Mutations affecting conformation or sequence of neutralizing epitopes identified by reactivity of viable plaques segregate from syn and ts domains of HSV-1(F) gB gene.
Mutations affecting conformation or sequence of neutralizing epitopes identified by reactivity of viable plaques segregate from syn and ts domains of HSV-1(F) gB gene.
复制标题
影响通过活斑块的反应性鉴定的中和表位的构象或序列的突变与HSV-1(F) gB基因的syn和ts结构域分离。
DOI：
10.1016/0042-6822(84)90194-6
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发表时间：
1984
期刊：
影响因子：
3.7
通讯作者：
Roizman,B
中科院分区：
文献类型：
--
作者：
Kousoulas,KG;Pellett,PE;Pereira,L;Roizman,B
选择三类HSV-1（F）突变体，其表达对糖蛋白B（gB）的两种高效力型普通单克隆抗体H126-5和H233的抗性表型。1类突变体，选择从非诱变病毒股票的中和抗性，表达了gB的反应，在生物素-亲和素增强的表面免疫测定和免疫沉淀试验与选择抗体。2类和3类突变体被选择为无反应性的生物素-亲和素-增强表面免疫测定从BUdR诱变，预中和病毒股票，但不同的是，选择抗体免疫沉淀的gB的2类，但不是3类。表达对一种单克隆抗体（H126-5或H233）的抗性表型的突变体在与异源抗体的所有测试中总是保持反应性，并且通过将一种突变体的完整DNA与来自另一种突变体的克隆DNA片段共转染来产生对两种抗体具有抗性的重组体。通过标记转移到1734-bp的DNA片段来定位1类突变。在生物素-抗生物素蛋白增强的表面免疫测定中，用一组HSV-1（F）BamHI G的DNA片段将2类和3类突变定位到最大377 bp和最小46 bp的区域，这些DNA片段在编码gB的区域上携带交错缺失。该区域不与T. C.霍兰德河M.桑德里-戈尔丁湖E.霍兰，S。D.马林，M. Levine和J. Glorioso（1983，J.Virol.46，649-652），并且位于HSV-1（HFEM）tsB 5的is病变的3′和该病毒的syn 3基因座的5′。结论是：（i）由于生物素-抗生物素蛋白增强的表面免疫测定法不破坏噬斑中所含的病毒，因此它是鉴定和选择与特异性单克隆抗体反应和不反应的突变体的快速和方便的方法。(ii)gB可以含有多个携带中和单克隆抗体的表位位点的结构域。(iii)抗性表型可由改变表位的构象或氨基酸序列的突变引起。这些突变可能是可区分的基础上的反应性突变gB选择单克隆抗体在非变性和变性环境中，分别。
Three classes of HSV-1(F) mutants expressing a resistance phenotype to two highly potent-type common monoclonal antibodies, H126-5 and H233, to glycoprotein B (gB) were selected. Class 1 mutants, selected for resistance to neutralization from nonmutagenized virus stocks, expressed a gB which reacted in biotin-avidin-enhanced surface immunoassays and in immune precipitation tests with the selecting antibodies. Class 2 and 3 mutants were selected for nonreactivity in the biotin-avidin-enhanced surface immunoassay from BUdR-mutagenized, preneutralized virus stocks, but differ in that the selecting antibodies immune precipitated the gB of Class 2 but not that of Class 3. Mutants expressing a resistance phenotype to one monoclonal antibody (H126-5 or H233) invariably retained reactivity in all tests with the heterologous antibody, and recombinants resistant to both antibodies were produced by cotransfection of intact DNA of one mutant with a cloned DNA fragment from another mutant. Class 1 mutations were mapped by marker transfer to a 1734-bp DNA fragment. Class 2 and 3 mutations were mapped to a region defined by a maximum of 377 by and a minimum of 46 bp, in a biotin-avidin-enhanced surface immunoassay with a panel of DNA fragments of HSV-1(F)BamHI G carrying staggered deletions across the region encoding gB. This region does not overlap the neutralizing antibody determinant site mapped by T. C. Holland, R. M. Sandri-Goldin, L. E. Holland, S. D. Marlin, M. Levine, and J. Glorioso (1983,J. Virol.46, 649–652) and is located 3′ to the is lesion of HSV-1(HFEM)tsB5 and 5′ to thesyn3 locus of that virus. It was concluded that (i) inasmuch as the biotin-avidin-enhanced surface immunoassay does not destroy the virus contained in the plaque, it is a rapid and convenient method for both identification and selection of mutants reactive and nonreactive to specific monoclonal antibodies. (ii) gB may contain multiple domains carrying epitopic sites of neutralizing monoclonal antibodies. (iii) The resistance phenotype may arise from mutations which alter the conformation or the amino acid sequence of the epitope. These mutations might be differentiable on the basis of reactivity of mutated gB with selecting monoclonal antibody in nondenaturing and denaturing environments, respectively.