抗牛病毒性腹泻病毒单克隆抗体的研制

将含有牛病毒性腹泻病毒1型(BVDV1)囊膜糖蛋白E2编码基因的真核表达质粒pEC143免疫BALB/c小鼠,应用淋巴细胞杂交瘤技术,以重组E2蛋白纯化产物为检测抗原,经间接ELISA法筛选和3次有限稀释法克隆,得到1株能稳定分泌抗BVDV1和BVDV2单克隆抗体的杂交瘤细胞株.该3D8株单抗能识别BVDV1标准毒株(NADL、OregonC24V)、BVDV2标准毒株(890、104)、2株BVDV1分离株和3株BVDV2分离株,而不能识别牛轮状病毒、牛疱疹病毒1型,显示出良好的特异性.阳性杂交瘤细胞的上清液、腹水ELISA抗体效价分别为2~9和10×2~7,为IgM亚类.其单克隆抗体杂交瘤细胞株的获得,为建立单抗介导的检测BVDV1和BVDV2抗原和抗体的血清学方法和实现标准化诊断奠定基础.

The development of a hybridoma cell strain secreting monoclonal antibody against BVDV1 and BVDV2

The glycoprotein E2 from bovine viral diarrhea virus genotype 1 or BVDV2 is an immunodominant antigen inducing the neutralizing antibodies after natural infection or following immunization with live or killed vaccines. In this study, E2 glycoprotein was focused and the BALB/c mice were immunized with the recombinant eukaryotic plasmid pEC143 encoding a secreted form of E2 glycoprotein according to the immune protocol for making monoclonal antibody. Using lymphocyte hybridoma technique, the mouse spleen cells were fused with myeloma cells. A hybridoma cell strain which can secrete stably monoclonal antibody against the E2 glycoprotein of the BVDV1 and BVDV2, was obtained by indirect ELISA using the coated antigen from the purified expression products of recombinant prokaryotic plasmid of pET28a-BE2 and was cloned through three times limiting dilution. The hybridoma cell strain was named 3D8. The mono-elonal antibody secreted by 3D8 strain reacted with not only the NADL and OregonC24V reference strains and two isolates of BVDV1, but also the 890 and 104 reference strains and three isolates of BVDV2, but not cross reaction with BRV and BHV-1. The hybridoma cell strain belongs to IgM subset. EIASA titer of monoclonal antibody which was existent in asci-tes of mice and supenatants of culture of hybridoma cells was 10 × 2~7 and 2~9 , respectively. The above preliminary data also makes a basis of serological diagnosis for BVDV1 and BVDV2.