Flag与GFP双标记人微粒体甘油三酯转移蛋白重组腺病毒载体的构建与表达

Construction of Recombinant Adenovirus for Microsomal Triglyceride Transfer Protein with Flag and GFP

目的利用重组腺病毒(adenovirus,ad)技术Adeasy系统构建带绿色荧光蛋白(Green fluorescence protein,GFP)和Flag双标记的人类微粒体甘油三酯转移蛋白(human microsomal triglyceridetransfer protein,hMTP)基因重组腺病毒载体,并包装成高滴度的腺病毒。方法自人HepG2细胞获得RNA,以RT-PCR和Nested PCR扩增hMTP-flag基因。PCR产物经测序证实后插入穿梭质粒GFP-Track。经PmeI酶切线性化后,共转化到含腺病毒骨架质粒Adeasy的感受态细菌BJ5183。筛取阳性克隆,经PacI酶切线性化后转染293A细胞,产生包装病毒颗粒,并传代扩増培养。结果经测序证实得到hMTP-flag-GFP基因,限制性内切酶证实同源重组腺病毒包含目的基因。荧光显微镜下可见GFP的高效表达,Western-blot检测可见97KD目的条带(hMTP分子质量为97KD)。结论成功构建含hMTP-flag-GFP基因的双标记重组腺病毒载体,并进行蛋白质表达,为进一步研究脂蛋白的功能打好了坚实基础。

Objective To construct the recombinant adenovirus of human microsomal triglyceride transfer protein with flag and GFP（hMTP-flag） with Adeasy system. Methods RNA is from HepG2 cell. The gene of hMTP-flag was amplified by RT-PCR and nested PCR. The PCR product was verified by sequencing. The gene was inserted into the shuttle vector： plasmid： GFP-Track. The recombinant plasmid confirmed by restriction enzyme digestion was linearized by PmeⅠ digestion and transformed into the competent cell： BJ5183 containing adenovirus backbone plasmid; Adeasy. The positive homologous recombinant was linearized with PacⅠ digestion then was transfected into 293A cells to get the packaged adenovirus followed by large scaled virus propagation. Results The gene of hMTP-flag-GFP was verified by the sequencing. Recombinant plasmid was confirmed by restriction enzyme digestion. High level GFP expression was observed under fluorescence microscope after the 293A cells were transfected with the recombinant adenovirus plasmid. Also, the hMTP band （97KD） was identified by Western-blot. Conclusion The recombinant adenovirus of hMTP-flag-GFP was fully constructed and protein expressed successfully. Based on this, the function of lipoprotein could be further discovered.