羊口疮病毒119蛋白表达、纯化和多克隆抗体的制备  

Expression and purification of orf virus encoded protein 119 and preparation a polyclonal antibody

作　　者：陈慧芹[1] 罗树红[2] 梁婉菲 陈瑜[1] 梁振明[1] 高其庆 郝文波[1] 

机构地区：[1]南方医科大学检验与生物技术学院抗体工程研究所,广东广州510515 [2]佛山科学技术学院口腔医学院医学检验学系,广东佛山528000

出　　处：《生物技术》2017年第2期112-116,173,共6页Biotechnology

基　　金：国家自然科学基金项目("羊痘病毒ORFV119蛋白与宿主细胞相互作用的分子机制";No.31170147;"羊口疮病毒ORFV118蛋白调控宿主细胞凋亡的分子机制研究";No.31672536)

摘　　要：[目的]克隆表达羊口疮病毒ORFV119蛋白,以纯化重组蛋白为免疫原制备鼠多克隆抗体,并鉴定抗体的特性。[方法]PCR扩增ORFV119基因,克隆入原核表达载体p ET-28a(+)中构建重组质粒p ET28a-119。经双酶切和测序正确后,转化感受态大肠杆菌BL21,IPTG诱导表达,SDS-PAGE鉴定融合蛋白表达情况。表达产物进行超声破碎和Ni柱纯化,之后目的蛋白免疫BALB/c小鼠,制备ORFV119多克隆抗体并对其通过中和实验进行鉴定。[结果]成功构建重组质粒p ET28a-119,在大肠杆菌BL21中ORFV119融合蛋白以部分可溶形式表达。可溶性目的蛋白纯化后作为免疫原制备鼠多克隆抗体,抗体效价达1∶12 800,中和实验显示该多抗具有保护作用,可减轻宿主细胞在病毒感染时的病变效应(中和效价66 ND50/m L)。[结论]成功诱导表达、纯化ORFV119蛋白并制备其多克隆抗体,为深入研究ORFV感染、发病机理及羊口疮疾病的临床诊断奠定基础。[ Objective] To express and purify recombinant orf virus encoded protein 119 （ORFV119 ） in E. coli, and prepare a polyclonal antibody （pAb） against ORFV119 for immunoassays. [ Methods ] The gene ORFV119 was amplified by PC R from orf viral genome,and subcloned into the expression vector pET28a（ ＋ ） to generate a recombinant plasmid pET28a- 119 and ex- press ORFV119 - His tagged fusion protein. Then the plasmid pET28a - 119 was transformed into E. coli BL2I. The transfor- mants were induced by IPTG. The His -tagged fusion protein（ORFV119） was purified through Ni -chelating affinity chroma- tography. The purified ORFVll9 was used as an immunogen to inject into BALB/c mouse to produce anti- ORFVll9 pAb which was characterized by neutralization test analysis. [ Results ] The gene encoded ORFVI 19 was successfully amplified from off viral genome. The His -tag fusion protein of ORFVII9 was expressed and purified from E. coli BI21. A pAb anti -OR- FV119 was produced and purified from immunized BALB/c mouse sermn with the titer of 1：12 800. The neutralization test re- suits demonstrate that anti -ORFVII9 can attenuate the viral virulence（ neutralization titer 66 ND50/mL）. [ Conclusion] The recombinant protein ORFV119 and a polyclonal anti - ORFVll9 antibody were successfully obtained. The recombinant OR- FV119 and pAb will be valuable tools for further study of viral infection biology, pathogenesis, and off clinical diagnosis.

关 键 词：羊口疮病毒 ORFV119 纯化 多克隆抗体 中和实验 

分 类 号：Q784[生物学—分子生物学]