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

摘要

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 pEGFP-C1-LMP1 was transiently transfected into CNE2 cells. The location and expression of LMP1 was detected by laser scanning confocal microscope. Results:Digested with SalⅠand 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.%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。