羊口疮病毒B2L蛋白抗原表位预测及重组蛋白设计与验证

Antigen Epitope Prediction and Recombinant Protein Design and Verification of ORFV B2L Protein

为得到羊口疮病毒(ORFV)B2L蛋白的可能抗原表位及截短后高效表达的表位蛋白,本试验采用DNAStar、Mega 7.0等软件对部分NCBI已登录的B2L基因序列进行分析,并应用不同在线服务器对其编码蛋白的信号肽、跨膜区、细胞毒性T细胞表位、辅助性T细胞表位、B细胞表位和二级结构进行预测,同时参考多模板进行三级结构预测,综合抗原表位、亲水性、表面可及性和抗原指数等主要预测数据进行抗原位点分析及融合His·tag抗原表位蛋白的设计,构建并原核表达截短后高效表达的表位蛋白。结果显示,得到了几个可能的优质抗原表位,分别为88-93、133-138、172-175、251-254、311-313和370-377位氨基酸;纯化并复性的蛋白以多聚体形式存在;Western blotting结果显示,其具有良好的反应原性。该研究确定了ORFV B2L蛋白抗原位点,构建并原核表达了截短后高效表达的表位蛋白,为羊口疮诊断制剂及亚单位疫苗的研究奠定了基础。

To obtain the possible antigenic epitopes of ORFV B2L protein and express the recombinant protein efficiently after truncation.DNAstar,Mega 7.0 and other softwares were used to analyze partial ORFV B2L gene sequences recorded by NCBI.The signal peptide,transmembrane region,cytotoxic T cell epitope,helper T cell epitope,B cell epitope and secondary structure of protein were predicted by different online servers.At the same time,tertiary structure prediction was carried out by referring to multiple templates.Antigenic site analysis and recombinant protein design were carried out by synthesizing major predictive data such as epitope,hydrophilicity,surface accessibility and antigen index,the fusion protein was double His·tag.Several possible high-quality epitopes were obtained,which were 88-93,133-138,172-175,251-254,311-313 and 370-377 amino acids,respectively.A recombinant protein containing major predictive epitopes was designed and expressed by fusing two His·tags to link flexible proteins.The purified and renatured expressed proteins existed in the form of polymers.Western blotting results showed that the purified and renatured recombinant proteins had good reactivity.This study acquired the ORFV B2L antigenic sites and constructed the highly efficient prokaryotic expression vector of recombinant B2L protein,providing the bases for further explore the diagnosis reagent and subunit vaccine.