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目的从小鼠肝脏中克隆出endostatin并在COS - 7细胞中分泌表达 ,为其在肿瘤抗血管基因治疗中的应用打下基础。 方法用RT -PCR从鼠肝脏扩增出内皮细胞抑制素cDNA并克隆入测序载体PUC -T测序证实后 ,装入分泌表达载体 pSEC -hygromycin ,用DEAE -葡聚糖转染COS - 7细胞 ,从mRNA水平及蛋白水平检测内皮细胞抑制素的表达。 结果测序结果显示所克隆的小鼠endostatincDNA与文献报道的完全一致 ,RT -PCR显示转染后的COS - 7细胞有endostatin的mRNA表达 ,Western -blot显示转染后的COS - 7细胞上清及包浆内均有目的蛋白的表达。 结论成功克隆并分泌表达了小鼠endostatin。
Objective To clone endostatin from mouse liver and express it in COS - 7 cells, which laid a foundation for its application in antiangiogenic gene therapy. Methods Endostatin cDNA was amplified from rat liver by RT-PCR and cloned into sequencing vector PUC-T. The recombinant plasmid was then inserted into secreted vector pSEC -hygromycin and transfected into COS-7 cells with DEAE-dextran. Endostatin expression was detected by mRNA and protein levels. Results The sequencing results showed that the cloned mouse endostatincDNA was completely consistent with that reported in the literature. The expression of endostatin mRNA was detected by RT - PCR and Western blotting showed that the transfected COS - 7 cells supernatant and Pulmonary plasma protein expression. Conclusion Mouse endostatin was successfully cloned and secreted.