摘要
采用RT PCR法得到人OPG的编码区cDNA ,克隆至穿梭质粒pShuttle,构建重组有OPG编码区cDNA的腺病毒DNA ,经PacI酶切线性化 ,在脂质体介导下转染HEK2 93细胞 ,制备重组腺病毒并测定病毒滴度约为 5× 10 6 ～1 5× 10 7pfu mL。体外感染小鼠成肌细胞C2C12 ,Westernblot及ELISA检测证实有OPG蛋白的表达 ,并可在细胞培养上清中持续表达 6周。感染OPG重组腺病毒的C2C12细胞生长状态良好、细胞周期无明显变化。将重组腺病毒加入体外培养的小鼠骨髓细胞的培养基中 ,诱导形成的破骨细胞数量及在象牙片上形成的吸收陷窝的数量显著减少 (P <0 0 1)。
Using the isolated total RNA from osteosacoma cell line MG63,the cDNA encoding human OPG was amplified by RT PCR. A recombinant adenoviral vector carrying cDNA of OPG was constructed and OPG expression in mouse myoblast C2C12 cells was confirmed by Western blot and ELISA The secreted expression of OPG protein persisted more than 6 weeks in vitro, and the growth of C2C12 cells infected by recombinant adenoviral were in good state Osteoclasts derived from mouse bone marrow cells infected with recombinant adenoviral made less number of TRAP positive cells and resorption pits formed on dentine slices
出处
《生物工程学报》
CAS
CSCD
北大核心
2003年第1期35-40,共6页
Chinese Journal of Biotechnology
