【摘要】 利用重组真核质粒pcDNA3-GP5免疫BALB/c小鼠,取脾细胞和骨髓瘤细胞SP2/0融合,经间接ELISA筛选和3次有限稀释法克隆,得到3株能稳定分泌抗猪繁殖与呼吸综合征病毒(PRRSV)GP5蛋白的单克隆抗体(McAb)杂交瘤细胞株,分别命名为4C9、5D10、5F12,其中5D10的Ig亚类为IgM。ELISA检测杂交瘤细胞培养上清液抗体的效价为1∶64~1∶1 024,腹水的效价为1∶3200~1∶10240,IFA和IPMA的检测结果均为阳性。Western-blot检测证明,这3株McAb均针对PRRSV的GP5蛋白。中和试验表明,5D10和5F12具有病毒中和活性,中和效价分别为1∶40和1∶80。5D10相对亲和力大于5F12,3株细胞连续培养20代后仍能稳定分泌抗体,表明抗GP5蛋白中和性McAb制备成功。更多还原

【Abstract】 To prepare monoclonal antibodies(McAb)against GP5 protein of porcine reproduction and respiratory syndrome virus(PRRSV),the plasmid encoding GP5 gene of PRRSV-S1 strain was used.BALB/c mice were immunized by subcutaneous injection.The induced spleen cells were fused with SP2/0.The McAbs anti-GP5 were obtained by screening with indirect ELISA and subcloned 4 times,and were preliminarily characterized.Three hybridoma cell strains steadily secreting monoclonal antibodies against GP5 protein of PRRSV were obtained,which were separately named 4C9,5D10 and 5F12.The isotype of the 5D10 was IgM.The ascites titers of PRRSV-specific ELISA antibodies obtained by mice were 1∶3 200 to 1∶10 240.Relative affinity of 5D10 was tighter than that of 5F12.It indicated that these McAbs were against different regions of GP5 protein of PRRSV.Moreover,the McAbs 5D10 and 5F12 had PRRSV-specific neutralization activity.The titers of the neutralizing antibodies were 1∶40 and 1∶80.Two monoclonal antibodies having virus neutralizing activity to GP5 protein of PRRSV were made in this study.It should be very useful to diagnose and prevent PRRSV infection.更多还原