title: Cloning and characterization of Clp protease proteolytic subunit 2 and its implication in clinical diagnosis of tuberculosis.
abstract: OBJECTIVE
  To clone, express, and characterize Mycobacterium tuberculosis (Mtb) ClpP2, and evaluated the potential usage of ClpP2 in clinical diagnosis of tuberculosis.
  
  
  METHODS
  Mtb ClpP2 was cloned into recombinant plasmid pET32a (+) and transformed into E. coli BL21 (DE3). SDS-PAGE and Western blot analysis were performed to detect the expression of the recombinant protein. The immunogenicity of Mtb ClpP2 was assessed with epitope prediction and antibody titer assay. Quantitative real-time PCR was performed to detect the influence of stress conditions on ClpP2 expression. ClpP2 antigen and antibody in patients with pulmonary diseases were detected by indirect ELISA. ROC curve was constructed to assess the diagnostic accuracy of Mtb ClpP2 for tuberculosis.
  
  
  RESULTS
  We had cloned and expressed recombinant Mtb ClpP2 in E. coli. Our results showed that Mtb ClpP2 had potent immunogenicity, and our own prepared polyclonal antibody could be used in detection and diagnostic tests. Results from Western blot showed that ClpP2 was mainly located in M. bovis BCG cytoplasm, and real-time PCR indicated that stress conditions could enhance the mRNA expression of ClpP2. Indirect ELISA suggested that, in tuberculosis patients, both the levels of ClpP2 antigen and antibody were increased, and the positive rates of ClpP2 were elevated. ROC curve had demonstrated satisfactory sensitivity and specificity of ClpP2-based diagnosis for tuberculosis.
  
  
  CONCLUSIONS
  Our results suggest that Mtb ClpP2 antigens would be used as a biomarker in tuberculosis pathogenesis. These findings highlight the feasibility of the application of Mtb ClpP2 in the clinical diagnosis of tuberculosis.
authors.affiliation: Department of Respiratory Disease, The First Affiliated Hospital of Chongqing Medical University Chongqing 400016, China ; Department of Pathology, Chongqing Medical University Chongqing 400016, China.
authors.forename: Dairong
authors.initials: D
authors.lastname: Li
authors.affiliation: Department of Pathogenic Organisms, Chongqing Medical University Chongqing 400016, China ; Molecular Medicine and Cancer Research Center, Chongqing Medical University Chongqing 400016, China.
authors.forename: Chunyan
authors.initials: C
authors.lastname: Zhang
authors.affiliation: Molecular Medicine and Cancer Research Center, Chongqing Medical University Chongqing 400016, China.
authors.forename: Nan
authors.initials: N
authors.lastname: Lu
authors.affiliation: Department of Pathogenic Organisms, Chongqing Medical University Chongqing 400016, China ; Molecular Medicine and Cancer Research Center, Chongqing Medical University Chongqing 400016, China.
authors.forename: Liuqing
authors.initials: L
authors.lastname: Mu
authors.affiliation: Department of Pathogenic Organisms, Chongqing Medical University Chongqing 400016, China ; Molecular Medicine and Cancer Research Center, Chongqing Medical University Chongqing 400016, China.
authors.forename: Yonglin
authors.initials: Y
authors.lastname: He
authors.affiliation: Department of Pathogenic Organisms, Chongqing Medical University Chongqing 400016, China ; Molecular Medicine and Cancer Research Center, Chongqing Medical University Chongqing 400016, China.
authors.forename: Lei
authors.initials: L
authors.lastname: Xu
authors.affiliation: Department of Pathogenic Organisms, Chongqing Medical University Chongqing 400016, China ; Molecular Medicine and Cancer Research Center, Chongqing Medical University Chongqing 400016, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Yang
authors.affiliation: Department of Pathogenic Organisms, Chongqing Medical University Chongqing 400016, China ; Molecular Medicine and Cancer Research Center, Chongqing Medical University Chongqing 400016, China.
authors.forename: Yu
authors.initials: Y
authors.lastname: Fan
authors.affiliation: Department of Pathogenic Organisms, Chongqing Medical University Chongqing 400016, China ; Molecular Medicine and Cancer Research Center, Chongqing Medical University Chongqing 400016, China.
authors.forename: Yuexi
authors.initials: Y
authors.lastname: Kang
authors.affiliation: Department of Pathogenic Organisms, Chongqing Medical University Chongqing 400016, China ; Molecular Medicine and Cancer Research Center, Chongqing Medical University Chongqing 400016, China.
authors.forename: Chun
authors.initials: C
authors.lastname: Yang
mesh_terms: D000818:Animals; D000907:Antibodies, Bacterial / Q000097:blood; D019540:Area Under Curve; D001426:Bacterial Proteins / Q000097:blood / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D015415:Biomarkers / Q000097:blood; D015153:Blotting, Western; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping; D000939:Epitopes; D006801:Humans; D008297:Male; D009163:Mycobacterium bovis / Q000201:enzymology* / Q000235:genetics / Q000276:immunology; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics / Q000276:immunology; D011237:Predictive Value of Tests; D012372:ROC Curve; D011817:Rabbits; D060888:Real-Time Polymerase Chain Reaction; D012697:Serine Endopeptidases / Q000097:blood / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D014397:Tuberculosis, Pulmonary / Q000097:blood / Q000175:diagnosis* / Q000382:microbiology*
chemical_list: D000907:Antibodies, Bacterial; D001426:Bacterial Proteins; D015415:Biomarkers; D000939:Epitopes; C119658:ClpP2 protein, bacteria; D012697:Serine Endopeptidases
keywords: Clp protease proteolytic subunit 2 (ClpP2); Mycobacterium tuberculosis (Mtb); serological diagnosis; tuberculosis