牛分枝杆菌MarP蛋白的表达纯化及其单克隆抗体制备

Expression and Purification of Marp Protein of Mycobacterium bovis and Preparation of Its Monoclonal Antibody

为制备能够特异性识别牛分枝杆菌(M.bovis)抗酸蛋白酶(MarP)的特异性单克隆抗体(MAb),本研究利用大肠杆菌表达系统表达了MarP蛋白的胞浆区,并以β-casein为底物、DTT为抑制剂检测MarP的丝氨酸蛋白酶活性。以具有活性的MarP蛋白免疫小鼠,取脾细胞与SP2/0细胞融合,并加入饲养层细胞轻轻摇匀分装于96孔培养板,置于37℃、5%CO2培养箱内培养10 d,通过IFA和间接ELISA筛选阳性细胞株进行亚克隆,制备能分泌针对MarP MAb的杂交瘤细胞株,并通过Western blotting和ELISA检测MAb与重组表达和天然的MarP蛋白的反应性。结果显示,重组表达的MarP具有良好的丝氨酸蛋白酶活性,能够在12 h内将30μg的β-casein酶解完全,DTT能够抑制MarP的酶活性。具有活性的MarP蛋白免疫小鼠后,获得5株针对MarP的MAb,均能够特异性识别重组和牛分枝杆菌天然表达的MarP蛋白的线性表位,而不与大肠杆菌(E.coli)和耻垢分枝杆菌(M.smegmatis)的蛋白反应。本研究成功制备了MarP蛋白及MAb,为进一步研究MarP的作用底物及其在牛分枝杆菌抗酸胁迫中作用机制提供了必备材料。

To prepare monoclonal antibodies(MAb)against Mycobacterial acid resistance protease(MarP)of Mycobacterium bovis(M.bovis),the cytoplasmic domain of MarP were expressed with Escherichia coli(E.coli)expression system.The serine proteinase activity of MarP were detected usingβ-casein and DTT as substrate and inhibitor,respectively.The mice were immunized with the active MarP protein,and the spleen cells were fused with SP2/0 cells,and mixed with the feeder cells.The cells were gently shaken and dispensed into a 96-well culture plate,and cultured in 37℃,5%CO2.On the 10 th day,the positive cell were screened by IFA and indirect ELISA for subcloning.Finally,the hybridoma cell lines which secreted monoclonal antibodies against MarP were prepared.The reactivity of monoclonal antibodies with recombinant expressed MarP and natural MarP protein was detected by Western blotting and ELISA.The results showed that the recombinant MarP had good serine proteinase activity,and could digeste 30μgβ-casein completely in 12 h.In addition,DTT could inhibit the activity of MarP.Five MAbs against MarP were obtained from the mice that immunized by MarP protein,and all these five antibodies could recognize the liner epitopes of recombinant MarP and natural MarP.The antibodies didn’t react with proteins from E.coli and M.smegmatis.The purified MarP protein and MAbs against MarP provided tools for further studies on the screening substrate of MarP and the mechanism of MarP in the resistance to acid stress of M.bovis.