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目的 研究构建重组表达体系pCKM mPTH质粒 ,寻找甲状旁腺功能低下症的基因治疗途径。方法 采用重叠聚合酶链反应 (PCR)法、巢式PCR法及粘端连接法构建pCKM mPTH质粒 ,以脂质体转染骨骼肌细胞 ,用 β actin半定量和放免法分别测定其在细胞内外的表达。结果 嵌合基因pCKM mPTH质粒测序完全符合已知核苷酸序列 ,并完成定点突变 ,转染骨骼肌细胞后 ,β actin半定量法测得mPTH在细胞内成功高效表达 ,放免法测得 2 4h克隆细胞培养液中甲状旁腺激素蛋白的含量为 2 6 3 7ng/L。结论 成功构建了SD (SpragueDaw ley)大鼠重组基因表达体系 pCKM mPTH质粒 ,并证实其在骨骼肌细胞中有高效表达
Objective To study the construction of recombinant plasmid pCKM mPTH and to search for the gene therapy pathway for hypoparathyroidism. Methods Plasmid pCKM mPTH was constructed by polymerase chain reaction (PCR), nested PCR and ligation method. The plasmid pCKM mPTH was transfected into skeletal muscle cells by liposome. Semi-quantitative PCR and radioimmunoassay expression. RESULTS: The chimeric gene pCKM mPTH plasmid was sequenced completely in accordance with the known nucleotide sequences and site-directed mutagenesis was performed. After transfected into skeletal muscle cells, mPTH was successfully and efficiently expressed in the cells by semi-quantitative method of β actin. The parathyroid hormone protein content in clonal cell culture medium was 2637ng / L. Conclusion The recombinant plasmid pCKM mPTH was successfully constructed in SD rats and was confirmed to be highly expressed in skeletal muscle cells