日本血吸虫SjIR3基因的克隆、表达和免疫保护力  

Cloning , Expression and Immunization of the Antigen Gene SjIR3 of Schistosoma japonicum

作　　者：伍小松[1] 黄复深[1] 郝知友[1] 袁鑫[1] 邱元[1] 陈尔曼[1] 

机构地区：[1]湖南农业大学动物医学院,长沙410128

出　　处：《农业生物技术学报》2007年第3期393-397,共5页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金项目(No.30571390);湖南农业大学稳定人才基金(No.03WD08)资助

摘　　要：为鉴定日本血吸虫重组蛋白SjIR(3rSjIR3)诱导小鼠抗攻击感染的免疫保护力,根据已报道的SjIR(3AY251481)的cDNA序列设计1对特异性引物,以日本血吸虫(Schistosoma japonicum)cDNA文库为模板扩增SjIR3基因,克隆到pMD-18-simple-T载体上,测序后进行生物信息学分析。然后将SjIR3基因克隆到pET-32a(+)原核表达载体中构建重组质粒pET-32a(+)SjIR3,转化Escherichia coli BL21,IPTG诱导表达,经SDS-PAGE及Western blot分析鉴定。结果表明SjIR3具有PS50204和SSF69572两个结构域(domain),属泛素样蛋白激酶家族,重组蛋白rSjIR3具有良好的免疫原性。动物保护试验采用昆明小鼠,结果表明rSjIR3及rSjIR3+FCA可诱导小鼠产生高水平的抗体应答及26.63%和36.38%的减虫率,以及34.79%和44.09%的减卵率。表明日本血吸虫rSjIR3可诱导小鼠产生一定的免疫保护作用。To test immune protection of recombinant protein rSjIR3 against challenge infection of Schistosoma japonincum （Sj） in mice, the special primers were designed according to the sequence of SjIR3in GenBank （AY251481）. SjIR3 was amplified from Sj adult worm eDNA library,then cloned into pMD-18-simple-T vector. The obtained sequence of SjIR3 was analyzed by bioinformaties methods and cloned into prokaryotic vector pET-32 a （＋）. The recombinant protein rSjIR3 was analysed by SDS-PAGE and Western blot, and results showed SjIR3 belonged to activating enzymes of the ubiquitin-like proteins superfamily and had 2 domains of PS50204 and SSF69572. SjIR3 gene was expressed in Escherichia coli BL21 and the expression products could react with sera from mice infected with S.japonicum. Kunming mice were divided into 4 groups and vaccinated by back subcutaneous multispot injection with 50 μg refolded rSjIR3, 50 μg refolded rSjIR3 ＋100μg FCA （Frund＇s adjuvant complete）, 100 μg PBS （phosphate buffer solution）and 100 μg FCA （Frund＇s adjuvant complete） per mice and per time respectively at 0～2nd and 4th week. Sera were collected before the every immunization and challenge infection. The serum IgG was detected by ELISA. The mice were challenged with 40±1 S.japonnicum. cereariae per mouse 2 weeks after the third vaccination. Forty-five days later, mice were killed and pcrfused, and the adult worms and eggs were counted. Results showed the level of IgG in sera increased after immunization in immunized groups with rSjIR3 and rSjIR3＋FCA. The worm reduction rate was 26.63% and 34.79% and the egg reduction rote was 36.38% and 44.09%, respectively.The data reveal that rSjIR3 can elicit immune response and partial protection against challenge infection of S. japonnicum.

关 键 词：日本血吸虫 SjIR3基因 原核表达 小鼠 免疫保护力 

分 类 号：S188[农业科学—农业基础科学]