人骨形态发生蛋白4在中国仓鼠卵巢细胞中的表达研究  

Expression of Human Bone Morphogenetic Protein 4 in Chinese Hamster Ovary Cells

作　　者：蒋梅琴 邓春梅[1] 曾永芬[1] 黄义德[1] 

机构地区：[1]福建省发育与神经生物学重点实验室,福建师范大学生命科学学院,福建福州350108

出　　处：《药物生物技术》2014年第4期302-305,共4页Pharmaceutical Biotechnology

基　　金：博士点基金联合资助项目(No.20103503120002)

摘　　要：为了在中国仓鼠卵巢细胞(CHO)中表达人骨形态发生蛋白4(hBMP4),构建hBMP4重组表达质粒并电转至CHO/DG44细胞中。经RT-PCR和Western blot检测,确定整合到CHO细胞基因组中的hBMP4序列能转录并表达hBMP4蛋白,表达的重组蛋白以二聚体和单体形式存在。利用氨甲喋呤逐步筛选具有较高拷贝数的细胞。抗氨甲喋呤的CHO细胞进一步通过有限稀释法筛选稳定高表达的细胞系。与筛选前相比,hBMP4在CHO细胞中的表达得到显著提高(从24 ng/mL提高到583 ng/mL)。碱性磷酸酶活性测定表明CHO细胞表达的hBMP4具有生物活性。成功构建了能表达hBMP4的CHO细胞株,这为hBMP4的结构和功能以及临床前的研究提供了另一种可供选择的途径。In order to express the biologically active Human bone morphogenetic protein 4 （hBMP4） in Chinese Hamster Ovary （CHO） cells, recombinant vector for hBMP4 expression was constructed and transfected into CHO/DG44 cells by electroporation. mRNA and protein expression levels of hBMP4 were analyzed by RT-PCR and Western Blot. Recombinant hBMP4 protein was suc- cessfully expressed in CHO cells in both monomeric and dimeric forms. The copy number of hBMP4 gene integrated in the genome was then amplified by subjecting the transfected CHO ceils to sequentially increased concentrations of methotrexate from 50 nmol/L to 2000 nmol/L. After selection, single clones in the methotrexate-resistant CHO cells were picked using series dilution method to find out CHO cell lines with stable and high hBMP4 expression. Expression levels of hBMP4 were significantly increased in the selected clones compared with the transfected CHO cells before selection （583 ng/mL vs 24 ng/mL）. Biological activity of recombi- nant hBMP4 protein was analyzed by the induction of alkaline phosphatase expression in NIH3T3 cells. The recombinant hBMP4 protein stimulated the expression of alkaline phosphatase in a dose-dependent manner. Taken together, our results have demonstrated the successful construction of CHO cell lines for the expression of biologically active hBMP4 protein. This will provide an alternative approach for structure and functional research of hBMP4 and preclinical studies.

关 键 词：人骨形态发生蛋白-4 中国仓鼠卵巢细胞 重组表达 氨甲喋呤 有限稀释 筛选 

分 类 号：Q81[生物学—生物工程]