采用原位杂交及电镜检测CR2转染小鼠细胞的EB病毒感染

EB病毒不能感染小鼠是因为小鼠CR2受体构像与人的不同，通过对小鼠CR2受体进行定点空变，然后将野生型和突变型小鼠CR2／1（MCR2／1）及人CR2（hCR2）用基因转移技术导入小鼠鼻咽上皮细胞系（TMNE）进行表达，观察转染阳性细胞是否具有结合EB病毒的能力，EBER－1杂交结果显示，只有转染hCR2和空变型MCR2（mtMCR2)的TMNE细胞可以感染EB病毒，但是前感染EB病毒的阳性率比后高的高得多。电镜结果也进一步证实EB病毒可以感染这两种细胞，这为进一步研究EB病毒进入细胞的机制及建立EB病毒相关的鼻咽癌动物模型奠定了良好的基础。

Detection of Epstein-Barr Virus in CR2 Transfected Mouse Cells by in situ Hybridization and Electron Microscope

Epstein-Barr virus(EBV) can not infect mouse naturally becausemouse CR2 receptor's conformation is different from human CR2 receptor. Method for site-directed mutagenesis was used to introduce two desired mutations into mouse complement receptor type Ⅱ gene(MCR2), which were confirmed by DNA sequencing. Then the constructed eukaryotic expression vectors containing wild type mouse CR2/1(wtMCR2/1) and mutant type mouse CR2/1 (mtMCR2/1) were transferred separately into mouse TMNE cells by LipofectAMINE as well as expression vector inserted with human CR2 (hCR2) cDNA. After long-term screening by G418, the stably transfected clones were obtained. Several ways including PCR, RT-PCR and immunohistochemistry were utilized to screen those clones with interested genes integrated and expressed. To explore whether these transfected cells could be infected by EBV, in situ hybridization method was utilized to detect the expression of EBV encoded RNAs (EBER-1) in the transfected mouse TMNE cells. The results showed that only hCR2 and mtMCR2 transfected TMNE cells could be infected by EBV, but the positive rate of EBER-1 in hCR2-expressed TMNE cells was much higher than that in mtMCR2-expressed TMNE cells. The observation results of electron microscope also showed that EBV could infect hCR2 and mtMCR2 transfected TMNE cells. This study sets groundwork for elucidating the mechanism by which EBV enters the cells and also provides possibility to establish EBV-related NPC animal model.