猪圆环病毒2型衣壳蛋白单克隆抗体的制备及鉴定

Preparation and Identification of Monoclonal Antibodies Specific for Capsid Protein of Porcine Circovirus Type 2

为获得猪圆环病毒2型(PCV2)衣壳蛋白单克隆抗体,将516 bp的ORF2基因片段克隆入pET-28a(+),转化入大肠杆菌,在IPTG的诱导下表达,用SDS-PAGE和Western blot对表达产物进行鉴定。将纯化的重组蛋白免疫小鼠,制备单克隆抗体,叠加ELISA方法检测两株单克隆抗体识别的抗原表位关系。SDS-PAGE和Western blot结果表明重组蛋白ORF2得到高效表达;筛选获得两株稳定分泌抗ORF2单克隆抗体的杂交瘤细胞系3H3和3F2,其分泌的单体均为IgG1亚型;且均具有较高的特异性和敏感性;叠加ELISA显示,两株单克隆抗体识别不同的抗原表位。本研究制备的单抗有较好的生物学特性,为PCV2的研究及快速准确诊断方法的建立提供了平台。

To produce monoclonal antibodies （MAbs） against capsid protein of porcine circoviru 2, a truncated ORF2 gene （516 bp） was cloned into pET-28a （ ＋ ） vector. The recombinant protein was trans- formed into Escherichia coli, expressed through induction of IPTG, and then identified by SDS - PAGE and western blot. Splenocytes from BALB/c mice immunized with purified target protein were fused with SP2/0 cells to produce MAbs, which were selected by indirect ELISA. The relationship between MAbs was detected by additive ELISA. The results showed that the recombinant ORF2 protein was successfully expressed and two MAbs （3H3 and 3F2） belonged to IgG1 subtype were produced. The additive ELISA results showed that the two MAbs recognized different epitopes. So this study supplied important tools for detecting and studying PCV2.