携带绿色荧光报告基因EBV-LMP2A真核表达载体构建及转染鼻咽癌细胞

Construction of an EBV-LMP2A eukaryotic expression plasmid with a green fluorescent protein reporter and transfection into nasopharyngeal carcinoma cells

目的构建携带绿色荧光基因的真核表达载体p IRES2-Zs-Green1-LMP2A,并转染至鼻咽癌CNE2细胞。方法从EB病毒阳性的狨猴淋巴瘤细胞B95-8中克隆EB病毒编码的EBV潜伏膜蛋白2A(latent membrane protein 2A,LMP2A)序列,并定向克隆入p IRES2-Zs-Green1载体,双酶切及测序鉴定重组的真核表达载体p IRES2-Zs-Green1-LMP2A;通过脂质体转染将重组的真核表达载体p IRES2-Zs-Green1-LMP2A转染至鼻咽癌CNE2细胞(实验组),同时另设转染p IRES2-Zs-Green1载体的阴性对照组及未转染的空白对照组。利用质粒所携带的绿色荧光蛋白表达计算细胞转染效率,RT-PCR检测目的基因LMP2A在鼻咽癌CNE2细胞中的表达。结果双酶切及测序鉴定证实真核表达载体p IRES2-Zs-Green1-LMP2A构建成功,荧光显微镜下发现实验组和阴性对照组细胞均发出绿色荧光,实验组细胞转染率约为75%;RT-PCR检测发现实验组细胞中有目的基因LMP2A表达,但阴性对照组和空白对照组均未检测到目的基因表达。结论成功构建了p IRES2-Zs-Green1-LMP2A真核表达载体并转染鼻咽癌CNE2细胞,目的基因LMP2A可在转染的鼻咽癌CNE2细胞中稳定表达。

objective To construct the eukaryotic expression plasmid pIRES2-Zs-Greenl-LMP2A encoding green fluorescent protein, and to transfect it into CNE2 nasopharyngeal carcinoma cells. Methods The LMP2A sequence from EBV-positive B95-8 cells was cloned into the vector pIRES2-Zs-Greenl. The identity of the resulting pIRES2-Zs-Greenl-LMP2A was confirmed using restriction enzymes and sequencing. The expression plasmid was transfected into CNE2 cells using a liposome-based method. The resulting transfectants were compared with negative controls transfected with pIRES2-Zs-Greenl and with untransfected cells in terms of expression of green fluorescent protein and LMP2A transcription. Results Restriction enzyme digestion and sequencing showed that pIRES2-Zs-Greenl-LMP2A was constructed successfully. Transfection of pIRES2-Zs-Greenl-LMP2A or empty vector pIRES2-Zs- Greenl led to expression of green fluorescent protein,and fluorescence microscopy indicated transfection efficiency of approximately 75%. Only cells transfeeted with the complete plasmid plRES2-Zs-Greenl-LMP2A produced LMP2A mRNA. Conclusion The eukaryotic expression plasmid pIRES2-Zs-Greenl-LMP2A has been successfully constructed,and it can drive steady expression of LMP2A in CNE2 cells, making this system useful for studies of nasopharyngeal carcinoma.