Cell death of THP-1 induced by puried Rv3671c protein of tuberculosis and the detection of TNF-α and IL-1β in Mycobacterium tuberculosis.
- Author:
Shou-gang KUAI
1
;
Hao PEI
;
Li-hua HUANG
;
Zhong-hua LIU
;
Guang-liang MAI
;
Jun LIU
;
Zhen-ling CUI
Author Information
- Publication Type:Journal Article
- MeSH: Bacterial Proteins; pharmacology; Cell Death; Cell Line; Humans; Interleukin-1beta; metabolism; Macrophages; cytology; metabolism; Mycobacterium tuberculosis; genetics; Tumor Necrosis Factor-alpha; metabolism
- From: Chinese Journal of Preventive Medicine 2013;47(5):444-447
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo assess the response in THP-1 treated with Rv3671c protein in Mycobacterium tuberculosis (M.tuberculosis).
METHODSThe 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-1β were detected by ELISA at each stimulating time.
RESULTSThe 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-1β 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.
CONCLUSIONThe 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-1β levels.