鼠内皮细胞抑制素的克隆及在COS-7细胞中的分泌表达

Cloning of Endostatin and Secreted Expression in COS-7 Cells

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 From the mouse liver, the endostatin cDNA was cloned and secretorily expressed in the recreted form in the COS-7 cells. This is a basis for tumor antiangiogenesis gene therapy. Methods Mouse endostatin cDNA was amplified from the liver cells of BABL/c mouse by means of RT-PCR, then cloned into sequencing vector puc-T. After verified by sequencing, secreting expression vector pSEC-endo was constructed. With the DEAE-dextran, the COS-7 cells were transfected by the plasmid pSEC-endo, monitoring the expression of endostatin gene by means of RT-PCR and Western analysis. Results The mouse endostatin cDNA sequence is identical to that reported in literature reference. And there's with mRNA transcribed in the transfected COS-7 cells. The results of Western blot showed that the supernatant and cytoplasmic of the COS-7 cells transduced with pSEC-endo had the expression of 20 kd objective protein. Conclusion The results suggested that cDNA of mouse endostatin could be expressed successfully cloned in COS-7 and secreted form COS-7.