结核杆菌分泌蛋白MPT64多克隆抗体的制备及初步应用  

Preparation and Preliminary Application of Polyclonal Antibody Against Mycobacterium Tuberculosis Secretory Protein MPT64

作　　者：黄震[1] 曾健 孙俊生[3] HUANG Zhen;ZENG Jian;SUN Junsheng(Office of Clinical Medicine,Longang Central Hospital,Shenzhen,Guangdong 518116,China;Departme of anesthesia,Longang Central Hospital,Shenzhen,Guangdong 518116,China;Department of Respiratory,Longang Central Hospital,Shenzhen,Guangdong 518116,China)

机构地区：[1]深圳市龙岗中心医院临床医学院办公室,广东深圳518116 [2]深圳市龙岗中心医院麻醉科,广东深圳518116 [3]深圳市龙岗中心医院呼吸科,广东深圳518116

出　　处：《九江学院学报（自然科学版）》2023年第1期120-124,共5页Journal of Jiujiang University：Natural Science Edition

基　　金：深圳市龙岗区经济与科技发展专项资金,医疗卫生科技计划(编号LGKCYLWS2020001)的研究成果之一。

摘　　要：目的鼠抗重组结核杆菌分泌蛋白MPT64多克隆抗体的制备及其在结核淋巴结组织中特性的鉴定及初步应用。方法通过PCR法从结核患者淋巴结组织中扩增MPT64基因,并构建到pET28a表达载体中。使用IPTG诱导重组蛋白的表达,通过镍柱亲和层析纯化获得MPT64蛋白。以纯化的MPT64蛋白混合福氏完全佐剂免疫Balb/C小鼠制备抗体,用ELISA法和免疫组化分别检测抗体的效价和特异性。结果成功的表达、纯化了MPT64重组蛋白,通过检测MPT64蛋白免疫的小鼠血清效价为1∶32000,Western blot显示该多克隆抗体能够与MPT64蛋白特异性的结合,同时还能够和天然的MPT64结合。通过结核病浸润期或进展期,溶解播散期,吸收好转期,硬结钙化期不同分期MPT64的表达情况分析,提示MPT64蛋白表达与临床病理因素之间有较好的相关性,对45例结核患者的血清进行MPT64检测,检测阳性率高达77.8%。结论以重组人MPT64为免疫原,成功地制备高效价、高特异性的鼠抗MPT64抗体,此研究提示MPT64能够作为活动性结核诊断的有效靶标,为进一步进行MPT64的检测及其临床初步应用奠定了基础。Objective To prepare the mouse anti recombinant human secretory protein MPT64 antibody and its characterization and preliminary application in tuberculosislymph node tissue.Method MPT64 gene was amplified from lymph node tissue of tuberculosis patients by PCR and constructed into pET28a expression vector.Then the recombinant plasmid pET28a/MPT64 was transformed into E.coli BL21(DE3)and expressed under IPTG induction.The recombinant MPT64 was purified through Ni 2+-NT agarose gel column and the purified MPT64 used as imunogen to imunize the mouse.The titer and specificity of the mouse anti-MPT64 antibody were analyzed by ELISA,WB and IHC repectively.Result The recombinant MPT64 was successfully expressed and purified,and the polyclonal ani-MPT64 antibody was successfully prepared.The titer of the antiserum was1∶32000 by ELISA.Western blot analysis showed that the antibody reacted specifically to MPT64.Analysis of MPT64 expression in different stages of tuberculosis infiltration or progressive stage,solubilization and dissemination stage,absorption improvement stage,sclerosis and calcification stage showed a good correlation between MPT64 protein expression and clinicopathological factors.The positive rate of MPT64 detection in serum of 45 tuberculosis patients was as high as 77.8%.Conclusion The mouse anti MPT64 antibody with high titer and specificity had been successfully prepared,This study suggested that MPT64 could be used as an effective target for the diagnosis of active tuberculosis,which layed a foundation for further research on the detection and clinical application of MPT64.

关 键 词：结核 MPT64 多克隆抗体 表达 检测 

分 类 号：R52[医药卫生—内科学]