医学毕业ebna2基因真核表达载体的构建及表达产物的功能.docxVIP

XX大学 毕业论文 EBNA2基因真核表达载体的构建及表达产物的功能 姓 名： 2014年6月25日 【关键词】EBNA2 Construction of eukaryotic expression vector of EBNA2 and functional detection of its product 【Abstract ] AIM: To construct the eukaryotic expression vector of EBNA2 and to express it in eukaryotic cells and detect the function of its product. METHODS: EBNA2 gene was obtained by PCR and DNA recombination from the plasmid pSG5EBNA2. PCR product of EBNA2b was then inserted into plasmid pMD18T and sequenced. The full length EBNA2 gene was cut and inserted into eukaryotic expression plasmid pCMVHA ? Using LipofectamineTM 2000, the plasmid pCMVEBNA2 was transfected into Cos7 and then detected by Western blot and immunofluorescence? The activity of EBNA2 was detected by luciferase reporter assay. RESULTS: The EBNA2 gene was successfully cloned and its sequence was identical to that in GenBank. After plasmid pCMVEBNA2 was transfected into Cos7, the expressed product of EBNA2 was detected by Western blot and immunofluorescence. The transcriptional activity of EBNA2 was demonstrated by reporter assay. CONCLUSION: The eukaryotic expression vector pCMVEBNA2 has been successfully constructed and successfully expressed in eukaryotic cell line. The EBNA2 protein has transcriptional activation. [Keywords ] EBNA2; clone; expression; genetic vector 【摘要】目的：构建EB病毒核心抗原2 (EBNA2)的真核表达载体， 在真核细胞中表达，并检测其表达产物的功能?方法：应用DNA重组和PCR方 法从质粒pSG5EBNA2中亚克隆EBNA2的编码基因，插入到真核表达载体 pCMVHA中.应用脂质体的方法将重组质粒pCMVEBNA2瞬时转染Cos7细胞， 通过Western blot和免疫荧光方法检测EBNA2的表达.应用荧光素酶报告实验检 测EBNA2蛋白的活性.结果：克隆EBNA2的编码基因，经序列分析证实与 GenBank中的序列完全一致.以含有EBNA2编码基因的真核表达载体瞬时转染 Cos7细胞，EBNA2基因的表达产物通过Western blot和免疫荧光方法得到证实. 荧光素酶报告实验显示所表达的蛋白具有转录激活活性.结论:成功构建EBNA2 的真核表达载体，证实其在真核细胞Cos7可以表达，并具有转录激活功能. 【关键词】EBNA2基因；克隆；表达；遗传载体 0引言 EB病毒核心抗原2 (EBNA2)的编码产物是EB病毒体外感染细胞时最先 表达的蛋白，作为一种转录因子，它可以调节病毒及细胞内多种基因表达，如 LMP1, LMP2A [1], CD21, CD23及cmyc等，同时还可以抑制细胞免疫球蛋 白重链的转录.Notch信号途径是体内一个非常保守的信号途径，在体内分布十 分广泛[2] . EBNA2可以与Notch信号途径中的重要分子RBPJk相结合，激活 下游靶基因，并通过模仿Notch信号途径对体内淋巴细胞的分化和发育方向起调 控作用[3]?同吋EBNA2还可以促进抑癌分子