关键词: 分枝杆菌, 结核, 血清学, 抗原, 免疫沉淀法, 串联质谱法

Abstract:

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.

Key words: Mycobacterium tuberculosis, Serology, Antigens, Immunoprecipitation, Tandem mass spectrometry