重组蛋白Rv2346c抑制巨噬细胞对结核分枝杆菌的免疫灭活效应

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

目的探讨重组蛋白Rv2346c对卡介苗(BCG)感染鼠巨噬细胞(RAW264.7)后的免疫效应的影响及其机制。方法通过DNA合成、基因扩增、载体构建、诱导表达、纯化等过程制备重组蛋白Rv2346c;利用Cell Counting Kit-8(CCK8)方法检测RAW264.7增殖水平;采用菌落形成试验评估BCG生长情况;运用ELISA方法检测BCG和RAW264.7共培养上清中的肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL)-6的浓度;采取Western blot方法检测RAW264.7细胞中核转录因子κB(NF-κB)p65的表达水平。结果 DNA测序及Western blot检测证实成功制备重组蛋白Rv2346c;BCG可以抑制RAW264.7细胞增殖(P<0.05),而RAW264.7细胞对BCG有灭活作用(P<0.05);重组蛋白Rv2346c可以增强BCG对RAW264.7细胞增殖的抑制作用(P<0.05),并降低RAW264.7对BCG的灭活效应(P<0.05);Rv2346c还可以抑制RAW264.7分泌TNF-α和IL-6(P<0.05),并抑制NF-κB p65的表达(P<0.05)。结论重组蛋白Rv2346c可以抑制小鼠巨噬细胞RAW264.7对BCG的免疫灭活效应,该作用可能与抑制细胞因子分泌和NF-κB p65活化有关,具体机制值得进一步深入探讨。

Objective To investigate the effect of recombinant protein Rv2346 c 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 Rv2346 c. 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 Rv2346 c 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 Rv2346 c 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). Rv2346 c 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 Rv2346 c 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.