含EB病毒核抗原1重组腺病毒的构建及其在小鼠中免疫效果研究  

Construction and Immunological Responses of Recombinant Adenovirus Containing Epstein-Barr Nuclear Antigen 1 in Mice

作　　者：仝艳艳[1] 李红霞[2] 张丽霞[1] 王湛[2] 周玲[2] 曾毅[1,2] 杜海军[2] 

机构地区：[1]北京工业大学生命科学与生物工程学院,北京100124 [2]中国疾病预防控制中心病毒病预防控制所传染病预防控制国家重点实验室,北京102206

出　　处：《病毒学报》2014年第4期429-435,共7页Chinese Journal of Virology

基　　金：国家"973"项目:病毒潜伏感染的分子机制(2011CB504800);国家"863"项目:病毒性鼻咽癌疫苗关键技术研究及产品开发(2012AA02A404);环境与病毒肿瘤学北京市重点实验室开放课题项目EBNA1特异性细胞免疫与鼻咽癌发生发展关系的研究(EV02012021)

摘　　要：构建含EB病毒核抗原1(ebna1)重组腺病毒疫苗防治鼻咽癌,并为鼻咽癌疫苗的研究提供药效学资料。PCR法扩增B95-8细胞株中ebna1的C-端部分片段,EcoRI和BglⅡ酶切后插入pDC316穿梭质粒,ebna1片段测序比对正确后,与腺病毒骨架质粒pBHG共转染293细胞。收集病变后细胞,通过流式细胞仪和Western blot检测EBNA1在293细胞中的表达。电镜下观察所获得的病毒颗粒大小,TCID50法确定重组腺病毒滴度。以2×108 VP/只的rAd-ebna1免疫BALB/c小鼠。在免疫结束后的第1、2、4和8周检测小鼠体内EBNA1特异性细胞免疫应答的水平变化。结果显示,经PCR扩增获得约900bp ebna1目的片段,测序结果与标准株序列一致,质粒共转染293细胞后,细胞出现病变,获得含EB病毒核抗原1重组腺病毒(rAd-ebna1),流式细胞仪和Western blot检测结果证实,ebna1在细胞中正确转录表达,收获病毒二代滴度可达108 TCID50/mL。rAd-ebna1免疫小鼠后,能够激活EBNA1的CD4+T细胞特异性细胞免疫应答。本实验成功构建了含ebna1片段的重组腺病毒,并具激活CD4+T细胞免疫应答活性。This study aimed to construct recombinant adenovirus expressing Epstein-Barr nuclear antigen 1（EBNA1）against nasopharyngeal carcinoma（NPC）.The C-terminal region fragment of the ebna1gene of Epstein-Barr virus was amplified from the standard strain B95-8by polymerase chain reaction（PCR）.The gene fragment was inserted into the pDC316shuttle plasmid using the EcoRI and BglII restriction enzyme sites.The pDC316-ebna1shuttle plasmid and pBHG helper plasmid were cotransfected into HEK293cells after sequencing.The soluble protein was extracted from HEK293cells,which caused apparent cytopathic effects.The transcription and expression of the ebna1gene were confirmed using flow cytometry and Western blotting.rAd-ebna1titers were measured by the TCID50.rAd-ebna1was injected into BALB/c mice at a dose of 2×108 VP per mouse,EBNA1epitope-specific responses were measured at 1st,2nd,4th and 8th weeks post-immunization.The target fragment of ebna1（939bp）was obtained by PCR,and was in consensus with the sequence from the standard strain B95-8.Cytopathic effects were observed after the pDC316-ebna1shuttle plasmid and pBHG helper plasmid were cotransfected into HEK293cells.rAd-ebna1was successfully recombined in HEK293cells.EBNA1protein was detected in HEK293cells.rAd-ebna1titers reached 108 TCID50/mL.Specific responses to CD4＋epitopes of EBNA1were detected in the immunized mice.In conclusion,rAd-ebna1was successfully constructed and induced specific responses to CD4＋epitopes of EBNA1in immunized mice.

关 键 词：EBNA1(EB病毒核抗原1) 重组腺病毒 疫苗 

分 类 号：R373.3[医药卫生—病原生物学]