【摘要】 经纯化的JSRV-CA融合蛋白乳化后免疫Balb/c小鼠,四免后,取其脾细胞与SP2/0骨髓瘤细胞经杂交瘤技术进行融合.最终获得了三株稳定分泌单克隆抗体的细胞系.同时,分段克隆绵羊肺腺瘤病毒ca基因并构建原核表达质粒,在E.coliBL21中诱导表达.以获得的三株抗JSRV-CA蛋白的单克隆细胞分泌的McAb为一抗,应用Western blot对分段表达的CA蛋白进行肽探针扫描,初步鉴定了三株单抗识别的线性表位,并应用非竞争性ELISA法测定了三株单抗的功能性亲和常数.为建立特异性的病原诊断方法、分析CA蛋白的功能及疫苗设计奠定了基础.更多还原

【Abstract】 Balb/c mice were immunized four times by the purified fusion CA protein which was emulsified with freund′s adjuvant. Then, cell fusion was conducted according to standard procedure. Positive hybridoma clones were screened by indirect ELISA and Western blot. Three hybridoma clones that stably secreted specific monoclonal antibody against JSRV-CA were developed. Meanwhile, JSRV ca gene was divided into four overlapping fragments and expressed in E. coli BL21 respectively. Pepscan technology was employed to screen the antigen epitope through the expressed fusion proteins were detected respectively with three McAbs by Western blot. Then three liner epitopes recognized by three McAbs were preliminary identified, and the functional affinities of anti-CA McAbs were assessed with non-competitive ELISA method. All this may be helpful in understanding molecular properties of JSRV-CA and may be useful for pathogenic diagnosis and vaccine design.更多还原