重组人骨形态发生蛋白2在CHO细胞中的表达、鉴定及活性分析  

Expression,Characterization and Biological Activity Analysis of Recombinant Human Bone Morphogenetic Protein 2 in CHO Cells

作　　者：张道永[1] 杨爽[1] 吕树军[1] 闫继东[1] 朱天慧[1,2] 

机构地区：[1]南开大学医学院分子遗传学实验室 [2]南开大学生物活性材料教育部重点实验室,天津300071

出　　处：《生物工程学报》2006年第6期968-972,共5页Chinese Journal of Biotechnology

基　　金：天津市自然科学基金资助(No.05YFJMJC01800)。~~

摘　　要：构建真核表达载体pCDNA3.1(+)-hBMP-2,与质粒pSV2-dhfr共转染CHO-dhfr-细胞,以含有700μg/mLG418的IMDM进行选择性培养,筛选抗性克隆,并用MTX扩增,提高rhBMP-2的表达量。收集的rhBMP-2蛋白进行Westernblot检测,还原蛋白样品电泳产生一条大小约为18kD的特异性条带,非还原蛋白样品电泳产生一条大小约为30kD的特异性条带,提示表达的rhBMP-2是经过糖基化修饰的,且以同源二聚体形式分泌表达。单细胞分离培养得到14株rCHO(hBMP-2)单克隆细胞株,ELISA法检测rhBMP-2表达水平,最高可达7.83μg/24h/106cells。活性分析结果表明,表达的rhBMP-2具有很强的生物学活性。Bone morphogenetic protein 2（BMP-2） is a member of the of BMPs family, its osteoinductive capacity has already been demonstrated. We tried to express hBMP-2 in CHO cell. In this study, we inserted hBMP-2 cDNA into vector pCDNA3.1（ ＋ ） to construct hBMP-2 eukaryotic expression vector pCDNA3.1 （ ＋ ）-hBMP-2. Recombinant Chinese hamster ovary （rCHO） cell line expressing high-level recombinant human bone morphogenetic protein 2（rhBMP-2） was constructed by co-transfecting the expression vectors pCDNA3.1 （ ＋ ）-hBMP-2 and plasmid pSV2-dhfr into dihydrofolate reductase （dhfr）-deficient CHO cells and the subsequent gene amplification in medium containing stepwise increments in methotrexate level such as 0. 1 and 1μmol/L. Western blot analyses showed a specific band of about 18kD in reduced sample lane and a specific band of about 32kD in non-reduced sample lane, this indicated that rCHO cells secret rhBMP-2 as a homodimeric glycoprotein form. Finally, we obtained a single clone cell strain expressing a high level （7.83μg/24 h/10^6cells） of rhBMP-2 tested by ELISA. Biological activity of rhBMP-2 was tested by the induction of alkaline phosphatase（ALP） activity in C2C12 cells. We treated C2C12 with different concentration of rhBMP-2 condition medium（CM） for 5d. The results showed that the rhBMP-2 could significantly increase the ALP activity of C2C12.

关 键 词：人骨形态发生蛋白2 CHO 共转染 C2C12 碱性磷酸酶 

分 类 号：Q78[生物学—分子生物学]