去除致癌基因的EB病毒潜伏膜蛋白1的重组腺病毒的构建及其免疫效果  

The Construction of Recombinant Adenovirus Inserted with Modified Ep stein-Barr Virus Latent Membrane P rotein 1 and the Study of Its Immu ne Effect

作　　者：贾俊岭[1] 周玲[1] 左建民[1] 王琦[1] 曾毅[1] 

机构地区：[1]中国疾病预防控制中心病毒病预防控制所,北京100052

出　　处：《病毒学报》2003年第3期245-248,共4页Chinese Journal of Virology

基　　金：国家"863"资助项目(2001AA217091);国家"973"资助项目(G1998051201);国家自然科学基金资助项目(30270520)

摘　　要：为进一步研究利用EB病毒潜伏膜蛋白1(LMP1)进行免疫治疗的可行性,构建了含有去除致癌基因的EB病毒LMP1片段(LMP1△)的穿梭质粒pAdTrack CMV LMP1△,将它与腺病毒骨架质粒pAdEasy 1用电转染的方法共同导入大肠杆菌BJ5183中,在宿主菌重组酶的介导下进行同源重组。通过抗性筛选,获得含有重组腺病毒基因的质粒;然后再通过脂质体将重组腺病毒质粒导入腺病毒包装细胞HEK293细胞中,在HEK293细胞E1蛋白的反式作用下,病毒被包装。将包装病毒的细胞裂解上清进行PCR鉴定证实,病毒DNA中含有目的基因的特异性片段。RT 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,LMP1△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3.0×109PFU/ml。为初步探讨其免疫效果,采用肌肉注射和滴鼻的方式感染Balb/c纯系小鼠,免疫酶检测其特异性抗体,LDH法检测特异性CTL的杀伤作用,结果发现,两种免疫途径均可诱发小鼠针对LMP1的特异性体液免疫和细胞免疫,而且Ad5作为免疫对照组的小鼠则没有引起相应的免疫反应。The LMP1△ gene was cloned and inserted into the shuttle vector pAdTrack-CMV.The resulting shuttle vector pAdTrack-CMV-LMP1△ was linea rized by digesting with restrictio n endonuclease Pme I,and subsequen tly cotransformed into E.coli.BJ51 83 cells with an adenoviral backbon e plasmid pAdEasy-1 by electroporoti on method.We acquired the recombin ants by selecting for kanamycin re sistance,and reombination was conf irmed by restriction endonuclease (Pac Ⅰ) analyses.Subsequently,the linearized recombinant plasmid was transfected into adenovirus packag ing cell line (the HEK 293 cell). The recombinant adenoviruses were typically generated within 7 to 12 days.The insertion of LMP1△ gene wa s confirmed by PCR method.The tran scription of the LMP1△ was also dem onstrated by RT-PCR.The expression of LMP1△ in HEK-293 cell was proved by both EIA and Western blot.The t itre of the recombinant virus test ed on HeLa cell reached 3.0×10 9PFU /ml.In order to know about the imm une effect of the recombinant viru s,Balb/C mice were infected by rAd 5-LMP1△ through muscle injection a nd nose dripping.The LMP1△ specifi c antibody was tested by EIA and t he LMP1△ specific cytotoxic T lymp hocyte response was by the method of LDH.We found that both the infe ction routes in Balb/C mice could stimulate LMP1△ specific lymphocytes and hum oral immunel responses and,in contr ast,the control group infected wit h Ad5 did not elicit the same res ponses.

关 键 词：致癌基因 EB病毒 潜伏膜蛋白1 重组腺病毒 免疫效果 

分 类 号：R730[医药卫生—肿瘤] R373.9[医药卫生—临床医学]