Abstract: Objective To clone and express a high mobility group box 1 （HMGB1）protein of Schistosoma japonicum（Main? land strain）and analyze its function. Methods The DNA fragment of open reading frame encoding Sj HMGB1 protein was ampli? fied by RT?PCR from the mRNA of S. japonicum worms，then it was subcloned into the expression vector pET28a （+）to form the recombinant expression plasmid SjHMGB1?pET28a. The recombinant expression plasmid was transformed into the component E. coli BL21 （DE3），and the tranformant containing recombinant expression plasmid was induced with IPTG to express the recombi? nant protein SjHMGB1. The recombinant SjHMGB1 protein was purified by affinity chromatography with nickel chelating affinity chromatography agarose gel. The Gel retard experiment and animal immunization were performed to analyze the DNA binding capacity and the immunologic property of recombinant SjHMGB1. The expression levels of HMGB1 in different life cycle stages of S. japonicum were analyzed by Western bloting and RT?PCR. Female ICR mice were immunized with the recombinant SjHMGB1 pro? tein and infected with 45±2 cercariae of S. japonicum after three immunizations. Forty?two days post?infection，the worms and eggs of S. japonicum were recovered from the portal vein and liver tissue，respectively. The worm and egg reduction rates were calculat? ed respectively. Results A 530 bp of specific DNA fragment was amplified from mRNA of S. japonicum by RT?PCR，which was the open reading frame（ORF）encoding SjHMGB1protein confirmed by DNA sequencing analysis. The recombinant expression plasmid SjHMGB1?pET28a was constructed by cloning the ORF of SjHMGB1 into a expression vector pET28a （+） . The bacterium transformants containing the recombinant plasmid expressed a soluble recombinant protein about 28 kDa after induced by IPTG， and the recombinant SjHMGB1 protein was purified by nickel chelating affinity chromatography. The gel retard experiment showed that the recombinant SjHMGB1 protein could bind to both supercoiled DNA and linear DNA，and the recombinant protein immu? nized mice produced high titers of antiserum IgG. Western bloting indicated that the recombinant SjHMGB1 protein was recognized specifically by the S. japonicum?infected mice serum. Above results showed that the recombinant SjHMGB1 protein possessed both functional activity and immunogenicity as the natural protein. RT?PCR and Western blot results showed that SjHMGB1 was abun? dantly expressed in the adult and egg stages whereas barely detectable in the cercaria stage. The immune protection experiment showed that the recombinant SjHMGB1 induced mice to produce high titers of specific antibody IgG but failed to conduct an effec? tive immune protection against S. japonicum. Conclusion The gene encoding HMGB1 from S. japonicum and the soluble recombi? nant SjHMGB1 protein with natural functional activity are obtained，and the recombinant SjHMGB1 has a high immunogenicity but is not able to induce an effective immune protection against S. japonicum.

Key words: Schistosoma japonicum；High mobility group box 1 （HMGB1）；Gene clone；Expression and distribution；DNA binding activity；Immunogenicity；Immune protection

摘要： 目的 目的 克隆、 表达日本血吸虫高速泳动家族B1蛋白 （SjHMGB1）， 并研究其功能特性。方法 方法 利用逆转录PCR （RT?PCR） 技术从日本血吸虫成虫mRNA中反转录扩增出编码SjHMGB1的完整开放阅读框DNA片段， 然后将其亚克隆至 原核表达载体质粒pET28a （+） 中， 构建重组表达质粒SjHMGB1?pET28a。将重组表达质粒转化大肠埃希菌BL21 （DE3）， 含 重组质粒的转化子细菌经异丙基硫代半乳糖苷 （IPTG） 诱导以表达重组SjHMGB1蛋白。通过镍螯合琼脂糖亲和胶亲和层 析法制备纯化的重组SjHMGB1蛋白。通过DNA迟滞实验和动物免疫鉴定重组SjHMGB1的DNA结合和免疫学特性。通 过免疫印迹试验 （Western blotting） 和RT?PCR确定SjHMGB1分子在日本血吸虫不同发育阶段的表达情况。以重组SjH? MGB1为抗原免疫小鼠， 3次免疫后每只小鼠感染45±2条血吸虫尾蚴， 42 d后剖杀， 计算减虫率与减卵率， 观察其诱导抗血 吸虫感染的免疫保护性作用。 结果 结果 采用RT?PCR法从日本血吸虫成虫mRNA中反转录扩增到长度约为530 bp的特异性 DNA条带， 序列分析证实为编码SjHMGB1蛋白的开放阅读框序列。将此片段亚克隆至表达质粒pET28a （+） 中， 成功构建 了重组表达质粒SjHMGB1?pET28a。含重组表达质粒SjHMGB1?pET28a的转化子细菌经IPTG诱导能表达分子量约28 kDa 的可溶性SjHMGB1蛋白。采用镍螯合亲和层法制备了纯化的重组SjHMGB1蛋白。DNA迟滞试验显示纯化的Sj HMGB1 蛋白可同超螺旋及线性DNA结合， 重组SjHMGB1免疫小鼠能产生高效价的抗体IgG， Western blotting分析证实重组SjH? MGB1蛋白能被重感染小鼠血清所识别， 表明重组表达的SjHMGB1分子具有天然蛋白的功能活性与免疫学特性。RT?PCR 和Western blotting分析显示SjHMGB1分子在血吸虫成虫和虫卵期大量表达， 而在尾蚴阶段表达量极低。免疫保护性试验 结果表明， 重组SjHMGB1分子免疫不能诱导有效的抗血吸虫感染免疫保护性作用。结论 结论 获得了编码SjHMGB1基因和 纯化的具有天然功能活性的重组SjHMGB1蛋白； 重组SjHMGB1蛋白具有强烈的免疫原性， 但不能诱导有效的抗血吸虫感 染免疫保护性作用。

关键词: 日本血吸虫； 高速泳动B1家族蛋白； 基因克隆； 基因表达； DNA结合活性； 免疫原性； 免疫保护作用