绿色荧光蛋白标记的人血管内皮生长因子165基因重组腺病毒载体的构建与鉴定

Construction and identification of green fluorescent protein labeled recombinant adenovirus containing human vascular endothelial growth factor 165 gene

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 identificate the recombinant adenovirus vector Ad5-human vascular endothelial growth factor（hVEGF） 165 gene with green fluorescent protein（GFP） carrying hVEGF165 and GFP labeled. Methods hVEGF165 gene was amplified and constructed into the expression plasmid pAV-MCMV-HA-P2A-GFP by homologous recombination and transformed into DH5 a cells, and the recombinant expression plasmid was obtained. After confirmed by colony polymerase chain reaction（PCR） and DNA sequencing, the recombinant expression plasmid were transfected into HEK293 cells by Ad Max adenovirus packaging system in which the virus was packaged and amplified. After completion of packaging, the morphological changes of HEK293 cells were observed via microscope. The recombinant adenovirus was determined titer, and detected expression by Western blot. Results The recombinant adenovirus carrier was constructed successfully, the infected HEK293 cells revealed significant cytopathic effect. The titer of recombinant adenovirus was 1.106 × 10~8IFU/mL, and recombinant adenovirus was observed to express about 23 000 by Western blot. Conclusion It is demonstrated that recombinant adenovirus Ad5-hVEGF165-GFP can be obtained successfully, which provides the basis for further study on gene therapy in septic shock and sepsis.