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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。
OBJECTIVE: To construct a mammalian expression vector pcDNA3.1 / DHFR-hTOPG with truncated human osteoprotegerin (hTOPG) and to obtain its expression in CHO-DHFR- cells with high biological activity. Methods: Recombinant expression vector pcDNA3.1 / DHFR-TOPG was constructed by gene recombination technique. CHO-DHFR- cells were transfected by LipofectamineTM2000 kit instructions and cultured in IMDM medium containing 50 mL / L dialysis serum. Methotrexate (MTX) The cells were screened by high pressure, the expression of recombinant protein and gene was determined by ELISA and RT-PCR, and the activity of recombinant protein was determined by using osteoclast-like cells (OLC) differentiation inhibition assay. Results: The expression of recombinant protein was up to 6 mg / L for 72 h, and could inhibit OLC production significantly (P <0.05). CONCLUSION: The truncated human OPG is highly expressed in CHO-DHFR- cells and has good biological activity, which provides the basis for further experimental research and clinical application.