EBV-LMP1 cDNA克隆与真核表达载体的构建及体外表达

目的：从鼻咽癌组织中克隆EB病毒潜伏膜蛋白1（LMP1）cDNA,构建真核表达质粒pIRES-LMP1,并检测其在体外的表达。方法：利用分子生物学技术，提取鼻咽癌总RNA，逆转录PCR获得LMP1 cDAN，克隆人pDrive Cloning Vector并测序，利用引物上设计的酶切位点Nhel和EcoRI将LMP1插入真核表达质粒pIRES中，测定序列后，用脂质体包裹转梁COS细胞，采用RT-PCR和Westrn blot 检测LMP1的表达。结果：扩增的LMP1 cDNA为1301bp，核酸序列测定证实本实验所构建的质粒正确，该表达质粒在体外转梁COS细胞后可表达LMP1分子。结论：实验所构建的pIRES-LMP1表达质粒能在体外表达LMP1分子。

Cloning EBV-LAqP1 cDNA and Constructing Eukaryon Vector pIRES- IAqP1 and Its Expression in Vitro

Objective:To clone Epstein-Barr virus latent membrane protein 1(LMP1) from nasopharyngeal carcinoma (NPC) and construct eukaryon vector pIRES-LMP1 and detect the expression of the plasmid in vitro.Methods:Using cloning technique,total RNA was isolated from human NPC and its cDNA fragments of LMP1 gene was gained by RT-PCR,its cDNA fragments was constructed into the eukaryon vector pIRES.The inserted target gene in the eukaryon vector was verified by nucleotide sequencing.COS cell line was transfected with this eukaryon vector using lipofectin reagent.The expression of LMP1 molecule was detected by RT-PCR and Western blot technique.Results:The eukaryon vector pIRES-LMP1 was obtained by cloning technique.The nucleotide sequences of LMP1 gene in this eukaryon vector had high homology with LMP1 B95-8 (100%).After transfection with this eukaryon vector,the LMP1 molecule was expressed in COS cells.Conclusion:The constructed eukaryon vector pIRES-LMP1 could express LMP1 molecule in vitro.