iRhom2及其突变基因重组慢病毒的包装和稳定表达细胞系的建立

Packaging of the recombinant lentivirus of iRhom2 and its mutant and establishment of a stable iRhom 2-expressing cell line

目的通过重组慢病毒感染,建立iRhom2及其突变基因的Vero细胞稳定细胞表达系,用于iRhom2的功能研究。方法将iRhom2及其突变基因克隆到慢病毒载体Lenti-OE-Flag,构建重组慢病毒载体Lenti-OEiRhom2和Lenti-OE-iRhom2mut,将重组质粒瞬时转染HEK-293T包装细胞,获得重组慢病毒。将重组病毒感染Vero细胞,利用puromycin进行加压筛选,获得二者的重组表达细胞系。结果构建了iRhom2及其突变基因的逆转录病毒载体,并获得了重组逆转录病毒,并利用该病毒获得了二者的稳定表达细胞系。Western-blot实验证实,二者能够在Vero细胞内稳定表达。结论利用重组逆转录病毒感染,成功获得了iRhom2及其突变系的Vero细胞稳定表达株,为进一步研究iRhom2的生物学功能及其机制奠定了良好的基础。

Objective To establish a stable transfection cell line of iRhom2 and its mutant through recombinant lentivirus infection. Methods The full-length gene of iRhom2 and its mutant were cloned into the lentivirus vector LentiOE-Flag,and got recombinant lentiviral vector of Lenti-OE-iRhom2 and Lenti-OE-iRhom2 mut. The constructed recombinant lentivirus vectors were transfected into HEK-293 T packaging cells to obtain the recombinant virus. Vero cells were infected with recombinant virus. The stable expressing cell lines were obtained by pressure screening with puromycin.Results The recombinant lentivirus vectors were constructed and the recombinant virus was obtained. The stable expressing cell lines were obtained using virus infection and the protein expression was testified with Western blotting. Conclusions Stable iRhom2-expressing Vero cell line and its mutant are achieved by recombinant lentivirus infection. It paves the way for future study on biological functions and mechanism of iRhom2.