猪圆环病毒重组Cap蛋白单克隆抗体的制备及初步应用

利用重组杆状病毒表达系统在昆虫细胞中表达PCV2-ORF2基因,并以构建的重组杆状病毒为免疫原,通过杂交瘤技术,研制针对PCV2的单克隆抗体,为建立准确快速的PCV2诊断方法奠定基础。首先将PCV2-ORF2基因克隆到杆状病毒的转移载体pFastBacTM1中,然后将其转化入大肠杆菌感受态细胞DH10Bac中,与DH10Bac中的穿梭载体Bacmid发生转座,通过抗性和蓝白斑筛选,得到重组杆状病毒质粒reBacmid-ORF2,通过脂质体介导reBacmid-ORF2转染Sf9细胞,成功获得了表达PCV2-ORF2基因的P1代重组杆状病毒。将所获得的重组杆状病毒经Vivaflow200浓缩后,免疫6~8周龄BALB/c小鼠,取其脾脏与Sp2/0细胞融合,以IFA进行筛选,经多次亚克隆后得到3株能稳定分泌抗体的杂交瘤细胞,分别命名为5H6、4E2和5F7。通过IFA、IPMA及Western-blot试验对3株单抗特异性进行分析鉴定。结果显示,3株单抗均能与PCV2-Cap蛋白产生特异性的反应,并利用流式细胞术初步建立了对PCV2毒株感染PK15细胞的检测方法,从而为快速检测PCV2病毒奠定了基础。

Development of monoclonal antibodies against porcine circorvirus type 2 and their primary application

ORF2 of porcine circovirus type 2（PCV2）was cloned into a baculovirus expression vector and the gene product was expressed in insect cells.Using the recombinant baculovirus as immunogen,by the hybridoma technology,we prepared the PCV2 specific monoclonal antibody,to lay the foundation of establishing an accurate PCV2detection method.The PCV2-ORF2 genes was subcloned into baculovirus transfer vector pFastBacTM1,E.coli DH10Bac containing baculovirus shutter vector was transformed with the recombinant plasmid,colonies of E.coli containing recombinant bacmid（reBacmid-ORF2）were selected by the resistance,blue and white selection.Through the lipofectamine TM2000,the reBacmid-ORF2 was transfected into insect Sf9cells,the recombinant baculovirus P1generation was obtained successfully.The recombinant baculovirus was concentrated through the Vivaflow200,immuned into 6-8-week-old female BALB/c mice,then the spleen cells of immunized mice were collected and fused with Sp2/0 myeloma cells,hybridoma were screened by indirect immunofluo rescence assay（IFA）.Three hybridomas consistantly secreting monoclonal antibodies（mAbs）were developed after three subcloning,named as5H6,4E2and 5F7 respectively.By IFA,IPMA and Western-blot analysis showed that three mAbs were able to react with PCV2-Cap.In this study,the flow cytometry based on mAbs was also developed to detect PCV2 in PK15 cells.