华支睾吸虫半胱氨酸蛋白酶的基因表达与在ELISA诊断上的应用研究

目的探讨重组华支睾吸虫半胱氨酸蛋白酶在华支睾吸虫病血清学诊断上的应用价值. 方法以成虫cDNA为模板PCR扩增GenBank中一个华支睾吸虫半胱氨酸蛋白酶(No. AF093242,简称CysB)的部分基因片段,亚克隆到pTrcHis表达载体,转化入大肠埃希菌DH5α.表达出的重组蛋白经亲合层析纯化后用于ELISA检测华支睾吸虫及其他寄生虫感染血清,评价其诊断应用价值. 结果成功克隆与表达CysB基因片段,纯化后的重组蛋白用于华支睾吸虫病诊断的敏感性达96.0%(48/50),与其他寄生虫病的总交叉反应为3.8%(1/26),而对比实验中可溶性粗抗原(CAA)用于检测的敏感性为88.0%(44/50),与其他寄生虫感染血清无交叉反应. 结论重组华支睾吸虫半胱氨酸蛋白酶(CysB)用于ELISA诊断技术具有较高的敏感性及特异性,有希望成为可溶性粗抗原的替代之一.

MOLECULAR EXPRESSION OF A CLONORCHIS SINENSIS CYSTEINE PROTEINASE AND ITS APPLICATION IN ELISA FOR CLONORCHIASIS DIAGNOSIS

Objective To evaluate the value of a recombinant Clonorchis sinensis cysteine proteinase for the serological diagnosis of clonorchiasis. Methods Based on the sequence (GenBank, No. AF093242), a cysteine proteinase (CysB) gene fragment of C. sinensis was cloned into pTrcHis expression vector and transformed into an Escherichia coli DH5α strain. The recombinant proteins expressed in the bacteria were purified by affinity chromatography and then probed by enzymed-linked immunosorbent assay (ELISA) with sera from patients with clonorchiasis and other parasitic infections. Results For the tested sera, ELISA showed that CysB, judged by a calculated cut-off value, had a sensitivity of 96.0% (48/50) for the diagnosis of clonorchiasis and a cross-reactivity of about 3.8% (1/26) with other parasitic diseases, while C. sinensis adult worm antigen (CAA) had a sensitivity of 88.0% and no cross-reaction was found. Conclusion The results suggested that the recombinant C. sinensis CysB, although having weak cross-reaction with some cases of other parasitic diseases, was valuable for the diagnosis of clonorchiasis and was promising to be developed as an alternative diagnostic antigen.