鸟分枝杆菌PPE25-MAV蛋白的原核表达及其对巨噬细胞凋亡的影响

Prokaryotic expression of Mycobacterium avium PPE25-MAV protein and its effect on apoptosis of macrophages

目的构建鸟分枝杆菌PPE25-MAV蛋白的重组质粒并诱导表达和纯化,观察其对Ana-1系巨噬细胞凋亡的影响。方法在GenBank中查找到鸟分枝杆菌编码PPE25-MAV蛋白的MAV-2928基因序列,合成全基因并构建质粒。将质粒亚克隆至表达载体pET-32a(+)上,构建重组表达质粒pET-32a-ppe25,转入感受态细胞BL21(DE3)后,通过IPTG诱导表达融合蛋白PPE25-MAV。用Ni Resin FF纯化蛋白,并作用于巨噬系Ana-1细胞,流式细胞术(FCM)检测巨噬细胞凋亡率并且分析其变化。结果鸟分枝杆菌PPE25-MAV蛋白表达载体构建成功,转化至BL21(DE3)后经IPTG诱导表达约59ku的蛋白,与预期大小相符;Western blot检测该蛋白能被His抗体识别。用纯化的PPE25-MAV蛋白作用小鼠巨噬系Ana-1细胞,与空白对照组相比,作用2h后细胞的早期凋亡率差异无统计学意义(P>0.05),细胞的晚期凋亡率和总凋亡率在0.5μg/ml与1μg/ml时均低于空白组(P<0.05),2μg/ml与5μg/ml的凋亡率与对照组比较差异无统计学意义;作用10h细胞的早期凋亡率仅在5μg/ml时升高(P<0.05),0.5、1和2μg/ml剂量组差异无统计学意义(P>0.05),细胞晚期凋亡率和总凋亡率在2μg/ml和5μg/ml时显著降低(P<0.05),0.5μg/ml和1μg/ml组凋亡率差异无统计学意义(P>0.05)。结论重组质粒诱导表达的PPE25-MAV蛋白存在于上清和包涵体中,但主要以包涵体形式表达。纯化的PPE25-MAV蛋白对巨噬细胞凋亡有抑制作用,且主要作用于巨噬细胞的晚期凋亡阶段。

Objectives To construct a recombinant plasmid of Mycobacterium avium(MAV)PPE25-MAV protein and induce its expression and purification,and to observe its effect on the apoptosis of Ana-1 macrophages.Methods The MAV-2928 gene sequence of MAV encoding the PPE25-MAV protein was found identified in GenBank,and the company synthesized the whole.The entire gene was synthesized and a plasmid was constructed a plasmid.The plasmid was subcloned into the expression vector pET-32 a(+),and the recombinant expression plasmid pET-32 a-ppe25 was constructed and transferred into competent cells BL21(DE3).Expression of tThe fusion protein PPE25-MAV was induced withby IPTG.After purifying the protein with Ni Resin FF and treating it with Ana-1 macrophages,flow cytometry(FCM)was used to detect the rate of macrophage apoptosis and changes in those cells were analyzed.Results An expression vector for expression of the recombinant M.avian protein PPE25-MAV was successfully constructed.The plasmid was transformed into BL21(DE3),and,protein expression was induced with IPTG.The expressed protein was about 59 ku,which was consistent with the expected size.Western blotting indicated that the protein was recognized by an anti-His-tag antibody.The purified PPE25-MAV protein was used to treat mouse Ana-1 macrophages.The rate of early apoptosis of cells did not differ after 2 hours of action compared to the blank control group.The rate of late apoptosis and the rate of total apoptosis of cells were significantly lower than rates in the blank group(P<0.05)at concentrations of 0.5μg/ml and 1μg/ml.The rate of late apoptosis and the rate of total apoptosis did not differ significantly from rates in the control group at concentrations of 2μg/ml and 5μg/ml(P>0.05).After 10 hours of treatment,the rate of early apoptosis of cells increased slightly at a concentration of 5μg/ml(P<0.05),and did not differ from the rate at concentrations of 0.5,1,and 2μg/ml(P>0.05).The rate of late cell apoptosis and the rate of total apoptosis decreased significantly at concentrations of 2μg/ml and 5μg/ml(P<0.05),but there was no significant difference in the rate of apoptosis at concentrations of 0.5μg/ml and 1μg/ml(P>0.05).Conclusion Expression of the recombinant plasmid was induced,and the PPE25-MAV protein was expressed in the supernatant and inclusion bodies but mainly in inclusion bodies.The purified PPE25-MAV protein has an inhibitory effect on macrophage apoptosis.It mainly affects the late phase of macrophage apoptosiss.