日本血吸虫SjIR3基因的克隆、表达和免疫保护力研究

鉴定SjIR3诱导小鼠抗攻击感染的免疫保护力。根据GenBank中的SjIR3（AY251481）序列设计特异性引物一对，以日本血吸虫cDNA文库为模板PCR扩增SjIR3基因，利用网上生物信息资源对SjIR3基因进行生物信息学分析。常规方法构建重组质粒pET-32a(+)/SjIR3，转化E.coliLB21，IPTG诱导表达重组蛋白SjIR3（rSjIR3），并利用SDS-PAGE及western-blot进行鉴定分析。动物保护试验用动物为昆明小鼠（雌性，20g），44只，随机分4组，每组11只：A组和B组为对照组，分别接种PBS和FCA；C组和D组为试验组，分别于背部多点注射接种50µg rSjIR3和50µg rSjIR3+FCA，免疫程序为0-2-4-8周。末次免疫后2 wK，每只小鼠攻击40±1条尾蚴，45天后剖杀，冲虫，计数减虫率和减卵率。免疫前和感染前尾静脉采血，ELISA检测IgG抗体水平。结果表明SjIR3 PCR扩增产物约为900bp，与GenBank 中的SjIR3（AY251481）100%的同源，为一跨膜蛋白，具有PS50204和SSF69572两个模体（Domain），为泛素样蛋白激酶家族。动物保护试验表明rSjIR3及rSjIR3+FCA免疫小鼠，可诱导产生26.63%、36.38%的减虫率和34.79%、44.09%的减卵率。结论：rSjIR3可诱导小鼠产生部分抗攻击感染的免疫保护力。

Cloning,Expression and Immunization in Mice of Sjir3

To test immune protection of SjIR3 against challenge infection of Schistosoma japonicum(Sj) in mice.The special primers were designed according the sequence of SjIR3 in GeneBank (Ay25148). SjIR3 was amplified using Sj adult worm cDNA library as temple. The obtained sequence of SjIR3 was analyzed in internet. The recombinant plasmid pET-32(a) / SjIR3 was constructed by routine methods. The recombinant protein rSjIR3 was expressed by induced with IPIG. rSjIR3 was analyzed by SDS-PAGE and western-blot. Forty-four Kun-Ming mice (female, 20g) were randomly divided into 4 group each with per 11. Group A.B were control immunized with PBS and FCA respectively. Groups C,D were immunized with 50µg rSjIR3,50µg rSjIR3 + FCA respectively in 0-2-4-6th week. Levels specific antibody were detected by ELISA. The mice were challenged with 40±1 S.j cercariae per mouse 2 after the fourth vaccination. Forty-five days later, mice were killed and perfused, and the adult worms and eggs were counted. Results showed DNA segment of SjIR3 is 890 bp long and 100% identify with SjIR3 in GeneBank (AY251481). Protein expressed by SjIR3 is trans-membrane, the worm reduction rate was 26.63% and 34.79%. The egg reduction rate was 36.38% and 44.09%.