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目的扩增、表达日本血吸虫Sjnanos编码基因并评估其重组蛋白诱导的免疫保护效果。方法从42d龄的日本血吸虫成虫中扩增了一个日本血吸虫性别差异表达的基因,GenBank登录号为AY814948,并对其进行生物信息学分析。将该基因亚克隆入原核表达载体pET28a,转化至大肠埃希菌(E.coli)BL21,用异丙基-β-D硫代半乳糖苷(IPTG)进行诱导表达,用纯化的重组蛋白免疫小鼠,ELISA检测其血清特异性抗体效价,蛋白印迹分析检测其抗原性,以看家基因NADH为内参,应用荧光定量PCR技术分析该基因在血吸虫各个阶段的表达状况。结果同源性分析表明,该基因为日本血吸虫的一个新基因,其编码序列的开放阅读框(ORF)为525bp,编码174个氨基酸,理论相对分子量为19.9,PI为8.2。荧光定量PCR技术分析显示该基因在7d、13d、18d、23d、32d、42d虫体均有表达,但在18d和13d虫体的表达量明显高于其他天数的虫体,在雄虫的表达量高于雌虫。成功构建了该基因的重组表达质粒pET28a(+)-Sjnanos,并在大肠埃希菌中获得表达,Western-blot分析表明重组蛋白具有较好的免疫原性,动物免疫保护试验获得了31.4%的减虫率和53.8%的肝脏减卵率。结论获得日本血吸虫新的抗原基因Sjnanos,该基因的重组蛋白在小鼠中诱导了部分免疫保护作用。
Objective To amplify and express Sjnanos encoding gene of Schistosoma japonicum and evaluate its immunoprotective effect induced by its recombinant protein. Methods A gene differentially expressed in Schistosoma japonicum was amplified from the adult 42 days old Schistosoma japonicum. The GenBank accession number was AY814948, and its bioinformatics analysis was performed. The gene was subcloned into the prokaryotic expression vector pET28a, transformed into E. coli BL21, induced with isopropyl-β-D thiogalactoside (IPTG), immunized with the purified recombinant protein Mice were tested for their serum-specific antibody titers by ELISA and Western blotting to detect their antigenicity. The housekeeping gene NADH was used as an internal control, and the expression of the gene in various stages of schistosoma japonicum was analyzed by fluorescence quantitative PCR. Results Homology analysis showed that the gene was a new gene of Schistosoma japonicum. The ORF of the gene was 525 bp, encoding 174 amino acids. The theoretical relative molecular weight was 19.9 and the PI was 8.2. Fluorescent quantitative PCR analysis showed that the gene was expressed on the 7th day, the 13th day, the 18th day, the 23rd day, the 32th day and the 42th day. However, the expression of the gene was significantly higher than that of the other days on the 18th day and the 13th day. The amount is higher than the female. The recombinant plasmid pET28a (+) - Sjnanos was successfully constructed and expressed in Escherichia coli. Western-blot analysis showed that the recombinant protein had good immunogenicity. The animal immunoprotection assay obtained 31.4% Worm reduction rate and 53.8% of the liver egg reduction rate. Conclusion Sjnanos, a novel antigen of Schistosoma japonicum, was obtained and the recombinant protein of this gene induced partial immunoprotection in mice.