日本血吸虫复合B细胞表位抗原的制备和鉴定  

Development and Identification of the Multiple B Cell Epitope Antigens of Schistosoma japonicum

作　　者：章辉[1] 朱荫昌[1] 司进[1] 赵松[1] 王晓婷[1] 殷旭仁[1] 曹利民[2] 曹国群[1] 华万全[1] 徐明[1] 梁幼生[1] 

机构地区：[1]江苏省血吸虫病防治研究所江苏省寄生虫分子生物学重点实验室江苏省寄生虫病学重点学科,无锡214064 [2]无锡市第三人民医院,无锡214041

出　　处：《中国寄生虫学与寄生虫病杂志》2007年第4期285-289,共5页Chinese Journal of Parasitology and Parasitic Diseases

基　　金：江苏省血吸虫病;寄生虫病;地方病基金(No.X200318.X2111);江苏省预防医学科研项目(No.Y20020027)~~

摘　　要：目的制备日本血吸虫复合B细胞表位抗原,并对其抗原性进行鉴定。方法用生物信息学软件(BioSun)预测已报告的日本血吸虫Sj22.6、Sj14-3-3、Sj26等3个B细胞表位片段的基因序列(P2、P6、P7),用随机顺序法连接3个片段,克隆入高效融合表达载体pET-32c(+)中,转化大肠埃希菌BL21感受态细胞,经酶切及基因测序鉴定重组子。阳性克隆经异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,表达产物用Ni2+螯合柱纯化,用蛋白质印迹(Westernblotting)分析该表达产物的抗原性。结果3个目的表位基因片段按P2-P6-P7和P6-P2-P7的顺序连接为2条复合表位片段(表位间由6个氨基酸组成的柔性接头连接),成功克隆入pET-32c(+)。其重组子均可表达相对分子质量(Mr)约20400的融合蛋白。亲和层析纯化的融合蛋白经十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)显示为单一条带(Mr20400),Western blotting分析显示,这2条重组复合表位蛋白均可被日本血吸虫病患者血清识别,而不与健康者血清反应。结论获得2个具有日本血吸虫病诊断潜在价值的复合B细胞表位抗原。　Objective To develop multiple B cell epitope antigens of Schistosoma japonicum and evaluate their antigenicity. Methods Bioinformatics software BioSun was used to predict B cell epitopes from Sj22.6,Sj14-3-3 and Sj26. The predicted epitopes P2,P6 and P7 were ligated to construct P2-P6-P7 and P6-P2-P7 multiepitope in random order,a 6 amino acid linker inserted between epitopes. Recombinant plasmids containing the two multiepitopes identified by enzyme digestion and sequencing were transformed into E.coli BL21. The expressed recombinant fusion proteins of E.coli BL21 induced with IPTG were purified with Ni^2＋ chelating HiTrap HP column. Their antigenicity was evaluated with Western-blotting. Result The two multiple B cell epitopes P2-P6-P7 and P6-P2-P7 were successfully cloned into pET-32c（＋） plasmid and fusion proteins were expressed. SDS-PAGE showed a single band and both of the recombinant fusion proteins were with Mr 20 400. The two proteins reacted with the sera of schistosomiasis patients but not with that of healthy people. Conclusion Two multiple B cell epitope antigens were developed with potential diagnosis value.

关 键 词：日本血吸虫 复合B细胞表位 基因克隆 基因表达 鉴定 

分 类 号：R383.24[医药卫生—医学寄生虫学]