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目的 寻找对华支睾吸虫病诊断有效的抗原基因,以便生物合成并研究其应用价值。方法 选取两个Genbank已报告的基因序列AF271091(CysA)及AF093242(CysB)分别克隆与表达融合蛋白,亲和层析纯化后进行免疫定性。结果 CysA与CysB均获得成功克隆与表达,Western blot分析显示CysA与华支睾吸虫病阳性血清有强反应,与日本血吸虫病阳性血清只有很弱的交叉反应,而CysA与华支睾吸虫感染血清无反应。免疫组化显示,被表达的两个半胱氨酸蛋白酶在华支睾吸虫体内的免疫定位无显著不同,主要在成虫肠道及虫卵。结论 成功表达的两个半胱氨酸蛋白酶中,CysB针对华支睾吸虫病血清有良好的抗原活性,值得视为诊断抗原候选做进一步的研究。
Objective To search for the diagnosis of Clonorchis sinensis diagnosis of effective antigen genes for biosynthesis and study its value. Methods Two genomic sequences AF271091 (CysA) and AF093242 (CysB) were cloned and expressed respectively in Genbank and purified by affinity chromatography. Results Both CysA and CysA were successfully cloned and expressed. Western blot analysis showed that CysA reacted strongly with Clonorchiasis positive sera and had only a weak cross-reaction with the positive serum of Schistosoma japonicum, while CysA and Clonorchis sinensis infected sera No reaction. Immunohistochemistry showed that there was no significant difference in the immunolocalization between the expressed two cysteine proteases in the adult Clonorchis sinensis, mainly in the intestine and eggs. Conclusion Among the two cysteine proteases successfully expressed, CysB has good antigenic activity against clonorchiasis serum and should be considered as a candidate antigen for further investigation.