潜伏膜蛋白1基因真核表达载体的构建及其在鼻咽癌细胞中的表达

目的：构建真核细胞中表达潜伏膜蛋白1（LMP1）基因的增强荧光表达载体,观察其在鼻咽癌细胞中的表达。方法：从鼻咽癌C15细胞中获取编码LMP1的cDNA片段,经双酶切亚克隆到pEGFP-C1质粒载体,构建pEGFP-C1-LMP1真核表达载体。重组质粒经双酶切和测序鉴定,利用脂质体介导的方法将pEGFP-C1-LMP1体外瞬时转染鼻咽癌CNE2细胞,激光共聚焦显微镜观察LMP1的表达及其细胞内定位。结果：重组载体经SalⅠ和BglⅡ双酶切后,行琼脂糖凝胶电泳,可见1条与理论预测值一致的目的条带。DNA序列鉴定证实插入片段与GenBank提供的序列一致。将pEGFP-C1-LMP1瞬时转染鼻咽癌细胞株CNE2后,LMP1基因表达产物定位于细胞膜和细胞质中。结论：成功构建LMP1真核表达载体,并可在鼻咽癌CNE2细胞株中表达。

Construction of eukaryotic expression vector containing latent membrane protein 1 and its expression in human nasopharyngeal carcinoma cells

Objective： To construct a eukaryotic expression vector containing latent membrane protein 1（ LMP1） and to detect its expression in human nasopharyngeal carcinoma CNE2 cells. Methods： The cDNA segment encoding LMP1 from human nasopharyngeal carcinoma cell line C15 was acquired and cloned into a eukaryote expression vector pEGFP-C1 after double digestion. The recombinant vector was identified by restriction enzyme analysis and nucleotide sequence determination. The eukaryotic expression plasmid pEGFPC1-LMP1 was transiently transfected into CNE2 cells. The location and expression of LMP1 was detected by laser scanning confocal microscope. Results： Digested with SalⅠ and BglⅡ,the recombinant vector was examined by agarose gel electrophoresis,and the result was in accordance with the anticipated objective strap size. The LMP1 was correctly inserted into pEGFP-C1; the sequencing results were identical with those reported in GenBank. GFP-LMP1 could be detected in both cell membrane and cytoplasm of CNE2.Conclusions： Recombinant plasmid pEGFP-C1-LMP1 is constructed successfully in vitro,which can also express in the cytoplasm of CNE2 in human nasopharyngeal carcinoma.