日本血吸虫SjIM基因克隆表达及动物免疫保护评估

Cloning,expression and immune function analysis of a gene encoding inositol monophosphate in Schistosoma japonicum

目的克隆并表达日本血吸虫肌醇单磷酸酶(SjIM)编码基因cDNA,评估该重组抗原抗血吸虫感染的免疫保护效果。方法以日本血吸虫42 d虫体cDNA为模板,经PCR扩增编码SjIM蛋白的开放阅读框(ORF)基因片段。采用荧光实时定量PCR分析该基因在童虫和成虫的表达情况。以pET28a(+)为载体构建重组表达质粒,经异丙基βD硫代吡喃半乳糖苷诱导,制备重组SjIM蛋白。Western blotting检测重组蛋白的抗原性与免疫原性,利用重组SjIM抗原免疫小鼠评估其免疫保护效果。结果获得了编码SjIM基因ORF的cDNA片段,ORF长度为834 bp,编码278个氨基酸。荧光实时定量PCR显示该基因在35 d虫体中表达量最高。获得了SjIM重组蛋白,其具有良好的免疫原性。免疫组小鼠获得48.76%的减虫率和41.29%的肝脏减卵率。结论获得了日本血吸虫SjIM基因ORF的cDNA序列,并制备了重组SjIM蛋白,该重组抗原在小鼠体内能诱导产生部分抗血吸虫感染的免疫保护效果。

Objective To clone and express a full-length cDNA encoding inositol monophosphate of Schistosoma japonicum（SjIM）,and to access its immunoprotection in BALB/c mice for schistosomisis.Methods A full-length cDNA encoding the S.japonicum inositol monophosphate was isolated from 42 d schistosomes cDNAs.The expression profiles in different developmental stages were detected by real-time quantitative RT-PCR.The open reading frame（ORF） was subcloned into a pET28a（＋） vector and transformed into BL21 and the recombinant protein was induced by IPTG.The immune characters of the purified recombinant protein were analyzed by Western blotting and immunoprotection in BALB/c mice.Results Bioinformatics analysis indicated that SjIM had an ORF of 834 base pairs that encoded 278 amino acids.Real-time quantitative RT-PCR analysis revealed that SjIM was upregulated in 35-day-old schistosomes,while the expression level in females was higher than that in male worms in 42nd day.Western blotting showed that the recombinant SjIM was immunogenic.An immunoprotection experiment in BALB/c mice showed that vaccination with recombinant SjIM could induce 48.76% and 41.29% reductions in the numbers of worms and eggs in the liver,respectively.Conclusions The gene of SjIM is obtained from schistosomes cDNAs and the recombinant SjIM protein is induced successfully in E.coli.These aforementioned results demonstrate that the recombinant SjIM cand induce partial protection against schistosomiasis in BALB/c mice.