Pathak Prachi, Kumar Ashu, Thavaselvam Duraipandian
Defence Research and Development Establishment, Jhansi Road, Gwalior, 474002, India.
BMC Infect Dis. 2017 Jul 11;17(1):485. doi: 10.1186/s12879-017-2588-1.
Brucellosis is an important zoonotic disease caused by different Brucella species and human brucellosis is commonly prevalent in different states of India. Among various Brucella species, B. melitensis is most pathogenic to human and included as category B biothreat which can cause infection through aerosol, cut, wounds in skin and contact with infected animals. The diagnosis of human brucellosis is very important for proper treatment and management of disease as there is no vaccine available for human use. The present study was designed to clone, express and purify immunodominant recombinant omp2a (rOmp2a) porin protein of B. melitensis and to evaluate this new antigen candidate for specific serodiagnosis of human brucellosis by highly sensitive iELISA (indirect enzyme linked immunosorbent assay).
Omp2a gene of B. melitensis 16 M strain was cloned and expressed in pET-SUMO expression system. The recombinant protein was purified under denaturing conditions using 8 M urea. The purified recombinant protein was confirmed by western blotting by reacting with anti-HIS antibody. The sero-reactivity of the recombinant protein was also checked by reacting with antisera of experimentally infected mice with B. melitensis 16 M at different time points. Serodiagnostic potential of recombinant porin antigen was tested against 185 clinical serum samples collected from regions endemic to brucellosis in southern part of India by iELISA. The samples were grouped into five groups. Group 1 contained cultured confirmed positive serum samples of brucellosis (n = 15), group 2 contained sera samples from positive cases of brucellosis previously tested by conventional methods of RBPT (n = 28) and STAT (n = 26), group 3 contained sera samples negative by RBPT(n = 36) and STAT (n = 32), group 4 contained sera samples of other febrile illness and PUO case (n = 35) and group 5 contained confirmed negative sera samples from healthy donors (n = 23).
The rOmp2a was found to be immunoreactive by iELISA and western blotting. The test showed a sensitivity of 93.75% and specificity of 95.83% when tested against 185 serum samples. For determination of statistical significance between experimental groups and control groups, Student's t test was performed on the data.
Omp2a emerges as a potential antigen candidate for serodiagnosis of human brucellosis.
布鲁氏菌病是一种由不同布鲁氏菌属引起的重要人畜共患病，人类布鲁氏菌病在印度不同邦普遍流行。在各种布鲁氏菌属中，羊种布鲁氏菌对人类致病性最强，被列为B类生物威胁因子，可通过气溶胶、皮肤切口、伤口以及与感染动物接触引发感染。由于尚无用于人类的疫苗，人类布鲁氏菌病的诊断对于疾病的恰当治疗和管理非常重要。本研究旨在克隆、表达和纯化羊种布鲁氏菌免疫显性重组外膜蛋白2a（rOmp2a）孔蛋白，并通过高灵敏度间接酶联免疫吸附测定（iELISA）评估该新抗原候选物用于人类布鲁氏菌病特异性血清学诊断的价值。
克隆羊种布鲁氏菌16M菌株的Omp2a基因，并在pET-SUMO表达系统中进行表达。使用8M尿素在变性条件下纯化重组蛋白。通过与抗HIS抗体反应，经蛋白质免疫印迹法确认纯化的重组蛋白。还通过与不同时间点经羊种布鲁氏菌16M实验感染小鼠的抗血清反应，检测重组蛋白的血清反应性。采用iELISA检测重组孔蛋白抗原针对从印度南部布鲁氏菌病流行地区采集的185份临床血清样本的血清学诊断潜力。样本分为五组。第1组包含布鲁氏菌病培养确诊阳性血清样本（n = 15），第2组包含先前通过传统虎红平板凝集试验（RBPT）和试管凝集试验（STAT）检测为阳性的布鲁氏菌病病例血清样本（n = 28和n = 26），第3组包含RBPT和STAT检测为阴性的血清样本（n = 36和n = 32），第4组包含其他发热性疾病和不明原因发热病例的血清样本（n = 35），第5组包含健康供体的确诊阴性血清样本（n = 23）。
iELISA和蛋白质免疫印迹法显示rOmp2a具有免疫反应性。针对185份血清样本进行检测时，该检测显示灵敏度为93.75%，特异性为95.83%。为确定实验组与对照组之间的统计学显著性，对数据进行了Student t检验。
Omp2a成为人类布鲁氏菌病血清学诊断的潜在抗原候选物。
