华支睾吸虫高迁移率族蛋白1同源蛋白的表达及其对小鼠巨噬细胞核转录因子-κB活化作用

Expression of the high mobility group box 1 homologous protein of Clonorchis sinensis and its effect on nuclear transcription factor-κB activation in mouse macrophages

根据华支睾吸虫死亡细胞释放的高迁移率族蛋白1(CsHMGB1)同源分子编码序列构建pET-28a(+)表达质粒并转化至Rosetta(DE3)感受态细胞,异丙基-β-D-硫代半乳糖苷(IPTG)诱导CsHMGB1-His融合蛋白表达;Ni-TED琼脂糖树脂纯化蛋白后,蛋白质免疫印迹(Westernblotting)分析鉴定His标签融合蛋白表达情况。用含不同浓度(1μg/ml和10μg/ml)CsHMGB1-159/127的培养液与小鼠巨噬细胞RAW264.7共培养,同时设立无刺激物的空白对照组,培养24h和48h。用pNiFty2-SEAP质粒和HEK-Blue^(TM)培养基检测SEAP活性代表核转录因子-κB(NF-κB)活化,用酶标仪检测培养上清吸光度(A_(620)值),并在显微镜下观察细胞内蓝色显色反应。结果显示,分别表达出编码159和127个氨基酸的CsHMGB1-159/127蛋白,相对分子质量(M_(r))约为23500和20000。1和10μg/mlCsHMGB1-159蛋白刺激小鼠巨噬细胞24h后的A_(620)值分别为0.66±0.08和0.65±0.03,均高于对照组(0.29±0.02)(t=11.1、28.5,P<0.01),且胞浆均出现明显蓝色;48h后的A_(620)值分别达1.02±0.08和1.07±0.08,均高于对照组(0.62±0.035)(t=11.2、12.9,P<0.01),且胞浆蓝色加深。用1和10μg/mlCsHMGB1-127蛋白刺激24h后的A_(620)值分别为0.52±0.08和0.56±0.08,均高于对照组(t=7.39、8.37,P<0.01),胞浆也出现明显蓝色;48h后的A_(620)值分别达到1.10±0.05和0.90±0.10,均高于对照组(t=20.30、6.26,P<0.01),胞浆蓝色也明显加深。1和10μg/mlCsHMGB1-159蛋白刺激24h后的A_(620)值高于相应浓度的CsHMGB1-127蛋白(t=2.98、2.75,P<0.05);48h后两种蛋白的A_(620)值的差异有统计学意义(t=-2.07,P<0.01;t=3.40,P<0.05)。CsHMGB1-159比CsHMGB1-127蛋白在体外均能够刺激小鼠巨噬细胞NF-κB活化,CsHMGB1-159比CsHMGB1-127蛋白的作用更强。

The PET-28a (+) expression plasmids were constructed according to the identified HMGB1homologous gene sequences (CDSs) of Clonorchis sinensis and cloned into Rosetta (DE3) competent cells.Isopropylβ-D-thiogalactoside (IPTG) was used to induce the expression of CsHMGB1-His fusion proteins.After purification by Ni-TED agarose resin,the expression of His -tagged fusion proteins was confirmed by Western blotting analysis.The mouse macrophage RAW264.7 cells were cultured with mediums supplemented with CsHMGB1 -159/127 at a concentration of 1 μg/ml and 10 μg/ml,for 24 h and 48 h,respectively.The cells cultured without stimulants were used as the blank control.The SEAP activity,which indicates NF -κB activation,was detected by using pNiFty2SEAP plasmid and HEK-Blue^(TM)culture medium.The A_(620) value of culture supernatant was detected by microplate a reader,and the intracellular NF-κB activation was also observed under a microscope.Our results showed that the CsHMGB1-159/127 proteins encoding 159 and 127 amino acids were expressed,and the relative molecular weights(M_(r)) were approximately 23 500 and 20 000,respectively.The A_(620) values of supernatant from mouse macrophage stimulated with CsHMGB1-159 protein (1 and 10 μg/ml) for 24 hours were 0.66 ± 0.08 and 0.65 ± 0.03respectively,which were higher than those in the control group (0.29 ± 0.02) (t = 11.1,28.5;P < 0.01),and blue staining was observed in the cytoplasm.The A_(620) values after 48 h reached 1.02 ± 0.08 and 1.07 ± 0.08respectively,which were higher than those in the control group (0.62 ± 0.035) (t = 11.2,12.9;P < 0.01),and the blu staining in the cytoplasm was stronger.Similarly,the A_(620) values after 24 h stimulation with CsHMGB1-127 protein (1 and 10 μg/ml) were 0.52 ± 0.08 and 0.56 ± 0.08,respectively,which were higher than those in the control group ( t =7.39,8.37;P < 0.01),and the blue staining was observed in the cytoplasm.The A_(620) values after 48 h reached 1.10 ±0.05 and 0.90 ± 0.10,respectively,which were also higher than those in the control group ( t = 20.30,6.26;P <0.01),and stronger blue staining was also observed in the cytoplasm.The A_(620) values of CsHMGB1-159 protein ( 1and 10 μg/ml) after 24 hours of stimulation were significantly higher than that of CsHMGB1 -127 protein (t = 2.982.75;P < 0.05).There were also significant differences in A_(620) value after 48 h stimulation ( t = 3.40,P < 0.01)The HMGB1 homologous proteins of Clonorchis sinensis encoding 159 and 127 amino acids can stimulate mouse macrophage NF-κB activation in vitro.The CsHMGB1-159 has stronger stimulatory effect than CsHMGB1 -127 protein.