大鼠重组骨桥蛋白在中国仓鼠卵巢细胞中的稳定表达和纯化

Expression and purification of recombinant rat osteopontin in Chinese hamster ovary cells

目的 构建包含大鼠骨桥蛋白（OPN）的真核表达载体,获得稳定表达重组蛋白的中国仓鼠卵巢（CHO）细胞株并对蛋白进行纯化,为OPN在大鼠模型实验中的进一步功能学研究奠定基础.方法 通过脂质体lipofectamine 2000将重组质粒pLVX-OPN转染到CHO细胞中,嘌呤霉素筛选出稳定克隆细胞.Real-time PCR法检测转染后CHO细胞中OPN的mRNA表达水平,酶联免疫吸附实验法（ELISA）检测上清中OPN分泌水平.取CHO细胞72 h无血清培养上清进行镍柱亲和层析纯化,ELISA法和Western blot法分别检测产量和纯度.结果 pLVX-OPN表达载体经DNA琼脂糖电泳鉴定为阳性,且质粒测序成功.经质粒pLVX-OPN转染的CHO细胞中OPN mRNA表达水平是转染前的上千倍,上清分泌蛋白水平较转染前增加.上清分泌的OPN蛋白通过镍柱亲和层析纯化后纯度有所提高,产量在30％以上.结论 pLVX-OPN真核表达载体的成功构建和在CHO细胞中的稳定表达及纯化为OPN的功能学研究提供了有效工具.

Objective To construct eukaryotic osteopontin （OPN） expression vector,to produce OPN clones in Chinese hamster ovary （CHO） cell line,and to obtain purified recombinant protein for further functional study in rat model.Method Recombinant plasmid pLVX-OPN was transfected into CHO cells by lipofectamine 2000.Ceils with stable clone strains were screened by puromycin.The relative OPN mRNA expression in CHO cells was detected by real-time PCR.The secretion of OPN in supernatant was detected by enzymelinked immuno sorbent assay（ELISA）.The recombinant protein from the supernatant of CHO cell culture undergoing 72 h serum starvation was purified by nickel affinity chromatography.The yield and purity were determined by ELISA and Western blot respectively.Results pLVX-OPN expression vector was positive by DNA agarose gel electrophoresis.DNA sequencing confirmed the successful construction of the expression vector.Relative OPN mRNA expression in transfected CHO cells was several thousand times more than that in non-transfected cells,and the secretion of OPN in supernatant of transfected CHO cells was also high.The purity of recombinant protein purified by nickel affinity chromatography was improved and the yield was higher than 30%.Conclusion Our expression system in CHO cell lines is capable of stably producing recombinant OPN and provides a useful tool for the functional study of OPN.