牛分枝杆菌MarP蛋白的表达纯化及其单克隆抗体制备

doi:10.16431/j.cnki.1671-7236.2019.06.025

Abstract

Abstract:

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 using β-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 10th 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 30 μg β-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.

Key words: Mycobacterium bovis; acid resistance; MarP; MAb; serine proteinase

CLC Number:

S852.61⁺8

LIN Weidong, SUI Xiukun, WANG Zhaoyang, JIA Hong, FANG Lichun, WANG Haichun, ZHU Liangquan, XIN Ting. Expression and Purification of Marp Protein of Mycobacterium bovis and Preparation of Its Monoclonal Antibody[J]. , 2019, 46(6): 1774-1782.

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

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