猕猴结核IFN-γ体外释放检测法的建立与初步应用  

Establishment and preliminary application of an IFN-γin vitro release assay for detection of macaque tuberculosis

作　　者：汪乾坤 姜勇[1,2] 童胜涛 郝丽影[1,2] 胡长敏[2] 陈颖钰[3] 李翔[3] 任习东 陈焕春[1,2] 郭爱珍[1,2] 

机构地区：[1]华中农业大学农业微生物学国家重点实验室,湖北武汉430070 [2]华中农业大学动物医学院,湖北武汉430070 [3]华中农业大学动物科技学院,湖北武汉430070 [4]湖北天勤猕猴繁育生物科技有限公司,湖北武汉430072

出　　处：《中国兽医学报》2015年第9期1488-1494,共7页Chinese Journal of Veterinary Science

基　　金：国家科技支撑计划资助项目(2013BAD03B01-02);农业科技成果转化资金资助项目(2012GB23600628)

摘　　要：旨在建立猕猴结核IFN-γ体外释放检测法,用于猴结核病的特异性诊断。从猕猴外周血白细胞克隆了猕猴IFN-γ(Macaca mulattaIFN-γ,Mm IFN-γ)基因,在大肠杆菌中表达,获得重组MmIFN-γ。利用杂交瘤细胞技术获得12株高效价的单克隆抗体。用所获得的单克隆抗体及兔抗IFN-γ多克隆抗体建立了检测猴IFN-γ的夹心ELISA,检测灵敏度达到62.5ng/L。用所建立的IFN-γ检测法与进口商品化试剂盒符合率为93.84%,具有很好的一致性(κ=0.779 3)。以眼睑皮内变态反应法(皮试法)为标准方法,先用皮试法检测146只猕猴,再用进口商品化的IFN-γ检测试剂盒(MABTECHTM)检测猕猴外周血淋巴细胞在牛和禽结核菌素刺激下释放的IFN-γ量,利用ROC曲线确定猕猴结核IFN-γ检测法的临界值为114.5ng/L,对皮试法的灵敏性为68.18%,特异性为91.94%。同时,本试验检测的猕猴的结核阳性率为17.12%,提示结核病对猕猴群的健康存在重大威胁。本试验建立了猕猴结核IFN-γ体外释放检测法,为猕猴结核病检测提供了新型技术手段。This study aimed to establish an IFN-γ in vitro release assay for detecting macaque tuberculosis.The gene encoding Macaca mulatta IFN-γ（MmIFN-γ）was cloned from peripheral blood lympbocytes and expressed in E.coli,and thereby the recombinant MmIFN-γ was obtained.By using hybridoma technology 12 monoclonal antibodies with high titers against MmIFN-γ were generated.A sandwich ELISA was established by using one of monoclonal antibodies 3H3 and the rabbit polyclonal antibodies against MmIFN-γ,The sensitivity of thes ELISA was 62.5ng/L.The resalts were compared with those detected by commercial MmIFN-γ detection ELISA kit （MABTECH^TM）and the agreement of these two methods was 93.84% with κ value of 0.7793.Then 146 rbesus macques were tesfed for tuberculosis by using eyelid tuberclin iniradermal skin test （TST）By using TST as the standard method,the commercial MmIFN-γdetection ELISA kit was used to detectIFN-γ in vitro release of the peripheral blood after the whole blood was stimulated with tuberculin of Mycobacterium boris （bPPD） and M. avittm （aPPD） ,and ROC curve determined the cut off value to be 114. 5 ng/L with sensitivity of 68. 18% and specificity of 91.94% in diagnosis of macaques tuberculosis. In addition,this study revealed a tuberculosis prev- alence of 17.12% for the tested macaques. In conclusion,this study established a mIFN-γ in vitro release assay for detecting macaque tuberculosis, and demonstrated tuberculosis might be a great threat to macaques existence.

关 键 词：猕猴 结核 IFN-Γ ELISA 结核菌素皮试检测 单克隆抗体 

分 类 号：S852.618[农业科学—基础兽医学]