日本血吸虫磷酸甘油酸变位酶SjPGAM基因的克隆、表达及功能分析

Cloning, Expressing and Characterizing of a Phosphoglycerate mutas Gene of Schistosoma japonncum

摘要:

磷酸甘油酸变位酶(Phosphoglycerate mutas)是糖酵解过程中的一种重要酶, 主要催化3-磷酸甘油酸转化为2-磷酸甘油酸。利用PCR技术从日本血吸虫19 d童虫中首次扩增到一个PGAM家族基因, 序列分析表明该基因的完整编码框含753 bp, 编码250个氨基酸, 理论分子量28.26 kD, 理论等电点7.01。同源性分析结果表明, 该基因的氨基酸序列具有典型PGAM家族特征, 推测为血吸虫的PGAM基因, 命名为SjPGAM(GenBank Accession No. EU374631)。实时定量PCR分析显示该基因在14 d和19 d童虫中的表达量明显高于其他发育阶段, 42 d雄虫中的表达量高于雌虫。构建了该基因的原核重组表达质粒pET-28a(+)-PGAM, 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。SjPGAM基因及其表达产物的获得, 为探索PGAM基因家族在血吸虫能量代谢过程中碳水化合物转运、新陈代谢调节和生长发育提供了重要基础。

Abstract:

Phosphoglycerate mutase (PGAM) is a key enzyme in glycolytic pathways. With PCR technique based on an EST identified in our lab, a novel gene named SjPGAM (GenBank Accession No. EU374631) was cloned. Sequence analysis revealed that the ORF of SjPGAM gene contained 753 nucleotides, encoding 250 amino acids, and the molecular weight was about 28.26 kD. Real-time PCR analysis showed that the mRNA level of SjPGAM was much higher in the 14 days and 19 days schistosomula than other stages, suggesting that the gene was a schistosomula stage differential expression gene. The SjPGAM cDNA fragment was subcloned into an expression vector pET-28a (+) and transformed into Escherichia coli BL21 cells. In the presence of IPTG, the 31 kD fusion protein was expressed in included bodies. Western blotting revealed that the fusion protein could be recognized by the rabbit serum anti-Schistosoma japonicum adult worm antigen preparation. The study provides important basis for investigating the mechanism of the PGAM in the glycolytic pathways of Schistosoma japonnicum.