鼻咽癌CNE1细胞株中EB病毒LMP1 cNDA的克隆及LMP1胞外段稳定性研究

目的:克隆鼻咽癌细胞株CNE1中EBV-LMP1 cDNA,载入质粒载体pGEM中,并检测CNE1细胞株LMP1胞外肽段表达的稳定性。方法:运用免疫组化的方法检测CNE1细胞潜伏膜蛋白(LMP1)的表达情况,通过RT-PCR技术从CNE1细胞中扩增EBV-LMP1基因全长,并载入质粒载体pGEM,转化JML09菌.提取质粒,利用引物上的BamH1与Hind3酶切位点限制性内切酶消化,琼脂糖凝胶电泳鉴定。扩增CNE1细胞中含有LMP1三段胞外段的序列并测序。结果:免疫组化显示CNE1细胞高表达LMP1,扩增的LMP1 cDNA长度为1158bp,测序结果与已知序列有部分变异。连续传代的CNE1细胞LMP1胞外段表达稳定。结论:成功克隆了EBV-LMP1 cDNA,并载入质粒载体,证实LMP1胞外段表达稳定,为研究LMP1在鼻咽癌致病机制中的作用,并进一步研制LMP1胞外段特异性单克隆抗体,为其在鼻咽癌放疗靶区功能显像的研究中奠定基础。

Cloning of NPC CNE1 cell strain EBV-LMP1 CDNA and the research of cell outside stability of LMP1

Objective:To clone CNE1 cell strain EBV-LMP1 cDNA,transfect into plasmid vector and detect CNE1 cell strain LMP1 expression outside cell stability.Methods: Immunohistochemical method was used to detect CNE1 LMP( ) cell expression of LMP1.Amplified EBV-LMP1 cDNA from CNE cell by RT-PCR technique was cloned into pGEM-T easy vector and sequenced.The constructed recombinant plasmid was transferred into E.coli JM109.and indentified by restriction analysis and eletrophrasis.Sequence including 3 sections LMP1 sequence out of CNE1 cell strain were amplified and sequenced.Results: LMP1 protein was expressed highly in the cell,and the result of sequence had some variations compared with the known sequence.The expression of 3 sections LMP1 sequence out of CNE1 cell strain was stable.Conclusions: The LMP1 gene is successfully amplified and cloned into plasmid vector.The stable expression of LMP1 out of CNE1 cell provides the basic pathogenesis for studying the oncogenic role of LMP1 and further study of antibody function using in comfirmation of NPC radiotherapy target.