日本血吸虫硫氧还蛋白-1的克隆、表达和功能分析

Cloning, Expression and Function Analysis of Thioredoxin-1 Protein of Schistosoma japonicum

目的克隆、表达日本血吸虫硫氧还蛋白-1(SjTrx-1)编码基因,测定重组SjTrx-1蛋白的酶催化活性,分析SjTrx-1基因在血吸虫不同发育阶段的转录本差异,观察其在日本血吸虫成虫体内的组织分布情况。方法从日本血吸虫cDNA文库中挑选SjTrx-1的基因序列,应用PCR技术对目的片段进行体外扩增,并克隆入原核表达载体pET28a,重组质粒转化E.coli BL21(DE3)细胞,异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,组氨酸标签亲和层析法纯化表达产物,二硫苏糖醇(DTT)/胰岛素还原法测定重组SjTrx-1蛋白的酶催化活性。纯化的重组SjTrx-1蛋白免疫新西兰白兔,以制备的免疫兔血清作为抗体,应用免疫荧光定位技术检测SjTrx-1在日本血吸虫成虫体内的分布情况。制备日本血吸虫各虫期的总RNA样品,利用RT-PCR方法扩增目的片段,分析SjTrx-1基因在各发育阶段的转录本丰度。结果构建了重组表达载体SjTrx-1/pET28a,在E.coli中实现了重组SjTrx-1蛋白的可溶表达,其相对分子质量(Mr)约为38 000,与预测的融合蛋白大小相符。经组氨酸标签亲和层析法获得纯化重组蛋白SjTrx-1,并测定了其体外的酶催化活性。虫期转录特异性分析显示,该基因在各个虫期均存在转录,其中胞蚴、童虫和成虫阶段的丰度较高,毛蚴和尾蚴中含量相对较低。荧光定位结果显示,SjTrx-1蛋白在虫体内分布广泛,且无组织特异性。结论 SjTrx-1在日本血吸虫虫体内分布广泛,在胞蚴、童虫和成虫阶段的丰度较高。

Objective To clone and express recombinant thioredoxin-1 protein of Schistosoma japonicum （SjTrx-1）, determine its enzyme activity, analyze the stage-specific transcription of SjTrx-1 gene and observe the distribution of SjTrx-1 protein in adult worm. Methods SjTrx-1 coding sequence identified from S. japonicum cDNA library was amplified by PCR. Subsequently, the obtained target fragment was subcloned into pET28a, and the recombinant plasmid was then transformed into E. coli BL21 （DE3） cells and induced with IPTG for protein expression. The recombinant SjTrx-1 protein was purified with Ni-NTA resin and the enzyme activity was determined by DTT/insulin reduction method. The rabbit antiserum was prepared by immunizing New Zealand white rabbit with purified recombinant SjTrx-1 protein. Indirect immunofluorescence staining with the prepared polyclonal antibody was used to investigate the distribution of SjTrx-1 protein in adult worm. Total RNA was extracted from different stages of S. japonicum and reverse-transcribed into cDNA samples. RT-PCR was used to analyze the SjTrx-1 transcription level of each development stages. Results The expression vector of SjTrx-1/pET28a was established and the recombinant SjTrx-1 protein was expressed as soluble form in E. coli （about Mr 38 000）. The purified SjTrx-1 protein was obtained through Ni-NTA affinity purification and was demonstrated with adequate enzyme activity in vitro. The stage-specific analysis showed that SjTrx-1 was expressed in each of the developmental stages studied, with increased level in sporocysts, schistosomula and adult worms, and relatively low level in miracidia and cercaria. Immunolocalization revealed SjTrx-1 was widely spread in the worm without tissue-specific. Conclusion SjTrx-1 is an abundant and widely distributed protein with increased level in sporocysts, schistosomula and adult worms.