摘要
CSFV囊膜糖蛋白E2是诱导中和抗体及激发保护性免疫应答的主要抗原蛋白.本研究应用抗CSFV(Alfort毒株)E2蛋白的单克隆抗体c2410、WH303及M1669,筛选噬菌体展示的12肽随机肽库,进行表位分析.结果发现:c2410、WH303针对同一线性表位,精确定位于E2蛋白的832～837位氨基酸SPTTLR,是CSFV特异性表位.同时发现WH303识别的2个主要拟位基序:(W)NKPxT、AxWPTA,M1669识别的3个主要拟位基序:DxMRLD、(SL)MPPF、MLLHxxPxL,但在E2蛋白中均未查找到与之相同(似)序列.应用ELISA、竞争性ELISA、免疫印迹分析不同表(拟)位对单克隆抗体的免疫反应性差异,结果发现:(1)c2410对WH303的3个噬菌体拟位(WNKPxT,AxWPxATA,SPSxLR)在ELISA、免疫印迹检测中均为阴性;(2)SPTxLR噬菌体拟位能与c2410、WH303发生特异性结合,且此结合能被病毒抗原阻断;(3)M1669的3个噬菌体拟位(DxMRLD,(SL)MPPF、MLLHxxPxL)在ELISA、免疫印迹中能与M1669发生特异性结合,但此结合不能被病毒抗原阻断.
Envelope glycoprotein E2 （gp55） of Classical swine fever virus （CSFV） is the most antigenic protein being responsible for eliciting neutralizing antibodies and conferring protective immunity. The mapping of the epitope of E2 by screening a 12-mer random peptide phage display library using the MAbs c2410, WH303, and M1669, raised against CSFV strain Alfort and reacted with the E2 protein. A linear epitope recognized by both MAbs, c2410 and WH303, which has strong specificity of CSFV, has been precisely defined and located at SP＇ITLR （aa832 - aa 837 of E2 protein） .Two major mimotope motifs （W） NKPxT, AxWPIATA and three major mimotope mo- tifs DxMRLD, （SL）MPPF, MILHxxPxL, can only be recognized by WH303 and M1669 respectively, have also been defined,but can not find the consensus or similar sequence among the amino acid sequence of CSFV E2 protein. Using ELISA, competitive ELISA and Westem blot to analysis immunoreactivities of different phage mimotopes to the MAbs, the results showed： a. （W）NKPxT,AxWqaIATA, and SPSxLR phage mimotopes, obtained by MAb WH303 screening, can not be recognized by MAb c2410 in ELISA and western blot. b. SPTxLR phage mimotopes can bind to both MAbs c2410 and WH303 with specificity and these binding can be inhibited or blocked by viral antigen, c. DxMRLD,（SL）MPPF,MLLHxxPxL phage mimotopes, obtained by MAb M1669 screening, can bind to MAb M1669 in ELISA and western blot with specificity, but these binding can not be inhibited or blocked by viral antigen.
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2005年第5期373-379,共7页
Chinese Journal of Preventive Veterinary Medicine
基金
国家"九五"科技攻关项目(96_120_10)
中
澳国际合作项目(ACAIR/ASI/9438)
