Recombinant protein Rv2346c inhibits the immunological response of macrophage against Mycobacterium tuberculosis

Abstract

Objective To investigate the effect of recombinant protein Rv2346c on murine macrophage-induced immunological response on Bacillus Calmette-Guerin (BCG) and the molecular mechanism related. Methods DNA synthesis, gene amplification, vector construction, induced expression and protein purification were used to synthesize recombinant protein Rv2346c. Cell Counting Kit-8 (CCK8) kit was applied to tested the proliferation of RAW264.7. Colony formation unit was observed to estimate the growth of BCG. Enzyme-linked immuno sorbent assay (ELISA) was utilized to detect tumor necrosis factor-α (TNF-α) and interleukin (IL)-6 in co-culture supernatant. Western blot was conducted to measure the expression of NF-κB (nuclear transcription factor-kappa B) p65. T test was applied to compare the means of two independent groups and P<0.05 was considered statistically significant. Results Recombinant protein Rv2346c was verified by DNA sequencing and Western blot. BCG inhibited the proliferation of RAW264.7 (P<0.05 ) while RAW264.7 inactivated BCG (P<0.05 ). Recombinant protein Rv2346c enhanced the BCG-induced inhibition on the proliferation of RAW264.7 (P<0.05 ) and reduced RAW264.7-medicated immunological killing effect against BGG (P<0.05 ). Rv2346c also suppressed the secretion of TNF-α and IL-6 by RAW264.7 (P<0.05 )and accelerated the protein expression of NF-κB p65 (P<0.05 ). Conclusion Recombinant protein Rv2346c could reduce macrophage-medicated immunological killing effect on BCG, which could be associated with the reduced secretion of cytokines and the suppression of NF-κB p65 expression. The exact mechanisms remain to be further explored. Key words: Recombinant protein Rv2346c; Macrophage; Mycobacterium tuberculosis