【摘要】 目的:构建截短型人骨保护素(hTOPG)哺乳动物表达载体pcDNA3.1/DHFR-hTOPG,实现其在CHO-DHFR-细胞中的高效表达,获取生物活性较高的重组蛋白。方法:利用基因重组技术构建重组表达载体pcDNA3.1/DHFR-TOPG,按LipofectamineTM2000试剂盒说明书转染CHO-DHFR-细胞,以含50 mL/L透析血清的IMDM培养基培养,氨甲喋呤(MTX)加压筛选高表达细胞株,ELISA法和RT-PCR法测定重组蛋白和基因的表达,并采用破骨细胞样细胞(OLC)诱导分化抑制实验测定重组蛋白的体外活性。结果:重组蛋白的表达量最高可达6 mg/L.72 h,且能够明显抑制OLC生成(P<0.05)。结论:截短型人OPG在CHO-DHFR-细胞中成功高效表达,并具有良好的生物学活性,为进一步的实验研究和临床应用提供了基础。更多还原

【Abstract】 AIM: To obtain high level expression of recombinant human truncated osteoprotegerin(TOPG) with higher bioactivity in CHO-DHFR-cells.METHODS: The recombinant vector pcDNA3.1/DHFR-TOPG was constructed and transfected into CHO-DHFR-Cells by the directions of LipofectAMINETM2000 for stable expression.The stable expression cell strains were screened by selective medium IMDM with 50 mL/L FCS,then serially passed in methotraxate(MTX) for gene amplification.The expression were analyzed by ELISA and RT-PCR.At last,the bioactivity analysis was performed in vitro.RESULTS: The expression level of recombinant truncated human OPG was up to 6 mg/L·72 h,and it had significant suppression effect on the formation of OLC(P<0.05).CONCLUSION: Recombinant truncated human OPG has high expression and bioactivity.The results make it possible for further studying and clinical implying of OPG.更多还原