新型人骨形成蛋白2逆转录病毒载体的构建及活性检测

目的 构建表达重组人骨形成蛋白2(BMP2)基因的重组逆转录病毒,对其在成骨细胞中的生物学作用进行探讨.方法 克隆BMP2基因,与pDNR-CMV连接构成pDNR-CMV-BMP2,然后将重组质粒pDNR-CMV-BMP2和逆转录病毒质粒pLP-LNCX以loxP位点进行同源重组,构成逆转录病毒载体pLP-LNCX-BMP2,转染包装细胞PT67进行病毒包装,NIH3T3细胞测定病毒滴度;将逆转录病毒感染人成骨细胞,噻唑蓝(MTT)法检测细胞生长变化,Western blot检测BMP2蛋白表达.结果 重组逆转录病毒载体pLP-LNCX-BMP2经鉴定连接正确;病毒载体pLP-LNCX-BMP2转染PT67后,上清液中病毒滴度可达到5×108pfu;MTT检测见逆转录病毒组与对照组比,48和72h细胞抑制率(5.1%比5.3%,8.5%比8.3%)差异无统计学意义(P>0.05),转染48 h后Westernblot可见BMP2蛋白高表达.结论 成功构建了BMP2逆转录病毒,为BMP2基因治疗及其功能研究提供了有效的手段.

Construction and identification of recombinant retroviral vector expressing BMP2 gene

Objective To construct recombinant retrovirus expressing human bone morphogenefic protein-2 gene ( BMP2 ) and investigate its biological function in osteoblasts. Methods BMP2 gene was amplified and reconstructed with pDNR-CMV into pDNR-CMV-BMP2 plasmid. Recombinant plasmid pD-NR-CMV-BMP2 and retroviral plasmid pLP-LNCX were recombinated homologously in loxP sites into pLP-LNCX-BMP2 plasmid transferred into packing cell line PT67. The viral titer was tested by NIH3T3 cells.Human osteoblasts were transfected with retrovirus. By using MTT assay, the changes in cell growth were measured. The expression of BMP2 protein was detected by Western blot. Results Recombinant retrovirus vector pLP-LNCX-BMP2 was constructed successfully. After transfection of pLP-LNCX-BMP2 into PT67,viral titer in the supernatant was up to 5 × 108 pfu. The cell growth inhibition rate at 48 and 72 h had no significant difference between retrovirus group and control group (5.1% vs 5.3% ,8.5% vs 8.3% ,P >0.05). After transfection for 48 h, the expression of BMP2 protein was up-regulated in retrovirus group. Conclusion BMP2 retrovirus vector was reconstructed and could express BMP2 protein correctly in vitro, which supplied an effective method for BMP2 gene therapy and further study on its mechanism.