华支睾吸虫半胱氨酸蛋白酶基因克隆表达和血清学诊断效果

Cloning, expression and evaluation on effect in serological diagnosis of cysteine protease of Clonorchis sinensis

目的克隆、表达华支睾吸虫半胱氨酸蛋白酶(CSCP),并评价其免疫学诊断效果。方法根据已知的CSCP(Gen Bank序列号:AF093242)基因序列设计引物,从华支睾吸虫成虫总cDNA中扩增该目的片段,克隆入真核表达载体pPIC9K,转化至巴斯德毕赤酵母(Pichia pastoris)GS115中,甲醇诱导表达、纯化;采用Westernblot技术鉴定该重组蛋白;用纯化的重组CSCP(rCSCP)免疫小鼠,ELISA法检测抗体效价;应用DotELISA法和Western blot法评估该重组蛋白的诊断效果。结果从华支睾吸虫成虫cDNA中扩增获得长度为927bp的CSCP基因,并成功进行了真核表达、纯化,纯化的rCSCP免疫小鼠后的血清能被华支睾吸虫成虫可溶性抗原识别,其血清抗体效价达1∶64000;采用该重组蛋白建立的DotELISA法检测华支睾吸虫病人的敏感性为91.7%(55/60),检测正常人的特异性为97.6%(82/84);Western blot分析显示,rCSCP与日本血吸虫病人血清无交叉反应,与卫氏并殖吸虫病人交叉反应率为20.0%(2/10)。结论 rCSCP具有较好的诊断效果,是华支睾吸虫重组蛋白中一个有潜在诊断价值的抗原。

Objective To clone and express the cysteine protease of Clonorchis sinensis and evaluate its effect on immunodiagnosis of human clonorchiasis. Methods Based on a cysteine protease gene fragment of C. sinensis（CS-CP, GenBank accession： AF093242）, a pair of primers were designed and amplified from total cDNA of C. sinensis, and the gene was cloned into plasmid pPIC9K and expressed in Pichia pastoris GS115. The expressed product was purified. Ten BALA/c mice were immunized with the purified CS-CP, and the anti CS-CP antibody in the sera of immunized mice was tested with ELISA. Finally, its effect on serodiagnosis was evaluated with Dot-ELISA and Western blot. Results The CS-CP gene （approximately 927 bp） was successfully amplified, cloned and expressed in Pichia pastoris GS115. The sera of mice immunized with purified rCS-CP could be recognized by the soluble antigen of C. sinensis adult worms and its special anti CS-CP antibody titer was high （1[∶]64 000）. The rCS-CP was probed by Dot-ELISA and Western-blot with sera from patients with clonorchiasis and other parasitic infections, and it had a sensitivity of 91.7% （55/60） in diagnosis of clonorchiasis and a specifity of 97.6% （82/84） for healthy population. The rCS-CP had no cross-reaction with the patients of schistosomiasis japonica, but had a cross-reactivity of 20.0% （2/10） with the patients with paragonimiasis westermani. Conclusions rCS-CP possesses a favorable diagnostic effect and it is a better serodiagnostic one among recombinant antigens.