日本血吸虫蛋白酶体α2亚基基因的克隆、表达及功能分析

Cloning, expression and characterization of a gene encoding α2 subunit of the proteasome in Schistosoma japonicum

摘要:

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18 d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708 bp，编码235个氨基酸，理论分子量25.84 kDa。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7 d、13 d、18 d、23 d、32 d和42 d虫体中都有表达，7 d和23 d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。

Abstract:

The 26S proteasome is a proteolytic complex responsible for the degradation of the vast majority of eukaryotic proteins. Regulated proteolysis by the proteasome is thought to influence cell cycle progression, transcriptional control, and other critical cellular processes. A novel Schistosoma japonicum gene (GenBank Accession No. AY813725) proteasome α2 subunit (SjPSMA2) was cloned. Sequence analysis revealed that the ORF of SjPSMA2 gene contains 708 nucleotides encoding 235 amino acids, and the molecular weight was estimated to be 25.84 kDa. Real-time PCR analysis showed that this gene expressed in 7 d, 13 d, 18 d, 23 d, 32 d and 42 d schistosoma. The mRNA level of SjPSMA2 was lower in 7 d and 23 d schistosomulum than that in other stages. The SjPSMA2 cDNA fragment was subcloned into an expression vector pET28a(+) and transformed into E. coli BL21 (DE3) cells. After induction with IPTG, the 30 kDa fusion protein was produced as included bodies. Western-blotting revealed that the fusion protein could be recognized by the rabbit serum anti-Schistosoma japonicum adult worm antigen preparation, and the protein in native could be detected. After immunization of BALB/c mice with the fusion protein, the reduction rates of worm counts and liver egg counts were 12.33% and 35.23%. ELISA results revealed that the vaccinated group showed a significant increase in the level of IgG antibody. This study provided an important basis for investigating the regulation mechanism of the proteasome during the development of Schistosoma japonnicum.