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

doi:10.3877/cma.j.issn.1674-6902.2017.06.005

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。

关键词: 重组蛋白Rv2346c, 巨噬细胞, 结核分枝杆菌

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

图1 重组表达的Rv2346c基因测序结果

图2 SDS-PAGE分析；注：泳道1: BSA (2.00 μg)，泳道2:重组蛋白Rv2346c (1.65 μg)；箭头指示重组蛋白Rv2346c条带

图3 Western Blot分析；注：泳道3:重组蛋白Rv2346c；箭头指示重组蛋白Rv2346c条带

图4 BCG对RAW264.7增殖的影响；注：^*与对照组相比，P<0.05；^#直线两端之间两组比较，P<0.05

图5 BCG和Rv2346c共同作用对RAW264.7增殖的影响；注：^*P<0.05，与相同培养时间下的对照组相比；^#P<0.05，与相同培养时间下的BCG ＋ Rv2346c (50 pg/ml)组相比；＋，P<0.05,与相同培养时间下的BCG＋Rv2346c (100 pg/ml)组相比；§P<0.05，与相同培养时间下的BCG ＋ Rv2346c (500 pg/ml)组相比

图6 Rv2346c影响BCG与巨噬细胞共培养后形成的菌落数量

表1 Rv2346c影响巨噬细胞对BCG的免疫抑制作用

组　别	例数	菌落数量(CFU)
组　别	例数	24 h	48 h	72 h
BCG组	3	134.00±15.52	56.67±10.79^b	39.33±6.43^b
BCG＋Rv2346c组	3	263.00±35.09^a	141.67±19.50^ab	71.67±9.45^abc

表1 Rv2346c影响巨噬细胞对BCG的免疫抑制作用

组　别	例数	菌落数量(CFU)
组　别	例数	24 h	48 h	72 h
BCG组	3	134.00±15.52	56.67±10.79^b	39.33±6.43^b
BCG＋Rv2346c组	3	263.00±35.09^a	141.67±19.50^ab	71.67±9.45^abc

表2 BCG与RAW264.7共培养上清中TNF-α和IL-6水平

组　别	例数	TNF-α	IL-6
对照组	3	76.42±3.49	75.61±3.27
BCG(24 h)组	3	208.27±24.98^a	248.10±6.10^a
BCG＋Rv2346c(24 h)组	3	94.35±10.73^b	155.72±6.77^ab
BCG(48 h)组	3	272.42±6.34^ac	317.64±7.95^ac
BCG＋Rv2346c(48 h)组	3	135.76±5.35^abc	220.24±6.14^abc
BCG(72 h)组	3	372.87±9.51^acd	362.16±9.75^acd
BCG＋Rv2346c(72 h)组	3	170.20±7.20^abcd	249.96±11.12^abcd

表2 BCG与RAW264.7共培养上清中TNF-α和IL-6水平

组　别	例数	TNF-α	IL-6
对照组	3	76.42±3.49	75.61±3.27
BCG(24 h)组	3	208.27±24.98^a	248.10±6.10^a
BCG＋Rv2346c(24 h)组	3	94.35±10.73^b	155.72±6.77^ab
BCG(48 h)组	3	272.42±6.34^ac	317.64±7.95^ac
BCG＋Rv2346c(48 h)组	3	135.76±5.35^abc	220.24±6.14^abc
BCG(72 h)组	3	372.87±9.51^acd	362.16±9.75^acd
BCG＋Rv2346c(72 h)组	3	170.20±7.20^abcd	249.96±11.12^abcd

图7 RAW264.7中NF-κB p65的表达

表3 BCG与RAW264.7共培养后细胞内NF-κB蛋白表达水平

组　别	例数	NF-κB p65
对照组	3	1
BCG(24 h)组	3	0.929±0.056
BCG＋Rv2346c(24 h)组	3	0.727±0.084^ab
BCG(48 h)组	3	0.556±0.071^ac
BCG＋Rv2346c(48 h)组	3	0.517±0.049^ac
BCG(72 h)组	3	0.441±0.051^ac
BCG＋Rv2346c(72 h)组	3	0.278±0.073^abcd

表3 BCG与RAW264.7共培养后细胞内NF-κB蛋白表达水平

组　别	例数	NF-κB p65
对照组	3	1
BCG(24 h)组	3	0.929±0.056
BCG＋Rv2346c(24 h)组	3	0.727±0.084^ab
BCG(48 h)组	3	0.556±0.071^ac
BCG＋Rv2346c(48 h)组	3	0.517±0.049^ac
BCG(72 h)组	3	0.441±0.051^ac
BCG＋Rv2346c(72 h)组	3	0.278±0.073^abcd

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

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