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  

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

作　　者：郑明慧[1,2] 胡坤华[3] 张彤[4] 刘炜[4] 余新炳[2] 

机构地区：[1]中山大学孙逸仙纪念医院检验科,广东广州510120 [2]中山大学中山医学院热带病防治重点实验室病原生物学教研室,广东广州510080 [3]中山大学中山医学院药理学教研室,广东广州510080 [4]中山大学第三附属医院广东省肝病重点实验室,广东广州510630

出　　处：《中国病理生理杂志》2014年第11期2059-2065,共7页Chinese Journal of Pathophysiology

基　　金：国家自然科学青年科学基金资助项目(No.81401689);广东省医学科研基金资助项目(No.B2014137);中央高校基本科研业务费专项资金资助(No.14ykpy23)

摘　　要：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Cs AP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 c DNA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 k D的重组蛋白Cs AP。Western blotting分析表明,Cs AP既是膜抗原又是分泌排泄抗原;免疫组化显示,Cs AP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Cs AP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,Cs AP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Ig G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:Cs AP具降解胶原能力。结论:上述结果表明,Cs AP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;Cs AP可能既是膜抗原,又是排泄分泌抗原。AIM： To clone,express and determine the biological characteristics and tissue localization of Clonorchis sinensis（ C. sinensis,Cs） acid phosphatase（ AP）,and to identify Cs AP as C. sinensis membrane antigen / excretory-secretory antigen. METHODS： The methods of bioinformatics,molecular biology,immunohistochemistry and gelatin zymography were used to analyze Cs AP. RESULTS： From c DNA library,a full-length 1 410 bp c DNA clone of Cs encoding a novel AP was selected,recombinated,expressed and purified in Escherichia coli. The molecular weight of the Cs AP recombinant was 55 k D. Cs AP was a membrane antigen as well as an excretory-secretory antigen. The fluorescence of Cs AP 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 Cs AP was observed. The recognition of Cs AP with C. sinensis crude antigens was not different among the mild,midrange or grave C.sinensis infection. After immuned by Cs AP recombinant,the total Ig G antibody titer of the rats went up to the peak value of over 1∶ 25 600 in the 3rd week. Cs AP had the ability to degrade collagen. CONCLUSION： Cs AP 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.

关 键 词：华支睾吸虫 酸性磷酸酶 排泄分泌抗原 膜抗原 

分 类 号：R383.22[医药卫生—医学寄生虫学]