结核分枝杆菌Rv3873抗原的同源表达及其在结核病血清学诊断中的应用  

Homologous expression of Rv3873 antigen of Mycobacterium tuberculosis and its application in serological diagnosis of tuberculosis

作　　者：顾雯菲 吴娟 胡志东 范小勇[1,2] GU Wen-fei;WU Juan;HU Zhi-dong;FAN Xiao-yong(School of Laboratory Medicine and Life Science,Wenzhou Medical Universily,Wenzhou325035,ZhejiangProvince,China;不详)

机构地区：[1]温州医科大学检验医学院生命科学学院,浙江温州325035 [2]上海市(复旦大学附属)公共卫生临床中心,上海201508,上海市(复旦大学附属)公共卫生临床中心,上海201508

出　　处：《中国生物制品学杂志》2022年第9期1069-1075,共7页Chinese Journal of Biologicals

基　　金：国家自然科学基金项目(82171815,31771004)。

摘　　要：目的在耻垢分枝杆菌(Mycobacterium smegmatis,Msm)中克隆并表达结核分枝杆菌(M.tuberculosis,Mtb)RD1区蛋白Rv3873,并评价其在结核病(tuberculosis,TB)血清学诊断中的价值。方法以Mtb H37Rv标准株全基因组DNA为模板,扩增得到rv3873基因完整序列,克隆至分枝杆菌表达载体pMF406,构建重组质粒pMF406-rv3873,电转化至Msm mc2155,加入乙酰胺诱导表达Rv3873同源重组蛋白(mRv3873),利用亲和层析进行纯化,Western blot分析抗原特异性;ELISA法检测27份TB患者和31份健康者血清IgG抗体,以大肠埃希菌表达的Rv3873(r Rv3873)和免疫优势抗原Ag85A作为对照抗原,评价mRv3873在TB血清学诊断中的作用。结果在Msm中成功表达了重组Mtb蛋白mRv3873,主要以可溶形式存在,经Ni2+亲和层析柱纯化可获得高纯度(95%)的可溶性重组蛋白,蛋白浓度约为2.0 mg/mL;TB患者组血清中抗mRv3873和Ag85A抗原特异性IgG水平均显著高于健康组(P<0.05),而两组血清r Rv3873特异性IgG水平差异无统计学意义(P>0.05);mRv3873抗原与对照抗原Ag85A的诊断效能相当,ROC曲线下面积分别为0.7760和0.7784,显著高于异源表达抗原r Rv3873的曲线下面积(0.5866)。结论在快速生长分枝杆菌Msm中可高水平表达并纯化Mtb RD1区抗原Rv3873,较之异源表达抗原,TB患者产生针对mRv3873的IgG水平显著高于健康人群,分枝杆菌同源表达的mRv3873具有作为TB血清诊断的潜力。Objective To clone and express Rv3873 protein of RD1 region of Mycobacterium tuberculosis(Mtb)in M.smegmatis(Msm),and evaluate its role in the serological diagnosis of tuberculosis(TB).Methods The complete sequence of rv3873 gene was amplified from the whole genomic DNA of standard strain of Mtb H37Rv,and cloned into mycobacterium expression vector pMF406.The constructed recombinant plasmid pMF406-rv3873 was electro-transformed to Msm mc2155,and induced with acetamide.The expressed homologous recombinant protein(mRv3873)of Rv3873 was purified by affinity chromatography and analyzed for antigen specificity by Western blot.IgG antibodies in 27 serum samples from patients with TB and 31 healthy human serum samples were determined by ELISA using Rv3873 expressed in E.coli(r Rv3873)and immune dominant antigen A g85A as control antigens to evaluate the role of mRv3873 in serological diagnosis of TB.Results Recombinant protein mRv3873 of Mtb was successfully expressed in Msm mainly in a soluble form,of which the purity reached 95%and the concentration was about 2.0 mg/mL after purification by Ni2+affinity chromatography.Both the antigen-specific IgG levels against mRv3873 and Ag85A in sera of patients with TB were significantly higher than those in healthy human sera(P<0.05),while the r Rv3873-specific IgG levels showed no significant difference(P>0.05).The diagnostic efficacy of mRv3873 antigen was equivalent to that of control antigen Ag85A.However,the areas under ROC curve of mRv3873 and Ag85A antigens were 0.7760 and 0.7784 respectively,which was significantly higher than that of expressed heterologous antigen r Rv3873(0.5866).Conclusion The Mtb RD1 antigen Rv3873 was highly expressed in rapidly growing Msm and purified.Compared with expressed heterologous antigen r Rv3873,mRv3873 induced significantly higher IgG level against mRV3873 in patients with TB than in healthy controls,indicating the potential of mRv3873 in serological diagnosis of TB.

关 键 词：结核分枝杆菌 RD1区抗原 同源表达 结核病 血清学诊断 

分 类 号：R378.912[医药卫生—病原生物学]