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

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.