[Cell death of THP-1 induced by puried Rv3671c protein of tuberculosis and the detection of TNF-α and IL-1ß in Mycobacterium tuberculosis].

ABSTRACT

OBJECTIVE: To assess the response in THP-1 treated with Rv3671c protein in Mycobacterium tuberculosis (M.tuberculosis). METHODS: The gene encoding Rv3671c protein of M.tuberculosis was cloned into pET-28a vector and then expressed in Escherichia coli. The Rv3671c was purified with Ni-NTA affinity and ion exchange chromatography. The detection of protein concentration was by Lowry method.THP-1 cell was stimulated with Rv3671c protein and cells were analyzed by Hochest staining under fluorescence microscopy to assay cell death (apoptosis and necrosis). TNF-α and IL-1ß were detected by ELISA at each stimulating time. RESULTS: The Rv3671c protein of M.tuberculosis was successfully expressed in Escherichia coli. The purity of recombinant Rv3671c protein was 95%, and the protein concentration was up to 0.4 mg/ml. The nucleus of THP-1 was isolated and necrosis-like under fluorescence when cells were stimulated by Rv3671c protein. The levels of TNF-α and IL-1ß in supernatant were 19 000 and 16 500 pg/ml respectively, and were significantly higher than control cells with the levels of 2100 and 3800 pg/ml separately. CONCLUSION: The necrosis of THP-1 cells could be stimulated by Rv3671c protein of M.tuberculosis and it was probably associated with high cytokines TNF-α and IL-1ß levels.