马立克病病毒Meq单克隆抗体的制备
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摘要
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.
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.
引文
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[15]许凯迪,高俊娜,侯天龙,等.H17N10亚型蝙蝠流感病毒NS1蛋白的表达及其特性[J].中国兽医科学,2016,46(04):424-429.XU Kai-di,GAO Jun-na,HOU Tian-long,et al.Expression and characterization of bat H17N10 influenza virus NS1 protein[J].Chinese Veterinary Science,2016,46(04):424-429.(in Chinese)
[16]薛庆善.体外培养的原理与技术[M].第1版.北京:科学出版社,2001:448-449.XUE Qing-shan.Principles and techniques of in vitro culture[M].1st ed.Beijing:Science Press,2001:448-449.(in Chinese)
[17]LIU J L,LEE L F,YE Y,et al.Nucleolar and nuclear localization properties of a herpesvirus b ZIP oncoprotein,MEQ[J].J Virol,1997,71(4):3188-3196.
[18]QIAN Z,BRUNOVSKIS P,RAUSCHER F 3rd,et al.Transactivation activity of Meq,a Marek's disease herpesvirus b ZIP protein persistently expressed in latently infected transformed T cell[J].J Virol,1995,69(7):4037-4044.
[19]XIE Q,ANDERSON A S,MORGAN R W.Marek's disease virus(MDV)ICP4,pp38,and meq genes are involved in the maintenance of transfor mation of MDCC-MSB1 MDV-transformed lymphoblastoid cells[J].J Virol,1996,70(2):1125-1131.
[20]韦平,崔治中,龙进学,等.马立克氏病病毒MEQ蛋白单克隆抗体的制备及应用研究[J].广西大学学报:自然科学版,2002,27(1):5-9.WEI Ping,CUI Zhi-zhong,LONG Jin-xue,et al.The development of monoclonal antibody(Mc Ab)against MEQ protein of Marek's disease virus(MDV)and its applications[J].Journal of Guangxi University:Nature Science Edition,2002,27(1):5-9.(in Chinese)
[2]VENUGOPAL K.Marek's disease:an update on oncogenic mechanisms and control[J].Res Vet Sci,2000,69(1):17-23.
[3]WITTER R L.Control strategies for Marek's disease:a perspective for the future[J].Poult Sci,1998,77(8):1197-1203.
[4]BARROW A,VENUGOPAL K.Molecular characteristics of very virulent European MDV isolates[J].Acta Virologica,1999,43(2-3):p.90-93.
[5]RAJA A,DHINAKAR R G,BHUVANESWARI P,et al.Detection of virulent Marek's disease virus in poultry in India[J].Acta Virologica,2009,53(4):255-260.
[6]ZHANG Y P,LIU C J,ZHANG F,et al.Sequence analysis of the Meq gene in the predominant Marek's disease virus strains isolated in China during 2006-2008[J].Virus Genes,2011,43(3):353-357.
[7]韦平.马立克氏病研究的最新进展[J].广西农业生物科学,2000,19(2):110-115.WEI Ping.Marek's Disease-An update review[J].Journal of Guangxi Agriculture and Biology Science,2000,19(2):110-115.(in Chinese)
[8]JONES D,LEE L,LIU J L,et al.Marek disease virus encodes a basic-leucine zipper gene resembling the fos/jun oncogenes that is highly expressed in lymphoblastoid tumors[J].Proc Natl Acad Sci USA,1992,89(9):4042-4046.
[9]付本懂,王鲁,韦旭斌.马立克病病毒致瘤相关基因的研究进展[J].中国兽医科学,2006,36(07):592-596.FU Ben-dong,WANG Lu,WEI Xu-bin.Advances in oncogenic genes of Marek's disease virus[J].Chinese Veterinary Science,2006,36(07):592-596.(in Chinese)
[10]DENG X,LI X,SHEN Y,et al.The Meq oncoprotein of Marek's disease virus interacts with p53 and inhibits its transcriptional and apoptotic activities[J].Virol J,2010,7:348.
[11]LIU J L,YE Y,LEE L F,et al.Transforming potential of the herpesvirus oncoprotein MEQ:morphological transformation,serum-independent growth,and inhibition of apoptosis[J].J Virol,1998,72(1):388-395.
[12]陈鸿军,宋翠萍,秦爱建,等.MDV-1 CVI988/Rispens疫苗毒株体外感染期间VP22的表达及细胞间转导作用[J].中国科学C辑:生命科学,2006,36(2):145-149.CHEN Hong-jun,SONG Cui-ping,QIN Ai-jian,et al.Expression of VP22 and cell transduction during infection of MDV-1 CVI988/Rispens in vitro[J].Science in China Series C:Life Science,2006,36(2):145-149.(in Chinese)
[13]JUNG Y S,QIAN Y,CHEN X.Pirh2 E3 ubiquitin ligase targets DNA polymerase Eta for 20S proteasomal degradation[J].Mol Cell Biol,2010,30(4):1041-1048.
[14]陈鸿军,沈欣悦,陈丹清,等.鸡毒支原体烯醇化酶单克隆抗体研制及黏附阻断[J].中国兽医学报,2012,32(6):862-870.CHEN Hong-jun,SHEN Xin-yue,CHEN Dan-qing,et al.Preparation and adherence inhibition by monoclonal antibodies to enolase of Mycoplasma gallisepticum[J].Chinese Journal of Veterinary Science,2012,32(6):862-870.(in Chinese)
[15]许凯迪,高俊娜,侯天龙,等.H17N10亚型蝙蝠流感病毒NS1蛋白的表达及其特性[J].中国兽医科学,2016,46(04):424-429.XU Kai-di,GAO Jun-na,HOU Tian-long,et al.Expression and characterization of bat H17N10 influenza virus NS1 protein[J].Chinese Veterinary Science,2016,46(04):424-429.(in Chinese)
[16]薛庆善.体外培养的原理与技术[M].第1版.北京:科学出版社,2001:448-449.XUE Qing-shan.Principles and techniques of in vitro culture[M].1st ed.Beijing:Science Press,2001:448-449.(in Chinese)
[17]LIU J L,LEE L F,YE Y,et al.Nucleolar and nuclear localization properties of a herpesvirus b ZIP oncoprotein,MEQ[J].J Virol,1997,71(4):3188-3196.
[18]QIAN Z,BRUNOVSKIS P,RAUSCHER F 3rd,et al.Transactivation activity of Meq,a Marek's disease herpesvirus b ZIP protein persistently expressed in latently infected transformed T cell[J].J Virol,1995,69(7):4037-4044.
[19]XIE Q,ANDERSON A S,MORGAN R W.Marek's disease virus(MDV)ICP4,pp38,and meq genes are involved in the maintenance of transfor mation of MDCC-MSB1 MDV-transformed lymphoblastoid cells[J].J Virol,1996,70(2):1125-1131.
[20]韦平,崔治中,龙进学,等.马立克氏病病毒MEQ蛋白单克隆抗体的制备及应用研究[J].广西大学学报:自然科学版,2002,27(1):5-9.WEI Ping,CUI Zhi-zhong,LONG Jin-xue,et al.The development of monoclonal antibody(Mc Ab)against MEQ protein of Marek's disease virus(MDV)and its applications[J].Journal of Guangxi University:Nature Science Edition,2002,27(1):5-9.(in Chinese)