结核病诊断性血清学特异抗原的筛选和鉴定  

Screening and identification of serological diagnostic antigens for tuberculosis

作　　者：杨双 郭婧玮 胡一敏 谭云洪 谭笑 袁仕善 YANG Shuang;GUO Jing-wei;HU Yi-min;TAN Yun-hong;TAN Xiao;YUAN Shi-shan(Clinical Laboratory,Hu’nan Provincial People’s Hospital,First Affiliated Hospital of Hunan Normal University,Changsha 410005,China)

机构地区：[1]湖南省人民医院湖南师范大学附属第一医院检验科,长沙410005 [2]湖南省结核病防治所检验科 [3]湖南师范大学医学院分子生物学研究室

出　　处：《中国防痨杂志》2019年第11期1217-1222,共6页Chinese Journal of Antituberculosis

基　　金：湖南省人民医院仁术科研发展基金(20121221)。

摘　　要：目的筛选和鉴定结核病患者血清抗体特异性识别的结核分枝杆菌抗原。方法于改良罗氏培养基中培养结核分枝杆菌标准株H37Rv 4周,收集菌体,通过冰浴超声法提取结核分枝杆菌菌体抗原。收集2013年2—7月湖南省结核病防治所首次入院的30例初治结核病患者的血清,以及收集2013年7月湖南省人民医院检验科接收的20名健康体检者的血清。通过饱和硫酸铵沉淀法提取纳入的20例结核病患者的血清IgG,从结核分枝杆菌菌体抗原中免疫共沉淀血清学抗原,以纳入的其余10例结核病患者血清免疫印迹(Western blot,WB)验证其免疫反应性;从十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)胶上切割阳性反应的蛋白质条带,通过串联质谱技术进行鉴定。结果免疫共沉淀获得1条相对分子质量为16000(16kDa)、能被结核病患者血清抗体特异性识别的蛋白质条带;经串联质谱鉴定,获得结核分枝杆菌热休克蛋白(heat shock protein,Hsp)16.3、糖基转移酶蛋白、铁调控Lsr2蛋白前体等3种蛋白抗原。结论采用免疫共沉淀偶联质谱技术获得3种结核分枝杆菌血清学抗原,可为结核病血清学诊断性抗原的选择进一步提供依据。Objective To screen and identify the antigens of Mycobacteria tuberculosis(MTB)that can be specifically recognized by serum of tuberculosis patients.Methods The standard strain H37Rv of MTB was cultured in L-J medium for four weeks,and then collected to extract MTB antigens using ice bath ultrasound.The serum of 30 newly treated tuberculosis patients admitted to Hunan Provincial Institute of Tuberculosis Prevention and Control for the first time from February to July 2013 was collected.Meanwhile,the serum of 20 health examinees received by the clinical laboratory of Hunan Provincial People’s Hospital in July 2013 was collected.Next,the IgG of 20 tuberculosis patients was extracted by saturated ammonium sulfate precipitation.The serological antigens were screened from the crude antigens of MTB by co-immunoprecipitation and verified in the remaining 10 tuberculosis patients by Western blot.The positive protein band was cut from sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE)gel and identified by the tandem mass spectrometry.Results One protein band in 16 kDa specifically recognized by serum of tuberculosis patients was screened from the crude antigens of MTB by co-immunoprecipitation.Based on the tandem mass spectrometry analysis,3 protein antigens were achieved as heat shock protein 16.3(Hsp16.3),glycosyltransferase and iron-regulated Lsr2 protein precursor.Conclusion Three serological antigens were obtained from the crude antigens of MTB by co-immunoprecipitation and the tandem mass spectrometry,which may provide further evidence for the selection of serological diagnostic antigens for tuberculosis.

关 键 词：分枝杆菌 结核 血清学 抗原 免疫沉淀法 串联质谱法 

分 类 号：R52[医药卫生—内科学]