羊布鲁菌体内诱导基因的筛选与初步鉴定

Screening and Preliminary Identification of in vivo-induced Genes of Brucella melitensis

本研究利用体内诱导表达抗原技术(IVIAT)来筛选和鉴定羊布鲁菌特异的体内诱导抗原,为筛选新的毒力分子,诊断靶标、疫苗候选抗原和药物靶点提供科学依据。通过收集临床上羊布鲁菌阳性血清,吸附后作为探针,构建羊布鲁菌16M基因组表达文库,进行体内诱导抗原的筛选,同时利用RT-PCR验证筛选得到的基因,对筛选得到的基因进行测序比对及生物信息学分析。结果显示:利用临床上感染布鲁菌的羊阳性血清作为探针,通过体内诱导抗原技术成功地从羊布鲁菌的基因组中筛选得到了14个体内诱导表达的基因。这些基因主要参与布鲁菌的糖代谢、tRNA修饰、离子转运和跨膜运输等生物过程,这些分子有的已经被证实是布鲁菌的药物靶点和毒力分子,有的可能与布鲁菌的毒力和免疫相关,可以作为疫苗候选抗原和诊断标识。体内诱导抗原技术(IVIAT)成功地应用到布鲁菌候选抗原和诊断标识的筛选和鉴定研究中,利用该技术成功获得14个体内诱导基因,这些基因将为布鲁菌疫苗的研制和诊断产品的研发提供候选抗原和诊断标识。

In this study,we aimed to screen and identify in vivo-induced antigens of Brucella melitensis via in vivo-induced antigen technology(IVIAT),which may provide a scientific basis for selecting of novel virulence molecules,diagnostic targets,vaccine candidate antigens and drug targets for Brucellosis control.The probe was prepared by absorbing the clinical positive serum collected from Brucella-infected animals onto PVDF membrane.Expression library of 16M gene of B.melitensis was constructed for identifying in vivo-induced antigens.The identified genes were confirmed by RT-PCR and the PCR products were sequenced and analyzed using bioinformatic software.The results showed that 14 of in vivo-induced genes were obtained from expression library of 16M gene of B.melitensis by IVIAT using the probe prepared in this study.Further analysis using bioinformatic software demonstrated that the products of these genes are mainly involved in the biological processes of glucose metabolism,tRNA modification,ion transport and transmembrane transport of B.melitensis.Some of these molecules have been identified as drug targets and virulence molecules in Brucella spp,while some may be related to Brucella virulence and immunity.The IVIAT was successfully used to the screening and identification of in vivo-induced antigens of B.melitesis.The in vivo-induced antigens of B.melitesis identified in this study are new candidate antigens and diagnostic markers for vaccine and diagnostic products of B.melitesis.