一株口蹄疫病毒非结构蛋白3A 单克隆抗体的表位肽段鉴定

摘要

为鉴定口蹄疫病毒非结构蛋白3A 的单克隆抗体3A24＃识别的表位区，对非结构蛋白3A 分两个肽段进行原核表达，其中3A1和3A2肽段分别包含3A 蛋白的第1位～89位和77位～153位氨基酸。分别针对两肽段基因设计并合成引物，PCR 扩增后定向克隆到原核表达载体 pET-30a（＋）-PES-SUMO，将重组质粒转化宿主菌 E．coli BL21（DE3）pLys，IPTG 诱导表达。用单克隆抗体3A24＃对表达的3A1和3A2肽段进行 Western blot 检测，以鉴定单克隆抗体3A24＃的识别表位肽。SDS-PAGE 分析表明，成功表达了3A1和3A2肽段，大小分别为26 ku 和25 ku。Western blot 分析表明，FMDV 牛阳性血清与两肽段均能识别，而单克隆抗体3A24＃只能与3A2肽段发生反应，与3A1肽段不发生反应。3A24＃单克隆抗体识别的抗原表位位于3A2肽段，即3A 蛋白的第89aa～第153aa 肽段上。%In order to identify the peptide region covering epitopes recognized by a monoclonal antibody 3A24# a-gainst non-structural protein 3A from foot-and-mouth disease virus,the protein 3A was prokaryotically expressed in two peptides,3A1 and 3A2,covering 1-89 and 77-153 amino acids.Primers targeting the genes 3a1 and 3a2 were designed and synthesized,respectively.Then the genes 3A1 and 3A2 were amplified by PCR and cloned into plas-mid pET-30a(+)-PES-SUMO.The recombinant plasmids were transformed into E.coli BL21(DE3)pLys and in-duced for expression by IPTG.The expressed peptides were examined by Western blot using monoclonal antibody 3A24# to identify the epitopes covering peptides.SDS-PAGE analysis showed that both 3A1 and 3A2 peptides were successfully expressed with the respective molecular weights as 26 ku and 25 ku.Western blot analysis showed that,both 3A1 and 3A2 can be recognized by FMDV infected cattle serum,but only 3A2 can react with monoclonal antibody 3A24 #.The epitopes recognized by monoclonal antibody 3A24 # are located on peptide 3A2,namely between the amino acids 89-153 of FMDV non-structural protein 3A.