华支睾吸虫膜抗原/排泄分泌抗原酸性磷酸酶的克隆、表达、生物学特征分析及组织定位

 目的:对华支睾吸虫(Clonorchis sinensis, Cs)成虫酸性磷酸酶 (acid phosphatase, AP)进行克隆、表达、生物学特征分析、组织定位及膜抗原/排泄分泌抗原鉴定。方法:对CsAP进行生物信息学、分子生物学、免疫组化及明胶酶谱分析。结果:从Cs cDNA文库中筛选出编码AP新基因，全长1 410 bp，重组并由大肠杆菌表达、纯化，得到分子量为55 kD的重组蛋白CsAP。Western blotting分析表明，CsAP既是膜抗原又是分泌排泄抗原；免疫组化显示，CsAP荧光显示于成虫的表皮层和肠支，在囊蚴也有显示，在雷蚴和尾蚴未显示荧光；ELISA分析表明CsAP识别华支睾吸虫病人和日本血吸虫病人存在吸虫间的交叉免疫反应，CsAP及粗抗原识别轻、中、重度感染程度华支睾吸虫病人的差别不明显。重组蛋白免疫大鼠后，总IgG抗体滴度于3周达较高峰，抗体效价大于1∶25 600。明胶降解实验表明：CsAP具降解胶原能力。结论: 上述结果表明，CsAP在大肠杆菌中高效表达，具有较好的免疫原性，但血清诊断价值不理想；CsAP可能既是膜抗原，又是排泄分泌抗原。

Cloning,expression, biological characteristics and tissue localization of a Clonorchis sinensismembrane antigen/excretory-secretory antigen,acid phosphatase

AIM:To clone, express and determine the biological characteristics and tissue localization of Clonorchis sinensis(C. sinensis, Cs) acid phosphatase (AP), and to identify CsAP as C. sinensismembrane antigen/excretory-secretory antigen. METHODS:The methods of bioinformatics, molecular biology, immunohistochemistry and gelatin zymography were used to analyze CsAP. RESULTS:From cDNA library, a full-length 1 410 bp cDNA clone of Cs encoding a novel AP was selected, recombinated, expressed and purified in Escherichia coli. The molecular weight of the CsAP recombinant was 55 kD. CsAP was a membrane antigen as well as an excretory-secretory antigen. The fluorescence of CsAP was located in metacercaria and intestinal cecum and tegument of adult, but not detected in redia or cercaria. The cross reactions between Clonorchis sinensis and Schistosoma japonicum in the recognition of their infective sera by CsAP was observed. The recognition of CsAP with C. sinensiscrude antigens was not different among the mild, midrange or grave C. sinensisinfection. After immuned by CsAP recombinant, the total IgG antibody titer of the rats went up to the peak value of over 1∶25 600 in the 3rd week. CsAP had the ability to degrade collagen. CONCLUSION: CsAP is highly expressed in Escherichia coli, showing its immunogenicity, but not a good diagnostic marker, and might be an excretory-secretory protein as well as a membrane protein.