山羊副流感病毒3型HN蛋白单克隆抗体的制备与鉴定

Preparation and characterization of monoclonal antibodies against hemagglutinin neuraminidase of caprine parainfluenza virus type 3

为制备山羊副流感病毒3型（caprine parainfluenza virus type 3,CPIV3）HN蛋白单克隆抗体,将CPIV3 HN基因1081～1725 bp片段（编码361～574 aa）克隆入原核表达载体p ET-32a（＋）,构建重组表达质粒p ET-32a（＋）-HN361-574 aa,转化至大肠杆菌Rosetta（BL21）感受态细胞并进行诱导表达,分别用SDS-PAGE和Western-blot鉴定重组表达蛋白。结果表明,CPIV3 HN蛋白361～574 aa片段被正确表达。用两段截短的HN蛋白混合免疫BALB/c小鼠,取小鼠脾与骨髓瘤细胞SP2/0融合制备单克隆抗体,分别用间接ELISA、Western-blot和IFA筛选和鉴定融合细胞上清。通过筛选获得3株杂交瘤细胞,分别命名为3F1、4A7、4H4,进一步研究表明,这3种单克隆抗体至少识别2个不同的抗原表位。本研究为CPIV3病原学检测方法的建立以及病理学诊断奠定了重要的基础。

To prepare monoclonal antibodies（MAbs） against hemagglutinin neuraminidase（HN）of caprine parainfluenza virus type 3（CPIV3）,the truncated gene of CPIV3 HN1081-1725 bp wascloned into p ET-32a（＋） vector,and a recombinant plasmid p ET-32a（＋）-HN361-574 aa was constructed.The protein was expressed in E.coli Rosetta（BL21）,and identified by SDS-PAGE and Western-blot.The results showed the recombinant protein was obtained.BALB/c mice were immunized with protein HN139-360 aa and HN361-574 aa,the spleens were fused together with SP2/0 to produce MAbs.The MAbs were selected and characterized by indirect ELISA,Western-blot and IFA.Three MAbs 3F1,4A7 and 4H4 were obtained,and recognized at least two independent epitopes,Which will be used for detection and diagnosis of CPIV3.