布鲁氏菌eryA基因的克隆、原核表达及生物信息学分析

Cloning,Prokaryotic Expression and Bioinformatics Analysis on eryA Gene of Brucella melitensis

为分析羊种布鲁氏菌Rev.I株的基因结构、功能及作为诊断抗原的可能性,从Rev.I株基因组中扩增eryA基因片段,构建重组质粒pET-30a-eryA并进行原核表达、Western-blot检测,同时对该基因进行生物信息学分析。结果显示:本试验成功克隆并表达了eryA基因;经Western-blot检测发现,该基因编码蛋白可与阳性血清发生特异性反应,具有良好的免疫原性;生物信息学分析显示,该蛋白没有跨膜区结构,无信号肽,二级结构中α-螺旋比重最大;抗原表位分析显示:该蛋白含有较多的抗原决定簇。因此,推测eryA蛋白有望作为布鲁氏菌的免疫诊断抗原,这为进一步探索布鲁氏菌基因工程疫苗的研制及iELISA诊断试剂盒的建立奠定了基础。

In order to analyze the genetic structure and function of Rev. I strain of Brucella melitensis and its possibility to be used as diagnostic antigen,the eryA gene fragment was amplified from Rev.I strain to construct the recombinant plasmid pET-30a-eryA with prokaryotic expression and Western-blot detection,meanwhile,bioinformatics analysis was also carried out. The results showed that the eryA gene was successfully cloned and expressed;it was found that,by Western-blot detection,the encoding protein of the gene showed good immunogenicity,which could react specifically with the positive serum;by bioinformatics analysis,there was no any transmembrane structure or signal peptide within the protein,but the proportion of α-helix was maximum in the secondary structure;and by antigenic epitope analysis,there were more antigenic determinants contained in the protein. Therefore,it was expected that the eryA gene could be used as the immuno-diagnostic antigen of Brucella,which would lay a foundation for further developing vaccines against Brucella genetic engineering and researching iELISA diagnostic kits.