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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SjHMGB1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SjHMGB1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。
Objective To clone and express SjHMGB1, a member of Schistosoma japonicum, and study its functional properties. Methods The complete open reading frame DNA encoding SjHMGB1 was reverse transcribed from mRNA of adult Schistosoma japonicum by RT-PCR and subcloned into the prokaryotic expression plasmid pET28a (+) to construct Recombinant expression plasmid SjHMGB1-pET28a. The recombinant expression plasmid was transformed into Escherichia coli BL21 (DE3), and the recombinant plasmid-containing transformant bacteria were induced by isopropylthiogalactoside (IPTG) to express the recombinant SjHMGB1 protein. Purified recombinant SjHMGB1 protein was prepared by nickel chelation agarose affinity gel affinity chromatography. The DNA binding and immunological properties of recombinant SjHMGB1 were identified by DNA lag test and animal immunization. The expression of SjHMGB1 in different developmental stages of Schistosoma japonicum was determined by Western blotting and RT-PCR. Mice immunized with recombinant SjHMGB1 were immunized with 45 ± 2 Schistosoma japonicum cercariae after 3 immunizations. The mice were sacrificed 42 days later, and the worm reduction rate and the oviposition rate were calculated. The protective immunity against schistosoma infection was observed effect. Results RT-PCR was used to reverse transcribe the mRNA of Schistosoma japonicum into a specific DNA fragment of about 530 bp in length. Sequence analysis confirmed the open reading frame (ORF) of SjHMGB1 protein. The fragment was subcloned into the expression plasmid pET28a (+), and the recombinant expression plasmid SjHMGB1-pET28a was successfully constructed. Transformant bacteria containing recombinant expression plasmid SjHMGB1-pET28a were induced by IPTG to express soluble SjHMGB1 protein with a molecular weight of about 28 kDa. Purified recombinant SjHMGB1 protein was prepared by nickel chelating affinity chromatography. DNA hysteresis test showed that the purified Sj HMGB1 protein can be combined with supercoiled and linear DNA. Recombinant SjHMGB1 can produce high titer antibody IgG. Western blotting analysis confirmed that the recombinant SjHMGB1 protein can be recognized by the sera of re-infected mice, indicating that recombination The expressed SjHMGB1 molecule has the functional and immunological properties of the native protein. RT-PCR and Western blotting analysis showed that the SjHMGB1 gene was highly expressed in adult and oviparous stages of Schistosoma japonicum and was extremely low in cercariae stage. Immunoprotective assay results show that recombinant SjHMGB1 molecular immunization can not induce effective anti-schistosome infection immune protective effect. Conclusion SjHMGB1 gene was obtained and the purified recombinant SjHMGB1 protein with natural functional activity was obtained. The recombinant SjHMGB1 protein had strong immunogenicity but could not induce effective protective effect against schistosoma infection.