结核分枝杆菌双组分系统PhoP蛋白的免疫学特性  

Immunological characteristics of the PhoP protein of two-component system in Mycobacterium tuberculosis

作　　者：李雪 宁唤唤 康健 徐铭泽 崔若囡 代婷 路延之 薛飒 柏银兰 LI Xue;NING Huan-huan;KANG Jian;XU Ming-ze;CUI Ruo-nan;DAI Ting;LU Yan-zhi;XUE Sa;BAI Yin-lan(College of Life Sciences,Northwest University,Xi’an 710069,China;Department of Microbiology and Pathogenic Biology,School of Basic Medicine,Air Force Medical University,Xi’an 710032,China;Military Medical Innovation Center,Air Force Medical University,Xi’an 710032,China)

机构地区：[1]西北大学生命科学学院,西安710069 [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]空军军医大学军事医学创新中心,西安710032

出　　处：《中国人兽共患病学报》2024年第4期352-358,共7页Chinese Journal of Zoonoses

基　　金：国家自然科学基金面上项目(No.82272343,No.81971560);陕西省重点研发项目(No.2022ZDLSF01-07)联合资助。

摘　　要：目的探究结核分枝杆菌双组分系统PhoP蛋白的免疫学特性。方法采用生物信息学方法预测PhoP蛋白的B细胞和T细胞表位。将Mtb H37Rv phoP基因克隆到pET-28a(+)原核表达载体上,并将重组质粒转化到E.coli BL21菌株中,经IPTG诱导表达及亲和层析法纯化获得PhoP重组蛋白。用ELISA分析Mtb感染小鼠和TB患者血清中PhoP特异性抗体水平。PhoP重组蛋白经肌肉注射途径免疫小鼠,分离小鼠多抗血清,用ELISA和Western blot分别检测抗体效价和抗体特异性。分离小鼠脾细胞,用ELISpot和ELISA法分别检测分泌IFN-γ细胞频数及其分泌水平。结果经生物信息学预测发现PhoP含有24个B细胞抗原表位和11个优势T细胞表位。成功构建PhoP原核表达载体并获得纯化的PhoP蛋白。Mtb感染小鼠和TB患者血清中的PhoP特异性抗体显著升高,查准率分别为99.9%和82.5%,特异度均为100%。PhoP免疫小鼠诱导产生高水平特异性抗体,免疫小鼠脾细胞经抗原体外刺激后,IFN-γ的分泌水平和细胞频数均显著增加,IL-2和IL-10分泌水平上调。结论PhoP蛋白可诱导体液免疫应答和Th1型为主的细胞免疫。PhoP蛋白有良好的免疫原性,且具有一定的TB诊断效能。本研究为阐明PhoP在Mtb感染中的作用及其用于TB诊断奠定了一定的基础。In this study,the immunological characteristics of the PhoP protein were explored with a two-component system of Mycobacterium tuberculosis(Mtb).Bioinformatics was used to predict the B and T cell epitopes of the PhoP protein.A recombinant expression plasmid was constructed by PCR analysis of the phoP sequence and cloning into the prokaryotic expression vector pET-28a(+).Competent Escherichia coli BL21 cells were transformed with the recombinant plasmid and expression was induced with IPTG.The recombinant PhoP protein was purified by affinity chromatography.Serum levels of PhoP-specific antibodies in Mtb-infected mice and tuberculosis(TB)patients were analyzed with an ELISA.BALB/c mice were immunized with the PhoP recombinant protein by intramuscular injection.Sera of mice were collected and antibody titers were detected with an ELISA and specificity was assessed by Western blot analysis.Mouse splenocytes were isolated and the proportions of IFN-γ-positive cells and cytokine levels were detected with an ELISpot and ELISA,respectively.Bioinformatics identified 24 B cell and 11 T cell epitopes of the PhoP protein.A prokaryotic recombinant vector of PhoP was successfully constructed and the recombinant PhoP protein was obtained by purification.Specific antibody levels to PhoP in sera of Mtb infected mice and TB patients increased significantly,with precision of 99.9%and 82.5%,and specificity of 100%,respectively.PhoP protein immunization successfully induced production of specific antibodies in mice.Stimulated by antigens in vitro,IL-2 and IFN-γlevels were significantly increased in the splenocytes of immunized mice.Immunization with the PhoP protein induce a humoral immune response and Th1-dominated cellular immunity,indicating that the PhoP protein was immunogenic with diagnostic efficacy for TB.These results lay a foundation to clarify the role of PhoP in Mtb infection and application for diagnosis and prevention of TB.

关 键 词：结核分枝杆菌 双组分系统 PHOP 体液免疫应答 细胞免疫应答 

分 类 号：R378.91[医药卫生—病原生物学]