一株A和B亚群禽白血病病毒特异性单克隆抗体的制备及其特性

Development and characterization of monoclonal antibody to subgroup A and B avian leukosis virus

目的:制备A和B亚群禽白血病病毒(Avian leukosis virus,ALV)特异性单克隆抗体。方法:用ALV-A-SDAU09C1株的env-gp85基因的PCR产物构建重组表达性质粒pET32a-SDAU09C1-gp85,经IPTG诱导后,表达分子量为53 kD的ALV-A囊膜gp85蛋白与GST的融合蛋白,将表达产物免疫BALB/c小鼠,取其脾脏细胞与骨髓瘤细胞SP2/0进行融合,筛选杂交瘤细胞株。结果:获得了1株(A6D1株)能与A和B亚群ALV发生反应但不与J亚群ALV反应的杂交瘤细胞株。Western blot试验结果表明,单克隆抗体识别的A和B亚群ALV囊膜糖蛋白的分子量为53 kD。在IFA中,这株单克隆抗体可以与所试验的3株ALV-A和1株ALV-B毒株反应,而与4株ALV-J亚群的毒株不反应。结论:A6D1株单克隆抗体可以用于A和B亚群ALV感染的诊断和流行病学调查,弥补了目前只有J亚群ALV特异性单克隆抗体可用的不足。

Objective：Development and characterization of monoclonal antibody to subgroup A and B avian leukosis virus.Methods：The env-gp85 gene of ALV-A-SDAU09C1 was subcloned into the prokaryotic expressing vector pET32a and recombinant vector pET32a-SDAU09C1-gp85 was further transformed into Escherichia coli strain BL21 for expression under the induction of IPTG.After IPTG induction,there was a new protein band about 53 kD on SDS-PAGE.The expressed proteins were vaccinated into Balb/c mice.Results：One Mab（A6D1）,which reacted with all exogenous subgroups of AL-AB but not with endogenous subgroup J,was obtained.Using immunofluorescence assay（IFA）,A6D1 reacted with 3 ALV-A and 1 ALV-B,but not with 4 ALV-J.Western Blot showed that molecular weight of ALV AB envelope glycoprotein or unglycosylated envelope protein recognized by Mabs was about 53 kD.Conclusion：A6D1 can be used for diagnosis and epidemology of ALV-AB,which compensated for only the subgroup J ALV could be used in clinical.