CAJ | 학술논문

目的：构建含有EB病毒潜伏膜蛋白2A（LMP2A）的减毒单核增生性李斯特菌重组疫苗（Lmdd-LMP2A），并检测其抑瘤效应，旨在为鼻咽癌的免疫治疗提供新的思路及理论依据。方法将EBV-LMP2A基因克隆入李斯特菌穿梭载体p1565中，再将重组穿梭载体p1565-LMP2A电转化至减毒单核增生性李斯特菌株Lmdd感受态细胞中，得到重组疫苗Lmdd-LMP2A。进而对HLA-A2转基因小鼠皮下接种CNE-1／HLA-A2／LMP2A鼻咽癌细胞株，建立皮下荷瘤鼠模型。利用尾静脉注射法接种李斯特菌疫苗免疫荷瘤鼠并检测其特异性CTL诱导情况及抑瘤效应。结果成功构建了重组减毒李斯特菌鼻咽癌疫苗Lmdd-LMP2A，该疫苗高度减毒，安全性较好且稳定表达LMP2A蛋白并诱导特异性T细胞免疫应答。荷瘤鼠抑瘤模型结果表明，与对照组相比，Lmdd-LMP2A能明显的抑制肿瘤生长。并且，Lmdd-LMP2A免疫组也能延长小鼠的荷瘤生存期，均显示了该种重组疫苗的有效抑瘤价值。结论成功构建的减毒重组李斯特菌疫苗Lmdd-LMP2A，可以在确保安全性的同时发挥良好的特异性抗肿瘤效应，对于肿瘤治疗具有潜在的临床意义。
목적：구건함유EB병독잠복막단백2A（LMP2A）적감독단핵증생성리사특균중조역묘（Lmdd-LMP2A），병검측기억류효응，지재위비인암적면역치료제공신적사로급이론의거。방법장EBV-LMP2A기인극륭입리사특균천사재체p1565중，재장중조천사재체p1565-LMP2A전전화지감독단핵증생성리사특균주Lmdd감수태세포중，득도중조역묘Lmdd-LMP2A。진이대HLA-A2전기인소서피하접충CNE-1／HLA-A2／LMP2A비인암세포주，건립피하하류서모형。이용미정맥주사법접충리사특균역묘면역하류서병검측기특이성CTL유도정황급억류효응。결과성공구건료중조감독리사특균비인암역묘Lmdd-LMP2A，해역묘고도감독，안전성교호차은정표체LMP2A단백병유도특이성T세포면역응답。하류서억류모형결과표명，여대조조상비，Lmdd-LMP2A능명현적억제종류생장。병차，Lmdd-LMP2A면역조야능연장소서적하류생존기，균현시료해충중조역묘적유효억류개치。결론성공구건적감독중조리사특균역묘Lmdd-LMP2A，가이재학보안전성적동시발휘량호적특이성항종류효응，대우종류치료구유잠재적림상의의。
Objective To prepare an attenuated Listeria vaccine Lmdd-LMP2A expressing the Ep-stein-Barr virus latent membrane protein 2A ( EBV-LMP2A) and evaluate its specific anti-tumor effects on nasopharyngeal carcinoma.Methods The gene fragment encoding EBV-LMP2A was amplified by PCR analysis and then subcloned into the shuttle vector p1565.PCR restriction enzyme digestion and DNA se-quencing were performed to identify the recombinant shuttle vector p1565-LMP2A.The p1565-LMP2A vector was then transformed into competent strains of the attenuated Listeria monocytogenes ( Lmdd) .The recombi-nant attenuated Listeria vaccine strain Lmdd-LMP2A was verified by Western blot assay.The histological sections of spleen and liver tissues were stained by Haematoxylin and eosin ( H&E) for analysis of inflamma-tion.A tumor-bearing HLA-A2 transgenic mouse model was established by subcutaneous injection of CNE-1/HLA-A2/LMP2A nasopharyngeal carcinoma cell line.The prepared Lmdd-LMP2A vaccine was inoculated into the mice via tail intravenous injection for the evaluation of specific CTL induction and the in vivo anti-tumor effects.Results The shuttle vector p1565-LMP2A and the recombinant attenuated Listeria vaccine strain Lmdd-LMP2A with stable expression of LMP2A protein were successfully constructed.The immunized mice showed mild inflammations with no structural damage and necrosis as indicated by H&E staining.The growth of tumors in tumor-bearing HLA-A2 transgenic mice was significantly inhibited by the immunization of Lmdd-LMP2A vaccine as compared with mice without inoculation.The survival time of mice was prolonged with the immunization of Lmdd-LMP2A vaccine.Conclusion The prepared attenuated Listeria vaccine Lm-dd-LMP2A showed specific anti-tumor effects with the safety advantage, suggesting the possibility of using it for anti-tumor therapy in clinic.