华支睾吸虫硫氧还蛋白过氧化物酶的表达定位及其酶活性的研究

目的克隆和表达有酶活性的华支睾吸虫硫氧还蛋白过氧化物酶(CsTPx),并进行组织定位研究,为进一步研究其功能奠定基础。方法利用生物信息学方法从华支睾吸虫基因组文库中识别硫氧还蛋白过氧化物酶(TPx)基因,并对蛋白的结构特征和相关生物学和免疫学功能进行预测。从华支睾吸虫成虫cDNA中扩增目的基因,克隆到原核表达载体pET-30a(+)中,进行蛋白纯化,免疫印迹鉴定、组织定位及其酶活性的检测。结果华支睾吸虫TPx基因全长2 175 bp,有一大一小的两个内含子,其编码区序列长度为636 bp,编码212个氨基酸;该蛋白第1~17位氨基酸为其信号肽序列,第51~74位氨基酸为跨膜区,属于经典的2-Cys过氧化物还原酶。重组蛋白可被感染了华支睾吸虫的大鼠血清识别,免疫组化结果表明该蛋白定位于成虫的皮层、肠支、卵黄腺、虫卵和囊蚴的体壁中。酶活性检测结果表明,在一定范围内,CsTPx还原H2O2呈剂量和时间依赖性。抗体对酶活性有影响,在合适的抗体水平可以促进酶活性。结论 CsTPx可在大肠杆菌中进行功能性表达,其产物具有良好的免疫学和生物学活性。

Expression,localization and enzymatic activity study on the thioredoxin peroxidase of Clonorchis sinensis

Objective To clone and express recombinant CsTPx for the studies of enzymatic activity and location in the worm, and lay foundation for further functional studies. Methods The structural and functional characteristics of CsTPx were predicted by bioinformatic software.The full-length cDNA of CsTPx was isolated from the adult C.sinensis cDNA library and the open reading frame（ORF） of CsTPx mature peptide was amplified by PCR and cloned into the prokaryotic expression vector pET-30a（＋）. Then the recombinant product was isolated by affinity chromatography and identified by western blot. The recombinant protein was used for preparing antibody for immunolocalization study, and enzyme activity. Results The whole sequence of CsTPx is 2 175 bp, containing a large and a small intron and with an ORF of 636 bp, coding for 212 amino acids.The purified recombinant protein could be recognized by C.sinensis-infected rat serum. The immunofluorescence assay showed that CsTPx was expressed in the tegument, intestine, vitellarium and eggs of adult worm, as well as body wall of metacercaria.The enzyme activity assays showed that the enzyme catalysed the reduction of H2O2 in a time-dependent and in a concentrationdependent fashion. Its antibodies can promote enzymatic activity at an appropriate concentration. Conclusion Recombinant CsTPx could be highly expressed in E.coli and retained its antigenicity and enzyme activity.