马立克病病毒Meq单克隆抗体的制备

Generation of monoclonal antibodies against Marek's disease virus oncoprotein Meq

为研制马立克病病毒RB1B超强毒株致瘤蛋白Meq单克隆抗体,本研究通过聚合酶链式反应（PCR）扩增马立克病病毒（Md5）meq基因5′端第1~507位核苷酸,将其克隆入原核表达载体p Cold-TF,转化到BL21（DE3）感受态中,经异丙基硫代半乳糖苷（IPTG）诱导表达,对表达产物用聚丙烯酰胺凝胶电泳（SDS-PAGE）进行检测。用融合蛋白His-Meq169腹腔免疫BALB/c小鼠,利用间接免疫荧光（IFA）和Western-blot检测多抗血清;血清检测阳性后,取小鼠脾脏与小鼠骨髓瘤细胞SP2/0融合,利用IFA和有限稀释法筛选阳性单克隆杂交瘤细胞株,最后筛选出了4株阳性杂交瘤细胞株,即1C11、3E4、3G10和5H11。将获得的单抗经IFA方法检测强毒株Md5或弱毒株CVI988感染的CEF细胞,结果显示,Meq单抗可特异性识别MDV感染形成的细胞空斑。这为进一步研究Meq的生物学功能奠定了基础。

The meq gene of Marek＇s disease virus strain RB1 B with a size of 507 bp was amplified and subcloned into the prokaryotic expression vector p Cold-TF.After being induced by IPTG, Meq was expressed in E.coli BL21（DE3） cells and analyzed by SDS-PAGE. BALB/c mice were immunized with His-Meq169 peritoneally.Indirect immunofluorescence assay（IFA）and Western-blot were performed to detect antiserum.After the detection results were positive, the mice spleen were isolated and fused with SP2/0 by PEG4000 reagent.IFA and limited dilution were used to screen hybridoma clones that produced antibodies against Meq.Finally, four positive hybridoma clones were obtained and designated as 1C11,3E4,3G10 and 5H11.The positive results by IFA on chicken embryonic fibroblast（CEF）cells infected by Md5 or CVI988strain indicated that Meq monoclonal antibodies could be used to detect classical cytopathic effects obviously.These monoclonal antibodies against Meq laid a foundation for further research on the biological functions of MDV Meq.