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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。
Objective To clone and express the enzyme-activated clonorchis sinensis thioredoxin peroxidase (CsTPx), and to study the tissue localization, which will lay the foundation for the further study of its function. Methods The gene of thioredoxin peroxidase (TPx) was identified from the genomic library of Clonorchis sinensis using bioinformatics methods, and its structural characteristics and related biological and immunological functions were predicted. The target gene was amplified from the cDNA of adult Clonorchis sinensis and cloned into the prokaryotic expression vector pET-30a (+) for protein purification, immunoblotting, tissue localization and enzymatic activity detection. Results The full-length TPx gene of Clonorchis sinensis was 2 175 bp in length with two small and one intron. The coding sequence of TPx gene was 636 bp in length and encoded 212 amino acids. The amino acids 1 to 17 of this gene were its signal peptide Sequence, amino acids 51-74 is a transmembrane region, belongs to the classic 2-Cys peroxidase reductase. The recombinant protein can be identified by the rat serum infected with Clonorchis sinensis, and immunohistochemistry results show that the protein is localized in the body wall of adult cortex, gut, yolk gland, egg and metacercariae. The results of enzyme activity test showed that CsTPx reduced H2O2 in a dose and time dependent manner. Antibodies have an effect on enzyme activity and promote enzyme activity at the appropriate antibody level. Conclusion CsTPx can be expressed in E. coli and its products have good immunological and biological activity.