Hyper-IL-6融合基因重组腺病毒的构建及其在肝细胞中的表达

Construction of the recombinant adenovirus ad-hil-6,and the express ion in euearyotic cell.

目的在扩增出融合基因Hyper-IL-6的基础上,构建重组腺病毒Ad-HIL-6。用Ad-HIL-6感染人肝细胞LO2,观察细胞内Hyper-IL-6 mRNA和蛋白的表达情况。方法将Hyper-IL-6克隆至穿梭质粒,线性化后与腺病毒骨架质粒在BJ5183细菌内同源重组,构建重组腺病毒质粒。线性化的重组腺病毒质粒转染293细胞,PCR进一步证实重组腺病毒的存在。RT-PCR检测Ad-HIL-6感染LO2细胞后Hyper-IL-6基因的表达情况,Western blot法检测Ad-HIL-6感染LO2细胞后Hyper-IL-6的蛋白表达情况。结果酶切及PCR鉴定表明同源重组成功,重组腺病毒质粒转染293细胞后可见绿色荧光,感染293细胞得到大量病毒。重组腺病毒Ad-HIL-6能在LO2细胞中表达出Hyper-IL-6基因和蛋白,条带分别为1600bp和60KD左右。结论重组腺病毒AdHIL-6构建成功,为以后进行基因治疗奠定基础。

Objective To construct the adenoviral vector that expresses fusion gene encoding Hyper-IL-6 on the basis of successfully amplifying Hyper-IL-6 by PCR.Detect the expression of Hyper-IL-6 in the human hepatic cell lines LO2 treated with Ad-HIL-6.Methods The Hyper-IL-6 gene was coloned to the shuttle plasmid pAdTrack-CMV,The linearized shuttle plasmids were co-transformed with adenoviral backbone vector to E.coli BJ5183 cells.Before transfection,the recombinant adenoviral plasmids were digested with PacI.Then the digested recombinant adenoviral plasmids were transfected to 293 cells with Lipofectamine 2000.Generation of recombinant adenovirus was monitored by GFP expression.Presence of recombinant adenoviruses was further confirmed by PCR.The expression of fusion gene encoding Hyper-IL-6 was detected in the Ad-HIL-6 treated LO2 cells by RT-PCR and western blot.Results The results show that we have successfully coloned the fusion gene encoding Hyper-IL-6 to the shuttle plasmid pAdTrack-CMV.The restriction analysis and the PCR conformed that correct recombinant adenoviral plasmid was constructed.Two days after transfection,the fluorescence was observed in 293 cells.the mRNA and protein of Hyper-IL-6 are successfully detected in the Ad-HIL-6 treated LO2 cells.Conclusion The success in construction of the adenoviral vector and expression of Hyper-IL-6 in LO2 cells make it possible to study further on Hyper-IL-6 mediated gene therapy.