Characterization of baculovirus p10 synthesis using monoclonal antibodies.
Characterization of baculovirus p10 synthesis using monoclonal antibodies.
复制标题
使用单克隆抗体表征杆状病毒 p10 合成。
DOI：
10.1016/0042-6822(87)90038-9
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发表时间：
1987
期刊：
影响因子：
3.7
通讯作者：
Beaudreau,GS
中科院分区：
文献类型：
--
作者：
Quant-Russell,RL;Pearson,MN;Rohrmann,GF;Beaudreau,GS
制备了一系列针对拟甜菜夜蛾多衣壳核型多角体病毒(OpMNPV)病毒粒子蛋白的单抗。这些抗体中的四个与蛋白质14kd在蛋白印迹上反应，这些蛋白质是经电泳法分离的OpMNPV病毒粒子蛋白。这些抗体用于从OpMNPVDNA的λgt11表达文库中鉴定免疫活性克隆。通过将来自λgt11克隆的插入DNA与OpMNPV基因组DNA的酶切斑点杂交，以及通过对λgt11插入片段末端的测序，这些克隆被证明包含部分p10基因。使用由trpE载体中的p10阅读框的选定部分制成的融合蛋白来定位包含四种单抗识别的表位的区域。其中一种p10抗体被用来通过免疫印迹和免疫荧光染色来鉴定感染的毒蛾细胞中p10的合成。感染后14小时免疫荧光显微镜检测到p10蛋白，20小时后p10蛋白形成染色较深的细胞质结构。在感染细胞的免疫印迹中，观察到两种形式的p10(约14kd和15kd)。其中一株p10单抗在未感染的昆虫细胞和大鼠成纤维细胞中表现出与细胞骨架结构的强烈交叉反应。
A series of monoclonal antibodies were produced against virion proteins of the multicapsid nuclear polyhedrosis virus ofOrgyia pseudotsugata(OpMNPV). Four of these antibodies reacted with a protein of 14 kd on Western blots of electrophoretically separated OpMNPV virion proteins. These antibodies were used to identify immunoreactive clones from a λ gt11 expression library of OpMNPV DNA. By hybridization of insert DNA from the λ gt11 clones to blots of digests of OpMNPV genomic DNA, and by sequencing the ends of the λ gt11 inserts, these clones were shown to contain a portion of the p10 gene. The regions containing epitopes recognized by the four monoclonal antibodies were located using fusion proteins made from selected portions of the p10 reading frame in a trpE vector. One of the p10 antibodies was used to characterize p10 synthesis in infectedLymantria disparcells by using Western blots and immunofluorescent staining. The p10 protein was detected with immunofluorescent microscopy at 14 hr postinfection and by 20 hr it formed intensely staining cytoplasmic structures. On Western blots of infected cells, two forms of p10 (of about 14 and 15 kd) were observed. One of the p10 monoclonal antibodies showed a strong cross-reaction with cytoskeletal structures in uninfected insect cells and rat fibroblasts.