重组rAAV-LMP诱导的特异性细胞毒T细胞对LMP阳性靶细胞的识别与杀伤

在正常个体中Ｅｐｓｔｅｉｎ－Ｂａｒ（ＥＢ）病毒是由病毒特异性细胞毒Ｔ淋巴细胞（ＣＴＬ）所控制，虽不能清除病毒，却对于控制细胞处于潜伏感染状态是必需的。抽取病人血分离淋巴细胞，在实验室制备ＥＢ病毒特异性ＣＴＬ，然后回输到病人体内，具有预防和治疗ＥＢ病毒相关疾病的意义。我们将ＥＢ病毒潜伏感染膜蛋白（ＬＭＰ）基因重组到腺病毒伴随病毒载体ｐＡＣＰ中去，与包装质粒Ａｄ８共转染已感染了Ⅱ型腺病毒的２９３细胞，获得重组病毒ｒＡＡＶ－ＬＭＰ，用此病毒感染淋巴细胞并表达ＬＭＰ，用高能Ｘ线照射灭活，与自体淋巴细胞共培养产生特异性ＣＴＬ。以ＥＢ病毒转化的类淋巴母细胞作靶细胞与ＣＴＬ反应，用ＢＬＴ活性法测定ＣＴＬ活性。结果表明，４株ＣＴＬ均能够识别和杀伤对应的靶细胞，并且随着ＣＴＬ数量的增加和反应时间的延长，上清中ＢＬＴ活性也增强。

Recombinant AAV LMP induced LMP specific cytotoxic response to autologous lymphoblastoid cell lines transformed by Epstein Barr virus

Epstein Barr virus is believed to be controlled in normal host by virus specific cytotoxic T lymphocytes (CTL). Although unable to eliminate EBV from the body, CTL seems to be essential in control of latently infected cells. Infusion of autologous EBV specific CTL, which can be produced in laboratory by separating lymphocytes from patients and stimulating them with EBV antigen, will provide an effective method of preventing and treating EBV related diseases. We inserted the LMP gene of EB virus into an AAV vector pACP and packed it in Ad2 infected 293 cells by co transfecting with plasmid Ad8, which produced the recombinant virus rAAV-LMP. The recombinant virus was used to infect stimulating cells and LMP antigen was expressed on the surface of these cells. Then the stimulating cells were irradiated and co cultured with T lymphocytes. The EBV specific CTLs were obtained. The target cells were autologous LCLs from EBV transformed B lymphocytes. The CTL activity was assayed by BLT activity method. The result indicated that all the four CTL strains could recognize and kill their target cells. This study has laid the technical basis for us to prevent and treat nasopharyngeal carcinoma in China with molecular biological methods.