MONOCLONAL ANTIBODY-DEFINED EPITOPE MAP OF EXPRESSED RUBELLA-VIRUS PROTEIN DOMAINS
MONOCLONAL ANTIBODY-DEFINED EPITOPE MAP OF EXPRESSED RUBELLA-VIRUS PROTEIN DOMAINS
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DOI：
10.1128/jvi.65.8.3986-3994.1991
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发表时间：
1991-08-01
影响因子：
5.4
通讯作者：
SIMMONS, D
中科院分区：
文献类型：
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作者：
WOLINSKY, JS;MCCARTHY, M;SIMMONS, D
用风疹病毒（RV）的Therien株感染BALB/C小鼠，利用纯化的病毒粒子靶点，采用酶联免疫吸附试验（ELISA）筛选分泌杂交体，建立了扩增的小鼠单克隆抗体文库。利用pGE374和pGE374衍生的表达载体，采用多种策略构建了含有特定RV cDNA片段的质粒。混合reca -RV- β -半乳糖苷酶(LacZ)-或reca -RV-截断的LacZ-含蛋白共同代表RV结构蛋白的整个开放阅读框在大肠杆菌中过表达。在变性条件下电泳分离后，用ELISA和免疫印迹对细菌裂解物进行检测。通过这种方法，与线性决定因素发生反应的单克隆抗体在以下区域内定义了表位：单克隆抗体C-1、C-2和C-8结合衣壳区C-9至C29的预测氨基末端21个氨基酸的表位；MAb C-9结合C64和C97结构域；单克隆抗体E2-1至E2-6结合E2糖蛋白骨干区E2(1)至E2(115)；单克隆抗体E1-18和E1-20与E1(202) ~ E1（283）糖蛋白区结合。MAb E1-18能中和RV传染性；MAb E1-20可中和感染性并适度抑制血凝。对选定的合成肽的分析证实了用表达蛋白推断出的几个分子结构域。这些质粒结构和多肽已被证明在开始解开这种人类病原体的几个抗原位点的分子组织方面是有用的。
An expanded library of murine monoclonal antibodies (MAbs) was generated by infecting BALB/C mice with the Therien strain of rubella virus (RV) and selecting secreting hybrids by enzyme-linked immunosorbent assay (ELISA) using purified virion targets. A panel of plasmids containing specified RV cDNA fragments was also constructed by using a variety of strategies with pGE374- and pGE374-derived expression vectors. Hybrid RecA-RV-beta-galactosidase (LacZ)- or RecA-RV-truncated LacZ-containing proteins collectively representing the entire open reading frame of the structural proteins of RV were overexpressed in Escherichia coli. Bacterial lysates were then probed by ELISA with selected MAbs and by immunoblot following separation by electrophoresis under denaturing conditions. With this approach, MAbs that appeared to react with linear determinants defined epitopes localized within the following domains: MAbs C-1, C-2, and C-8 bind epitopes within the predicted amino-terminal 21 amino acids of the capsid region C-9 to C29; MAb C-9 binds to a domain bounded by C64 and C97; MAbs E2-1 through E2-6 bind to the E2 glycoprotein backbone region from E2(1) to E2(115); MAbs E1-18 and E1-20 bind to the E1 glycoprotein region from E1(202) to E1(283). MAb E1-18 neutralizes RV infectivity; MAb E1-20 neutralizes infectivity and modestly inhibits hemagglutination. Analyses with selected synthetic peptides have confirmed several of the molecular domains deduced with the expressed proteins. These plasmid constructions and peptides have proven useful in beginning to unravel the molecular organization of several antigenic sites of this human pathogen.