中文摘要：

目的制备可溶性重组日本血吸虫信号传导蛋白14-3-3（rSj14-3-3）,并研究其免疫学特性。方法采用反转录聚合酶链反应方法从日本血吸虫成虫mRNA中制备Sj14-3-3基因cDNA片段,将此cDNA片段亚克隆至表达载体pGEX-4T-3的谷胱甘肽还原酶的基因下游,构建重组表达质粒Sj14-3-3/pGEX-4T-3。重组质粒转化大肠埃希菌BL21,转化子细菌采用异丙基-β-D-硫代吡喃半乳糖苷（IPTG）进行诱导,表达产物经SDS-PAGE以观察重组GST-Sj14-3-3表达情况。GST-rSj14-3-3经凝血酶消化后,经过Glutathione Sepharose-4B胶亲和层析制备纯化的rSj14-3-3。rSj14-3-3免疫家兔制备免疫兔血清,经免疫印迹试验分析rSj14-3-3免疫原性和反应性。结果日本血吸虫江苏株Sj14-3-3蛋白基因编码序列被成功克隆,开放阅读框的DNA序列与基因库中的登录序列同源性为99.08%。构建的含Sj14-3-3蛋白基因的重组表达质粒经IPTG诱导能表达分子量约为55 kDa的融合蛋白,融合蛋白经凝血酶切割和亲和层析获得纯化的rSj14-3-3。rSj14-3-3能诱导家兔产生高效价的特异性抗体,该抗体可识别重组和天然的14-3-3蛋白。结论本研究成功制备了可溶性rSj14-3-3,其具有良好的免疫原性与反应性。

英文摘要：

Objective To prepare soluble recombinant signal transduction protein 14-3-3 of Schistosoma japonicum（rSj14-3-3） and investigate its immunologic features.Methods The cDNA fragment of signal transduction protein 14-3-3 gene was prepared from Schistosoma japonicum adult worm mRNA,and was subcloned to the downstream of glutathione S transferase gene of expression vector pGEX-4T-3 to construct a recombinant expression plasmid 14-3-3/pGEX-4T-3.The recombinant plasmids were transferred into E.coli BL21,the transforments containing recombinant plasmid were induced by IPTG,and the expression situation of fusion protein GST-14-3-3 was observed by SDS-PAGE.The pure recombinant 14-3-3 protein was prepared by digesting the fusion protein GST-rSj14-3-3 with thrombin and affinity chromatography.The specific antibody against rSj14-3-3 protein was prepared by an immunized rabbit with rSj14-3-3 protein.The immunogenicity and immune reactivity of rSj14-3-3 protein were analyzed by Western blotting.Results The gene encoding signal transduction protein 14-3-3 of Schistosoma japonicum Jiangsu strain was cloned successfully,the homology of open read frame sequence to the registrated sequence of Sj14-3-3 protein in genbank was 99.08%.A 55 kilo Dalton fusion protein GST-rSj14-3-3 was expressed by transferring the recombinant plasmid 14-3-3/pGEX-4T-3 into E.coli BL21 and induced with IPTG.The high titer antibody against rSj14-3-3 was produced by the immunized rabbit with rSj14-3-3,and could recognize the nature and recombinant rSj14-3-3 proteins.Conclusions The soluble rSj14-3-3 protein has been prepared successfully in this study,and it has good immunogenicity and reactivity.