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目的构建与鉴定携带绿色荧光蛋白标记的人血管内皮生长因子165(hVEGF165)基因的重组腺病毒载体Ad5-hVEGF165-GFP。方法将hVEGF165基因进行扩增,并同源重组入表达质粒pAV-MCMV-HA-P2A-GFP,转化入DH5a感受态细胞,得到的转化子经菌落聚合酶链反应(PCR)鉴定和测序鉴定后,通过Ad Max腺病毒包装系统转染HEK293细胞,进行病毒的包装和扩增。包装后镜下观察HEK293细胞,测定腺病毒滴度,Western blot检测重组腺病毒的表达。结果成功构建了重组腺病毒载体Ad5-hVEGF165-GFP,重组腺病毒转染的HEK293细胞出现了明显的细胞病变效应;获得重组腺病毒的滴度为1.106×10~8 IFU/mL;Western blot检测重组腺病毒表达在23 000左右。结论成功获得重组腺病毒载体Ad5-hVEGF165-GFP,为进一步研究基因治疗脓毒症/感染性休克奠定了实验基础。
Objective To construct and identify a recombinant adenovirus vector Ad5-hVEGF165-GFP carrying green fluorescent protein-labeled human vascular endothelial growth factor 165 (hVEGF165) gene. Methods The hVEGF165 gene was amplified and homologously recombined into pAV-MCMV-HA-P2A-GFP and transformed into DH5a competent cells. The obtained transformants were identified by colony PCR and sequenced The HEK293 cells were transfected with the Ad Max packaging system for virus packaging and amplification. HEK293 cells were observed under the microscope and the titer of adenovirus was determined. Western blot was used to detect the expression of recombinant adenovirus. Results The recombinant adenovirus Ad5-hVEGF165-GFP was successfully constructed. The recombinant adenovirus transfected HEK293 cells showed obvious cytopathic effect. The titer of recombinant adenovirus was 1.106 × 10 ~ 8 IFU / mL. Western blot The recombinant adenovirus is expressed at around 23,000. Conclusion The recombinant adenovirus Ad5-hVEGF165-GFP was successfully obtained, which laid the foundation for further study of gene therapy for sepsis / septic shock.