ABSTRACT
Latent tuberculosis infection (LTBI) lacks diagnostic gold method. Effective method is important to the control of tuberculosis. IFN-γ responses in 600 military recruits were tested by ELISA using whole blood incubation with latent protein Rv2029c, Rv2659c and recombinant protein CFP10-ESAT6 (rCE) respectively. They also received tuberculin skin test. Their BCG vaccination status was recorded. When 30.7% (184/600) of recruits gave higher IFN-γ responses (≥470pg/mL) to rCE as LTBI, the rests as healthy control, the AUC of rRv2029c was 0.856 and rRv2659c was 0.827 for LTBI diagnosis. IFN-γ responses to rCE were higher in PPD-positive group (≥5mm) than negative group (<5mm) (p<0.05), while for rRv2029c and rRv2659c were not (p>0.05). IFN-γ responses induced by rRv2029c and rRv2659c were higher in the moderately-positive group (≥5, <15mm) than the strongly-positive group (≥15mm) (p<0.05), while for rCE were not (p>0.05). IFN-γ levels to three antigens were not related to BCG vaccination status (p>0.05). Rv2659c and Rv2029c are good candidate antigens to complement the role of rCE for LTBI diagnosis, which provide a basis for developing cost-effective LTBI screening methods in the army.
Subject(s)
Interferon-gamma/blood , Latent Tuberculosis/diagnosis , Peptide Fragments/metabolism , Adolescent , Adult , Antigens, Bacterial/genetics , Antigens, Bacterial/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , China , Enzyme-Linked Immunosorbent Assay , Humans , Male , Mass Screening , Military Personnel , Mycobacterium bovis , Mycobacterium tuberculosis/metabolism , Peptide Fragments/genetics , Recombinant Proteins/genetics , Skin Tests , Young AdultABSTRACT
We constructed a recombinant vaccine of Mycobacterium tuberculosis rBCG-Rv2029c,and then identified it.Rv2029c antigen encoding gene was amplified by PCR.The enzyme digestion products were ligated into rpMV261-2029c recombinant plasmid,after double digestion of Rv2029c and pmv261 vector,and then we introduced the plasmid into BCG to construct rBCG-Rv2029c recombinant vaccine by electroporation method.Finally,we analyzed the expression of the recombinant protein by SDS-PAGE and Western blotting.A total of 1 020 bp Rv2029c gene successfully amplified by PCR was inserted into the plasmid pmv261,then the fusion gene was successfully transduced into BCG.After identified by double enzyme digestion,confirmed by gene alignment and by thermally induced with Western blotting,the recombinant protein had a free primary.The recombinant live vaccine of M.tuberculosis rBCG-Rv2029c is successfully constructed,which lay a foundation for the study of the immune mechanism of recombinant vaccine.
ABSTRACT
Latent tuberculosis infection (LTBI) constitutes the main reservoir for reactivation tuberculosis. The finding of potential biomarkers for differentiating between TB and LTBI is very necessary. In this study, the immunological characteristics and potential diagnostic utility of Rv2029c, Rv2628 and Rv1813c proteins were assessed. These three proteins stimulated PBMCs from ELISPOT-positive LTBI subjects produced higher levels of IFN-γ in comparison with TB patients and ELISPOT-negative healthy subjects (p<0.05). BCG vaccination and non-TB respiratory disease had little influence on the immunological responses of Rv2029c and Rv2628 proteins (p>0.05). The LTBI diagnostic performance of Rv2029c was higher than Rv2628 and Rv1813c by ROC evaluation. But Rv2628 had much higher specificity than Rv2029c in active TB patients and uninfected healthy subjects. The IgG level against Rv1813c was higher in the TB group than in LTBI and uninfected healthy subjects (p<0.05). These results suggest that T cell response to Rv2628 and antibody against Rv1813c might be applicable as biomarkers to distinguish TB from LTBI and uninfected individuals.
