鸭坦布苏病毒单克隆抗体的制备和治疗效果分析  

Preparation and Therapeutic Efficacy Analysis of Monoclonal Antibodies against Duck Tembusu Virus

作　　者：王丹娜 乌兰 李三 WANG Danna;WU Lan;LI San(Beijing Biomedical Science and Technology Center,Zhaofenghua Biotechnology(Nanjing)Co.,Ltd.,Beijing 102600,China)

机构地区：[1]兆丰华生物科技(南京)有限公司北京生物医药科技中心,北京大兴102600

出　　处：《中国兽医杂志》2024年第2期27-31,共5页Chinese Journal of Veterinary Medicine

摘　　要：为了制备、筛选和评价治疗鸭坦布苏病毒(DTMUV)感染的中和抗体,本试验克隆DTMUV E基因,连接至原核表达载体pET-30a(+),转化至大肠杆菌(E.coli)DH5α感受态,提取质粒,鉴定重组质粒pET30-E,转化E.coli BL21感受态,经异丙基-β-D-硫代半乳糖苷(IPTG)诱导后,通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)分析E蛋白表达,使用镍柱亲和层析纯化E蛋白,将E蛋白与弗氏佐剂混合、乳化,免疫6周龄雌性BALB/c小鼠3次后,取小鼠脾脏,分离脾细胞,在聚乙二醇(PEG)的作用下,将脾细胞与SP2/0细胞融合,采用酶联免疫吸附测定(ELISA)筛选分泌抗E蛋白抗体的杂交瘤细胞株,通过Western blot进行单克隆抗体鉴定,通过间接免疫荧光技术检测DTMUV,病毒中和试验评价单克隆抗体中和DTMUV感染的能力,并评价单克隆抗体治疗DTMUV感染小鼠的能力。结果显示,含质粒pET30-E的E.coli BL21经诱导后可在裂解的沉淀物中表达E蛋白;制备的E蛋白单克隆抗体4A1可以与E蛋白发生免疫反应,不与鸭A型肝炎病毒、呼肠孤病毒、新城疫病毒、细小病毒和H9亚型禽流感病毒发生交叉反应;4A1株杂交瘤细胞株分泌的抗体对DTMUV具有中和活性,且能够很好地识别DTMUV并与之发生中和反应,可完全清除感染小鼠血液中的DTMUV。结果表明,本试验制备的单克隆抗体4A1在应对DTMUV感染方面具有潜在治疗价值。In order to prepare,screen,and evaluate neutralizing antibodies for the treatment of duck tembusu virus(DTMUV)infection,this study cloned the DTMUV E gene,ligated it into the prokaryotic expression vector pET-30a(+),transformed into Escherichia coli(E.coli)DH5αcompetent cells,extracted the plasmid,identified the recombinant plasmid pET30-E,transformed into E.coli BL21 competent cells,induced by isopropyl-β-D-thiogalactoside(IPTG),analyzed E protein expression by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE),purified the E protein by nickel column affinity chromatography,mixed and emulsified the E protein with Freund's adjuvant,immunized 6-week-old female BALB/c mice three times,isolated spleen cells,fused spleen cells with SP2/0 cells in the presence of polyethylene glycol(PEG),screened hybridoma cells secreting antibodies against E protein using enzyme-linked immunosorbent assay(ELISA),identified monoclonal antibodies by Western blot,detected DTMUV by indirect immunofluorescence assay,evaluated the ability of monoclonal antibodies to neutralize DTMUV infection,and assessed the therapeutic ability of monoclonal antibodies in DTMUV-infected mice.The results showed that E.coli BL21 containing the plasmid pET30-E could express the E protein in the precipitate after induction;the prepared E protein monoclonal antibody 4A1 could immunoreact with the E protein and did not cross-react with duck hepatitis A virus,reovirus,Newcastle disease virus,parvovirus,and H9 subtype avian influenza virus;the antibody secreted by hybridoma cell line 4A1 exhibited neutralizing activity against DTMUV,effectively recognized and neutralized DTMUV,and completely cleared DTMUV from the blood of infected mice.The results indicate that the monoclonal antibody 4A1 prepared in this study has potential therapeutic value against DTMUV infection.

关 键 词：鸭坦布苏病毒 单克隆抗体 中和活性 治疗效果 

分 类 号：S834[农业科学—畜牧学]