重组腺病毒Ad-Endostatin载体构建与表达检测

Construction of Recombinant Adenovirus-endostatin Vector and Detection of Its Expression

目的构建含有人内皮抑素(Endostatin)基因的重组腺病毒Ad-Endostatin,检测其转染HEK293细胞的表达。方法用RT-PCR扩增Endostatin基因,并将其克隆到pShuttle2载体,再将Endostatin基因克隆到腺病毒载体Adeno-X重组为Ad-Endostatin重组腺病毒,感染HEK293细胞使病毒扩增,然后纯化和滴定腺病毒,用Western blot法分析Ad-Endostatin感染HEK293细胞后Endostatin蛋白的表达。结果 PCR和测序方法证明重组腺病毒载体Ad-Enostatin被成功构建,能感染HEK293细胞,Western blot分析,细胞Endostatin蛋白高表达。感染HEK293细胞8d后,观察到细胞出现细胞病变效应现象。结论重组腺病毒内皮抑素载体Ad-Endostatin感染HEK293细胞后可有效表达Endostatin蛋白。

Objective To construct a recombinant adenovirus vector expressing human endostatin and to detect the expression of Ad-Endostatin in HEK293 ceils. Methods Endostatin gene was cloned into plasmid pShuttle2 vector and pAdeno-X vector. Recombinant pAd-Endostatin was tansformated into HEK293 cells and Ad-Endostatin viruses were obtained, purified and titered. Endostatin expression was analyzed by Western Blot. Results pAd-Endostatin was successfully constructed and verified by PCR and sequencing. Ad-Endostatin was reproduced and endostatin was highly expressed in HEK293 cells. 8 days after transfection, CPE could be observed in HEK293 ceils by microscope. Conclusion The recombinant plamid pAd-Endostatin with endostatin gene was constructed successfully. After HEK293 ceils are infected by AdEndo recombinant viruses, Endostatin can be expressed in the ceils.