摘要
为筛选鸡毒支原体(MG)膜蛋白,利用生物信息学软件对其进行序列分析后,选择不含跨膜区的714 bp基因片段作为靶标蛋白进行原核表达(因其分子量约31 kDa,故而命名为P31蛋白),表达P31蛋白并进行纯化后制备兔多克隆抗体,运用ELISA方法检测抗体效价,提取MG液体培养物总蛋白、胞浆蛋白、膜蛋白,利用Western blot进行亚细胞定位.结果显示,P31蛋白具有跨膜结构和良好的B细胞抗原表位,原核表达后获取融合蛋白rP31,大小约为31 kDa,ELISA检测兔血清效价为1∶25 600,Western blot分析显示,rP31蛋白分布在MG膜表面.本研究证实了P31蛋白确为MG膜蛋白且抗原性良好,可做为研发MG新型诊断方法、基因工程疫苗及治疗药物的候选蛋白.
Abstract
In order to investigate the function of Mycoplasma gallisepticum(MG)membrane protein,the transmembrane area 714 bp was analyzed using bioinformatics software and for prokaryotic expression of a target protein with molecular weight about 31 kDa.The expressed recombinant P31 protein was purified and used for preparation of rabbit polyclonal antibodies.The rabbit polyclonal antibodies were titrated to be 1:25 600 by ELISA.The P31 protein was distributed on the surface of MG membrane as visualized in Western blot.In this study,P31 protein was confirmed to be a MG membrane protein with good antigenicity,which could be used as a candidate protein for developing novel MG diagnostic methods,genetic engineering vaccines and therapeutic drugs.
基金项目
国家自然科学基金项目(32060786)
国家自然科学基金项目(31660723)
贵州省科学技术项目(黔科合基础[2019]1181号)
贵州省科技支撑计划项目(黔科合支撑[2021]一般161)
贵州省优秀青年科技人才项目(黔科合平台人才[2021]5646)
国家科技期刊平台
NSTL
万方数据