携带人内皮抑素基因的重组腺病毒构建及表达

Construction and expression of recombinant adenovirus carrying human endostatin gene

目的:内皮抑素是目前发现的作用最强的内皮细胞抑制因子,但内皮抑素蛋白极不稳定,难以制备及应用,故需要借助基因治疗发挥其抑制血管生成的作用。实验利用AdEasy-1系统构建并鉴定人内皮抑素重组腺病毒,并在人血脐静脉内皮细胞中表达出内皮抑素蛋白。方法:实验于2006-03/10在中山大学生命科学院完成。以Pshuttle-Endostatin质粒为模板,应用特异引物,通过聚合酶链反应扩增纯化得到Endostatin基因片断,经测序鉴定后,酶切插入穿梭质粒pAdTrack-CMV中,再与骨架质粒AdEasy-1在大肠杆菌BJ5183中进行同源重组,重组质粒经筛选、提取、纯化后,经酶切及测序鉴定正确,再大量扩增为腺病毒载体,线性化后利用脂质体2000转染AAV293细胞包装并扩增,得到的病毒液纯化扩增后,感染人脐静脉内皮细胞,反转录-聚合酶链反应检测感染细胞中目的基因的表达,蛋白免疫印迹检测感染细胞中蛋白的表达。结果:获得的Endostatin基因片段经酶切及测序鉴定正确,重组腺病毒质粒经酶切及测序鉴定正确,成功转染AAV293细胞,包装并扩增出病毒液,纯化后测滴度为2.06×1010pfu/mL,进一步感染人脐静脉内皮细胞,在其中检测到目的基因及蛋白表达。结论:人内皮抑素重组腺病毒pAd-Endo构建成功,且可在人脐静脉内皮细胞中表达出相应的目的基因及蛋白。

AIM： So far, endostatin is found playing the strongest effect to inhibit endothelial cells. Because endostatin protein is instable, it is difficult to prepare and apply. Gene therapy is used to assist endostatin to inhibit angiogenesis. This study constructed recombinant adenovirus of human endostatin gene using AdEasy-I system to investigate the feasibility of expressing endostatin protein in ECV304 cells （human umbilical vein endothelial cells）. METHODS： The experiment was performed at College of Life Science, Sun Yat-set University from March to October 2006. Endostatin was amplified from Pshuttle-Endostatin plasmid with PCR and subcloned into the pAdTrack-CMV shuttle vector after sequencing and identification. The resultant plasmid was co-transduced into E. coli BJ 5183 cells with pAdEasy-I plasmid for homologous recombination. The recombinant plasmid was detected by restriction analysis and DNA sequence analysis. The linearized recombinant plasmid was transfected into AAV 293 cells with LipofectAMINE2000. The recombinant adenovirus was detected by GFP, PCR and restriction analysis. The virus was used to infect ECV304, and the expression of endostatin in vitro was detected by the techniques of RT-PCR and Western blot. RESULTS： The positive clones of the recombinants were constructed and confirmed by restriction endonuclease analysis and DNA sequence analysis, AAV293 cells were transfected. The titer of viral liquid was 2.06× 10^10 pfu/mL. The transcription of endostatin mRNA in ECV304 cells was checked by RT-PCR and endostatin protein could be detected in the culture by Western blot analysis. CONCLUSION： The recombinant adenoviral vector pAd-Endo carrying human endostatin is successfully constructed, and effective expression is found in ECV304 cells in vitro.