2+亲和层析柱纯化重组蛋白,获得纯度达90%以上的PCSK9重组蛋白;用纯化的重组蛋白免疫小鼠制备了高效价特异性PCSK9多克隆抗体。所建立的CHO细胞表达体系PCSK9的平均表达量为5.6 mg/L,为开发PCSK9抑制剂和深入研究PCSK9分子结构和功能奠定了基础。To prepare human proprotein convertase subtilisin/kexin type 9 （PCSK9）, the plasmids named PCSK9-pPICZaA and PCSK9-pcDNA4.0 were constructed with cordon optimized, synthesized human PCSK9 genes. Then they were introduced into GSII5 yeast ceils by electroporation and suspension cultured Chinese hamster ovary （CHO） cells by transient transfection respectively to express his-tag PCSK9 protein. Human PCSK9 recombinant protein was successfully expressed in both Pichia pastoris and CHO cells. With the SDS-PAGE and Western-blot methods, it is easy to find that the size of the recombinant protein expressed in GS115 was larger than expected and the protein was also found degraded, while the protein ex- pressed in CHO cells can be purified easily with a purity up to 90%, and the size is nearly the same as human native PCSK9 protein. After purification with a His/Ni^2＋ affinity column, the high specific anti-PCSK9 serum was successfully prepared by immunizing mice with the purified PCSKg. A rapid method of producing human PCSK9 recombinant protein has been developed with a yield of 5.6 mg/L, which laid the foundation for specific PCSK9 inhibitors development and further study of molecular structure and function as well as the relationship between them.

关 键 词：人前蛋白转化酶枯草溶菌素9(PCSK9) 毕赤酵母表达 CHO细胞表达 重组PCSK9蛋白 

分 类 号：Q291[生物学—细胞生物学]