结核分枝杆菌分泌蛋白MPT64单克隆抗体的制备及其特异性  

Preparation and specificity of monoclonal antibody to the Mycobacterium tuberculosis secreted protein MPT64

作　　者：谢燕玲 宁唤唤 梁璇 康健 任瑞 张慧 张娜娜 路延之 柏银兰 XIE Yan-ling;NING Huan-huan;LIANG Xuan;KANG Jian;REN Rui;ZHANG Hui;ZHANG Na-na;LU Yan-zhi;BAI Yin-lan(School of Life Sciences,Yan’an University,Yan’an 716000,China;Department of Microbiology and Pathogen Biology,School of Basic Medicine,Air Force Medical University,Xi’an 710032,China;Medical school,Xi’an Peihua University,Xi’an 710065,China)

机构地区：[1]延安大学生命科学学院,延安716000 [2]空军军医大学基础医学院微生物与病原生物学教研室,西安710032 [3]西安培华学院医学院,西安710065

出　　处：《中国人兽共患病学报》2022年第5期387-393,共7页Chinese Journal of Zoonoses

基　　金：国家“十三五”重大传染病专项课题(No.2018ZX10302302002004);国家自然科学基金面上项目(No.81971560,No.81671638)联合资助。

摘　　要：目的制备结核分枝杆菌(Mycobacterium tuberculosis,Mtb)分泌蛋白MPT64的单克隆抗体(Monoclonal antibody,mAb),并分析mAb的特异性。方法PCR扩增mpt64基因并克隆入pET28a(+)构建原核表达载体;将获得的重组菌株在IPTG作用下诱导表达MPT64蛋白,Western blot验证蛋白表达;亲和层析法纯化目的蛋白。重组MPT64蛋白免疫小鼠,取脾细胞与杂交瘤细胞SP2/0融合,筛选得到阳性杂交瘤细胞系。以该杂交瘤细胞制备小鼠腹水,中压液相色谱仪纯化腹水获得mAb。ELISA法检测获得的mAb的相对亲和力和亚类,Western blot检测mAb的特异性。结果本研究成功构建原核表达载体pET28a(+)-mpt64并诱导表达了MPT64蛋白。亲和层析法获得纯化的重组MPT64蛋白。该重组蛋白免疫小鼠的脾细胞和SP2/0细胞融合后,经筛选获得能够稳定分泌抗MPT64 mAb的杂交瘤细胞系MPT64-A5B2。中压液相色谱仪纯化小鼠腹水中的MPT64-A5B2 mAb。该mAb的亲和力为2.813×10^(-7) g/mL,属于IgG 1亚类。MPT64-A5B2 mAb能特异性识别Mtb中的MPT64蛋白。结论本研究制备了MPT64重组蛋白,并获得了抗MPT64 mAb,为MPT64用于结核病诊断和治疗制剂的研制奠定了基础。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 asci tes.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.

关 键 词：结核分枝杆菌 MPT64 分泌蛋白 单克隆抗体 

分 类 号：R378.91[医药卫生—病原生物学]