绿色荧光蛋白-EGFRvⅢ真核表达载体的构建及在Tca8113细胞中的表达

Construction of Eukaryotic Expression Vector pEGFP-N 1 -EGFRv Ⅲ

目的构建含有表皮生长因子受体Ⅲ型变异体（EGFRvⅢ）的增强型绿色荧光蛋白表达载体，转染到舌癌Tca8113细胞中，建立稳定表达的细胞系，为研究EGFRvⅢ在舌癌细胞传导通路中的作用奠定实验基础。方法以从人乳腺癌细胞中提取的总RNA为模板经反转录为cDNA，用RT-PCR的两步法扩增得出合有HindⅢ和Xhol的EGFRvm目的基因连接到绿色荧光蛋白pEGFP-N1载体上。将重组质粒通过脂质体转染的方法转染到舌癌细胞中观察，以G418筛选表达目的基因的单细胞系并通过RT-PCR的方法进行目的基因的表达水平检测。结果经过PCR引物扩增得到1218bp基因片段，生物公司测序正确，重组质粒pEGFP-N1-EGFRvⅢ经双酶切分析检测无误；质粒转染舌癌细胞可见荧光信号表达，经过G418筛选后成功获得细胞克隆株。结论成功构建pEGFP-N1-EGFRvⅢ表达载体，并能够在舌癌细胞中高度表达，为研究EGFRvⅢ在舌癌MAPK传导通路中的作用建立细胞模型奠定了实验基础。

Objective To study the effect of EGFRv Ⅲ on conduction path of tongue cancer cell through constructing eukaryotic expression vector pEGFP-N 1-EGFRv Ⅲ and establish ing a functional cell expression. Methods The cDNA coding sequence of EGFRv Ⅲ containing the digestion site of Hind Ⅲ and Xhol was amplified by RT-PCR from the human breaster cancer cell and then was inserted into pMD18-T plasmid and pEGFP-N1 vector. The recombi- nant vector was transfected into Tca8113 through lipofection and then chosen by G418. At last RT-PCR was used to check the level of tbe expression of the recombined vector. Results The amplified gene fragment was 1218bp and con- finned by sequencing which was consistent with the human EGFRvm sequence in Genbank. Restriction enzyme digestion and sequencing showed that pEGFP-N1-EGFRv Ⅲ was constructed correctly. Tea8113 cell transfected could been seen bright green signal, achieved chosen cell clone after G418 sereening. Conclusion The enkmyotic expression vector pEGFP-NI-EGFRv Ⅲ is construced sucessfully,highly expressed in the Tca8113 cells,which the exprimental foundation for studying the effet of E（,FRv Ⅲ in the MAPK conduction pass has been established.