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为了进一步研究以EB病毒潜伏膜蛋白2穴LMP2)为靶抗原制备EB病毒相关肿瘤的治疗性疫苗,利用AdEasy系统构建了EBV鄄LMP2的重组腺病毒。PCR鉴定结果证实病毒DNA中含有目的基因的特异性片段;RT鄄PCR证明了外源基因在真核细胞中得以转录;免疫酶和Westernblot的结果也显示LMP2蛋白在真核细胞得到表达。将扩增后的病毒感染Hela细胞测定病毒滴度为1.5×109pfu/ml。将重组病毒以灌胃、肌肉注射和滴鼻的方式感染小鼠,经免疫荧光检测其特异性抗体,LDH法检测特异性CTL的杀伤作用,结果发现三种给药途径均可诱发小鼠针对LMP2的特异性体液免疫和细胞免疫,而用Ad5作为对照组的小鼠则没有引起相应的免疫反应。该研究将有益于进一步探索抗肿瘤特异性主动免疫作用在阻止肿瘤的生长、扩散或复发中的作用。对诱导机体产生特异性抗瘤活性有重要意义。
In order to further study the therapeutic vaccine for Epstein-Barr virus-associated tumors using Epstein-Barr virus latent membrane protein 2 (LMP2) as the target antigen, a recombinant adenovirus of EBV-LMP2 was constructed by AdEasy system. The results of PCR showed that the DNA contained the specific fragment of the target gene. RT-PCR proved that the exogenous gene was transcribed in eukaryotic cells. The results of immunoenzyme and Western blot also showed that LMP2 protein was expressed in eukaryotic cells. The amplified virus was used to infect Hela cells to determine the virus titer of 1.5 × 109 pfu / ml. The recombinant virus was inoculated intragastrically, intramuscularly and intranasally inoculated in mice, and its specific antibodies were detected by immunofluorescence. LDH was used to detect the killing effect of CTL. The results showed that all the three routes of administration induced mice LMP2-specific humoral and cellular immunity, whereas mice treated with Ad5 as a control did not elicit the corresponding immune response. This study will be helpful to further explore the role of antitumor-specific active immunization in preventing the growth, spread or recurrence of tumors. The induction of specific anti-tumor activity of the body is of great significance.