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26S蛋白酶体是一种能够降解大多数内源性蛋白的多亚基复合物,它的蛋白降解作用能够影响细胞周期、转录控制和其他一些重要的细胞进程。本实验利用PCR技术从日本血吸虫18d童虫中首次扩增到蛋白酶体α2亚基基因(GenBank Accession No.AY813725),序列分析表明该基因的开放阅读框(ORF)含708bp,编码235个氨基酸,理论分子量25.84kDa。同源性分析结果显示,该基因为日本血吸虫蛋白酶体α2亚基,命名为SjPSMA2。实时定量PCR分析显示该基因在7d、13d、18d、23d、32d和42d虫体中都有表达,7d和23d虫体表达量低于其他几个时期。构建了该基因的原核表达质粒pET28a(+)-SjPSMA2,在大肠杆菌系统中成功获得了表达,重组蛋白以包涵体形式存在,Western blotting显示表达产物能被日本血吸虫成虫粗抗原免疫血清所识别,并且能检测到天然状态下该蛋白的存在。应用重组蛋白免疫BALB/c小鼠后,诱导产生了较高的特异性抗体水平及12.33%的减虫率和35.23%的肝脏减卵率。SjPSMA2基因及其表达产物的获得,为探索蛋白酶体在血吸虫生长发育中的作用提供了重要基础。
The 26S proteasome is a multisubunit complex that degrade most endogenous proteins. Its proteolysis can affect the cell cycle, transcriptional control, and other important cellular processes. In this study, GenBank Accession No.AY813725 was amplified from the 18D Schistosoma japonicum Schistosoma japonicum for the first time by PCR. Sequence analysis showed that the open reading frame (ORF) of the gene contained 708bp and encoded 235 amino acids, The theoretical molecular weight of 25.84kDa. Homology analysis showed that the gene was named as SjPSMA2, a proteasome α2 subunit of Schistosoma japonicum. Real-time quantitative PCR analysis showed that the gene was expressed in the worms at 7d, 13d, 18d, 23d, 32d and 42d, and the expression levels at 7d and 23d were lower than those at other stages. The prokaryotic expression plasmid pET28a (+) - SjPSMA2 was successfully constructed and expressed in E.coli. The recombinant protein was expressed in inclusion bodies. Western blotting showed that the expressed product was recognized by the crude antigen of Schistosoma japonicum, And can detect the presence of this protein in its native state. Immunization of BALB / c mice with the recombinant protein induced a higher specific antibody level and a reduction rate of 12.33% and a reduction rate of 35.23% of the liver. The acquisition of SjPSMA2 gene and its expression products provide an important basis for exploring the role of proteasome in the growth and development of Schistosoma japonicum.