基于2-DE和蛋白质组技术筛选的日本血吸虫鸟氨酸氨基转移酶的表达及其诊断应用  

The two-dimensional electrophoresis-and proteomics-based screening and expression of ornithine aminotransferase from Schistosoma japonicum(SjOAT) for the diagnosis of schistosomiasis

作　　者：胡梅梅[1] 钟政荣[2] 罗庆礼[1] 方婷娜[1] 沈继龙[1] 

机构地区：[1]人兽共患病安徽省重点试验室,安徽病原生物学省级实验室(安徽医科大学),合肥230032 [2]蚌埠医学院第一附属医院检验科,蚌埠233004

出　　处：《中国人兽共患病学报》2011年第6期484-488,共5页Chinese Journal of Zoonoses

基　　金：国家“863”计划(2006AA02A251);安徽省自然科学基金资助项目(090413089)联合资助

摘　　要：目的扩增并表达日本血吸虫鸟氨酸氨基转移酶(SjOAT),评价其在日本血吸虫病免疫诊断中的应用价值。方法构建pET28a/SjOAT质粒,转化至E.coliBL21,IPTG诱导表达;应用Ni2+亲和层析法纯化OAT;SDS-PAGE和Western blot分析、鉴定表达产物;应用ELISA法检测待检患者血清,比较SjOAT与日本血吸虫可溶性虫卵抗原(SEA)对血吸虫和其它寄生虫感染的检测结果。结果成功克隆表达了SjOAT,Western blot检测结果表明rSjOAT能被血吸虫病患者血清识别;以rSjOAT-ELISA和SjSEA-ELISA分别检测急/慢性血吸虫病患者和其它寄生虫感染者血清的结果经校正χ2检验,差异无统计学意义(P>0.05);对于正常对照者血清和疫区粪检阴性者血清,两种检测方法的特异性比较差异有统计学意义(P<0.05)。结论 rSjOAT在体外获得表达,用于诊断人血吸虫病具有高度敏感性和特异性。In order to clone and express ornithine aminotransferase of Schistosoma japonicum （SjOAT） that screened by proteomic techniques and to access its potential value in the immunodiagnosis of S. japonica, SjOAT was amplified by PCR and ligated to vector pET28a（＋）. After the recombinant plasmid was transformed into E. coli BL21, the expression was induced by IPTG. The fusion protein was purified by Ni^2＋-NTA chromatography and identified by SDS-PAGE. Western blotting confirmed the immunoreactions of SjOAT with the positive sera of schistosomiasis patients. SjOAT and soluble egg antigen （SEA） were further applied in the diagnosis of human Schistosomiasis japonica using ELISA. The positivity rates were 90.4% in rSjOAT-ELISA and 94.2% in SEA-ELISA in 52 cases of acute schistosomiasis. The positive rates in 81 cases of chronic schistosomiasis were 86.4% in rSjOAT-ELISA and 82.7% in SEA ELISA . Statistical analysis revealed no significant difference in sensitivity （P〉0. 05） between rSjOAT and SEA. The false positive reaction rates were found to be 1.1 % in rSjOAT- ELISA and 9.6 % in SEA-ELISA when detected in 94 cases of normal control sera. When rSjOAT-ELISA and SEA-ELISA were subjected to the detection in 94 cases of sera from people in endemic area with negative eggs in the feces, the positive reaction rates were found to be 3.2% and 12.8 %. Statistical analysis revealed significant difference in specificity between rSjOAT and SEA（P〈0.05）. The rSjOAT-ELISA and SEA-ELISA were used to detect the sera of patients with clonorchiasis, paragonimiasis and hookworm infection. Statistical analysis revealed no significant difference in cross reactivity between rSjOAT and SEA（P〉0.05）. The result indicated SjOAT was promising antigen for serological diagnosis of S. japonica instead of crude SEA.

关 键 词：日本血吸虫 鸟氨酸氨基转移酶 ELISA 诊断 

分 类 号：R383.2[医药卫生—医学寄生虫学]