肺炎衣原体ompA基因VD2-VD3区重组蛋白在血清学诊断中的初步应用

Application of the recombinant protein MOMPVD2-VD3 from Chlamydia pneumoniae in sero diagnosis

摘要:

应用聚合酶联反应(PCR)技术,从肺炎衣原体Chlamydia pneumoniae的主要外膜蛋白(Major Outer Membrane Protein,MOMP)编码基因(ompA)上扩增出抗原优势表位VD2-VD3区基因,构建原核表达系统并诱导表达重组蛋白,经Ni-NTA亲和层析法纯化表达产物。间接酶联免疫吸附试验(Enzyme link immunosorbent assay,ELISA)检测人血清中特异性IgG抗体。试验表明,转化入BL21大肠杆菌的重组质粒,能表达并纯化出相对分子质量(Mr)为24KD的重组蛋白。Western blot证实重组蛋白只与Cpn MOMP mAb发生特异性反应;重组蛋白用作ELISA包被抗原检测Cpn阴阳性参比血清,特异性和灵敏度均为100%;对126位冠心病患者血清进行的检测中,该间接ELISA法与晶美公司Cpn IgG ELISA诊断试剂盒的检测结果相比,符合率达到96.3%。结果证实,制备的重组蛋白MOMP_VD2-VD3具有良好的免疫活性,在Cpn血清学诊断的应用中具有较大的利用价值。

Abstract:

To express the recombinant protein MOMP_VD2-VD3 of Chlamydia pneumoniae，and research on the immunocompetence of the MOMPVD2-VD3 to support serodiagnosis，PCR and gene recombinant technique was used to clone the targeted DNA fragment from a strain AR-39. The recombinant plasmid was induced in E.coli BL21 after having constructed the prokaryotic expression system，then the immunocompetence of the expression product was analyzed by Western blot and indirected ELISA which is based on the animal experimentation. A group of control sera and 126 sera from patients with coronary heart disease were examined by using ELISAs based on the recombinant protein (MOMP_VD2-VD3)，and then the results were evaluated comparing with a commercial ELISAs kit. The results of the Western blot and indirected ELISA showed ompAVD2-VD3 gene inserted in pET30a could express a recombinant protein with the molecular weight of 24kDa in BL21 and specifically reacted with the antibodies against the MOMP. Specific humoral response was elicited after immune the BALB/c mouse with protein and the specific antibody titer was more than 1∶20480. Using a panel of control sera，the participation of the recombinant antigen，the sensitivity and the specificity of the indirected ELISAs were 100% respectively. Comparisons between two methods in detecting 126 sero samples，the concordance of two tests was 96.3%. The results reported here show that the recombinant protein with excellent immunocompetence could benefit the research on the serodiagnosis to Chlamydia pneumoniae.