弓形虫多表位基因重组抗原在弓形虫免疫检测中的应用  

Application of the Recombinant Antigen of MAG in Immunodiagnosis of Toxoplasmosis

作　　者：杨培梁[1] 李华[2] 周晓红[2] 彭鸿娟[2] 吴焜[2] 陈晓光[2] 

机构地区：[1]南方医科大学南方医院实验动物研究中心,广州510515 [2]南方医科大学公共卫生与热带医学学院病原生物学系,广州510515

出　　处：《热带医学杂志》2007年第9期853-855,共3页Journal of Tropical Medicine

基　　金：国家自然科学基金(No.30671837);广东省自然科学基金(No.20020041)

摘　　要：目的评价弓形虫多表位基因重组抗原在弓形虫感染诊断中的效果,探索多表位抗原在弓形虫免疫检测中的应用前景。方法以Ni-NTA Agarose和割胶电渗两步纯化法,获取弓形虫多表位基因的原核表达纯化产物rMAG,以适宜浓度的rMAG包被ELISA微孔板,分别检测弓形虫急性和慢性感染血清,评价试剂盒检测的敏感性和特异性。结果经Ni-NTAAgarose和割胶纯化,得到了纯度为95.86%的弓形虫多表位重组可溶性抗原。以3!g/ml的抗原浓度包被ELISA微孔板,对兔和小鼠的急慢性弓形虫感染血清进行了检测。结果检测小鼠血清141份,其中慢性感染弓形虫小鼠血清117份、正常小鼠血清24份,检测体系的敏感性为88.88%,特异性为91.67%,一致性为89.36%。检测兔血清24份,其中急性感染弓形虫兔血清18份,正常兔血清6份,阳性血清检出率为94.4%,总一致性为91.5%。结论以弓形虫多表位重组抗原构建的ELISA试剂盒既可以检测弓形虫急性感染血清,又可以检测弓形虫慢性感染血清,具有一定的应用前景。Objective To evaluate the potential application of the recombinant multiple epitope antigen in diagnosis of toxoplasmosis. Methods Purifed recombinant MAG （rMAG） was collected by both immobilized metal affinity chromatography （Ni-NTA Agarose） and electroelution from SDS-polyacrylamide gels. rMAG was used to coat the 96-well plates for the screening of mice antisera against chronic infection of T.gondii and rabbits antisera against acute infection of T.gondii by ELISA. Results rMAG was prepared with a purity of 95.86%. 3 μg/ml of purifed rMAG was used to coat the 96-well plates. A number of 117 sera from 17 mice infected by T.gondii B36 strain and 24 from normal ones were detected by rMAG-ELISA kit. The results showed that the sensitivity and specificity of the kit for detecting chronic infection of T.gondii were 88.88% （104/117）and 91.67% respectively,with total consistency of 89.36%. A number of 24 rabbit sera, in which 18 were derived from rabbits infected with RH T.gondii and the other 6 from normal rabbits, were also detected by rMAG-ELISA kit. 94.4% （17/18） of the positive sera were detected by the kit, with total consistency of 91.5% [ （17＋5）/24]. Conclusion Purified rMAG can be used as good coating antigen to construct ELISA kit for diagnosis of both acute and chronic infection of T.gondii.

关 键 词：弓形虫 多表位基因 重组抗原 免疫诊断 ELISA试剂盒 

分 类 号：R382.5[医药卫生—医学寄生虫学]