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目的 构建表达人内皮细胞抑制素 (Endostatin)的重组腺病毒载体 ,并进行活性检测。方法 将含有IL3分泌肽的人Endostatin全长cDNA插入穿梭质粒pAdCMV产生重组质粒pAd IL3 Endo ,通过脂质体介导与 pBHG1 0共转染 2 93细胞 ,经同源重组产生重组腺病毒Ad IL3 Endo。体外转染人结肠癌SW62 0细胞并观察其上清对人脐静脉内皮细胞 (HUVEC)生长的影响。结果 扩增到的Ad IL3 Endo的滴度为 4 .3× 1 0 1 2 空斑形成单位 (pfu) /L ,多聚酶链反应 (PCR)显示在转染的细胞中有人EndostatincDNA的存在 ,Westernblot显示转染细胞上清中有人Endostatin蛋白表达 ,台盼蓝染色显示其上清作用 72h后HUVEC的生长被抑制了 67% (P <0 .0 5)。结论 所制备的重组人Endostatin腺病毒载体能有效表达具有生物学活性的人Endostatin ,为进一步的研究奠定了基础
Objective To construct a recombinant adenovirus vector expressing human endostatin and test its activity. Methods The full-length human Endostatin cDNA containing IL-3 secreted peptide was inserted into the shuttle plasmid pAdCMV to generate the recombinant plasmid pAd IL3 Endo, which was cotransfected with pBHG10 by liposome into 293 cells. The recombinant adenovirus Ad IL3 Endo . Human colon cancer SW62 0 cells were transfected in vitro and the effects of supernatants on the growth of human umbilical vein endothelial cells (HUVECs) were observed. Results The amplified Ad IL3 Endo had a titer of 4.3 × 10 12 PFU / L. The polymerase chain reaction (PCR) showed the presence of human Endostatin cDNA in the transfected cells. Western blot showed that The expression of Endostatin protein was detected in the supernatant of transfected cells. The growth of HUVEC was inhibited by 67% (P <0.05) by trypan blue staining after 72 hours. Conclusion The recombinant human endostatin adenovirus vector can effectively express human endostatin with biological activity, which lays the foundation for further research