携带恶性疟原虫ama1基因的重组MVA构建及免疫性分析  

Construction of recombinant MVA carrying Pf ama1 gene and evaluation of immune response in vaccinated mice

作　　者：李珣[1] 缪军[1] 薛采芳[1] 刘忠湘[1] 雷俊川[1] 王宪锋[1] 

机构地区：[1]第四军医大学病原生物学教研室,西安710032

出　　处：《中华微生物学和免疫学杂志》2003年第10期792-795,共4页Chinese Journal of Microbiology and Immunology

基　　金：军队医药卫生基金课题 (0 1L0 95 ) ;联合国发展计划/世界银行 /世界卫生组织热带病研究与培训特别规划 (TDR)基金课题 (A3 0 12 5 )

摘　　要：目的　构建稳定表达恶性疟原虫 (Pf)顶端膜抗原 1(AMA1)的重组改良安卡拉痘苗病毒 (MVA) ,对其诱导小鼠免疫应答的特性进行分析。方法　构建包含PfAMA1编码基因的重组载体pⅢdHR .ssp E ,转染MVA感染的BHK2 1细胞 ,经同源重组插入MVA基因组的deletionⅢ位点 ,利用宿主范围基因k1l在RK13细胞中对rMVA进行瞬时筛选 ,用获得的rMVA E(K1L)感染BHK2 1并进行传代 ,经内部同源重组去除k1l后获得rMVA E。采用PCR、IFA和Westernblot对rMVA E的基因组序列和重组抗原的表达情况进行鉴定。用不同剂量的rMVA E经腹腔接种免疫BALB c小鼠 ,ELISA检测特异性抗体应答的水平并分析IgG亚类 (IgG1和IgG2a) ,取脾细胞用重组抗原在体外进行刺激增殖。结果　获得了可稳定表达PfAMA1的重组MVA ,经 3次腹腔接种不同剂量 (10 7～ 10 8TCID50 )后的rM VA E诱导BALB c小鼠产生了水平相当 (P >0 .1)的抗AMA1抗体 ,其IgG亚类以IgG2a为主 ,各免疫组脾细胞在体外对AMA1抗原的再刺激均产生了明显的回忆应答。结论　成功构建了携带Pfama1基因的重组MVA。rMVA E可诱导小鼠产生AMA1特异的免疫应答。Objective To construct recombinant MVA carrying Plasmodium falciparum ( Pf ) ama1 gene and evaluate its immune characteristic in mice. Methods MVA recombination vector pⅢdHR.ssp/E which contains Pf ama1 gene segment was built and used to transfect MVA infected BHK21 cells, recombinant viruses carrying host range gene k1l were selected by serially infecting RK13 cells. rMVA/E(K1L) was used to reinfect BHK21 under nonselective growth conditions, k1l was removed by intra-genomic homologous recombination and the final rMVA/E was harvested. rMVA/E genome sequence and AMA1 expression were identified by PCR, IFA and Western blot. Groups of BALB/c mice were immunized with different dosages of purified rMVA/E, level of anti-AMA1 IgG antibodies and their sub-isotypes were then determined by ELISA, and in vitro proliferation of splenocytes was examined. Results Stable recombinant MVA carrying Pf ama1 gene segment had been selected. After three intraperitoneal administration of different doses (10 7-10 8 TCID_ 50 ) rMVA/E, similar levels of IgG were induced in BALB/c mice of which IgG2a was dominant. After re-stimulating with AMA1 antigen all the immune splenocytes produced significant reminiscent reaction. Conclusion Recombinant MVA stably carrying Pf ama1 gene had been successfully constructed and rMVA/E could elicit AMA1 specific immune response in mice.

关 键 词：恶性疟原虫 ama1 基因重组 MVA构建 免疫性 顶端膜抗原1 安卡拉痘苗病毒 

分 类 号：R392[医药卫生—免疫学]