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| Preparation and specificity of monoclonal antibody to the Mycobacterium tuberculosis secreted protein MPT64 |
| XIE Yan-ling1,2, NING Huan-huan2, LIANG Xuan2, KANG Jian2, REN Rui2, ZHANG Hui3, ZHANG Na-na3, LU Yan-zhi2, BAI Yin-lan2 |
1. School of Life Sciences, Yan’an University, Yan’an 716000, China;
2. Department of Microbiology and Pathogen Biology, School of Basic Medicine, Air Force Medical University, Xi’an 710032, China;
3. Medical school, Xi’an Peihua University, Xi’an 710065, China; |
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Abstract This study prepared monoclonal antibody (mAb) to the Mycobacterium tuberculosis secreted protein MPT64 and analyzed its specificity. The mpt64 gene was amplified by PCR and cloned into pET28a(+) to construct the prokaryotic expression vector. The obtained recombinant strain was induced with IPTG to express MPT64 protein, which was identified by western blotting. The recombinant MPT64 protein was purified by affinity chromatography and used to inoculate mice subcutaneously. Splenocytes were separated and fused with SP2/0 hybridoma cells. A positive hybridoma cell line was obtained by screening. The hybridoma cells were used to prepare mouse ascites. The mAb was purified from the ascites with medium pressure liquid chromatography. The relative mAb affinities and subclasses were detected with ELISA, and the specificity was detected with western blotting. The prokaryotic expression vector pET28a (+)-mpt64 was constructed, and recombinant MPT64 protein was expressed and purified with affinity chromatography. After fusion of splenocytes from MPT64 protein immunized mice with SP2/0 cells, the hybridoma cell line MPT64-A5B2, secreting anti-MPT64 mAb, was obtained by screening. MPT64-A5B2 mAb was purified from mouse ascites with medium pressure liquid chromatography. The affinity of MPT64-A5B2 mAb was 2.813×10-7 g/mL, and it belonged to the IgG 1 subclass. MPT64-A5B2 mAb specifically recognized MPT64 protein in Mycobacterium tuberculosis. In this study, MPT64 recombinant protein was prepared, and anti-MPT64 mAb was obtained, thus providing a foundation for the development of MPT64 for diagnostic and therapeutic applications for tuberculosis.
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Received: 21 January 2022
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Corresponding Authors:
Bai Yin-lan, Email: yinlanbai@fmmu.edu.cn; Ning Huan-huan, Email: ninghuanhuan2014@163.com
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2022, Vol. 38 
