Pallas D C, Schley C, Mahoney M, Harlow E, Schaffhausen B S, Roberts T M
J Virol. 1986 Dec;60(3):1075-84. doi: 10.1128/JVI.60.3.1075-1084.1986.
Polyomavirus small t antigen was purified from genetically engineered Escherichia coli and used as the immunogen for the production of polyclonal and monoclonal antibodies. A new series of plasmids for increased expression of polyomavirus T antigens or a T antigen-beta-galactosidase fusion protein was constructed by replacing sequences coding for the ribosome-binding site of previously published plasmids with a chemically synthesized sequence that has a higher degree of complementarity to the 3' end of the 16S rRNA. Cells expressing the fusion protein from the plasmid with the synthetic sequence contained 5- to 10-fold more fusion protein after a 3-h induction than did control cells. Pulse-labeling of cells bearing the new plasmids revealed that the T antigens were synthesized at high levels after induction: 10% of total synthesis for small t; 15% for Py-1387T middle T, a truncated mutant of middle T; and probably 1 to 5% for middle T. Small t and Py-1387T middle T, but not wild-type middle T, were seen as minor bands in total cell protein analyzed on sodium dodecyl sulfate-polyacrylamide gels stained with Coomassie blue. A simple, rapid procedure for purification of bacterial small t from the pellet of sonicated bacteria yielded 1 to 2 mg of small t per liter of bacterial culture at 80 to 90% homogeneity. High-titer polyclonal rabbit antisera raised against purified small t recognized all three T antigens and were suitable for immunoaffinity purification of middle T. Mouse monoclonal antibodies raised against bacterial small t were of four classes, immunoprecipitating either all three polyomavirus T antigens, small t and middle T only, primarily small t, or middle T and large T in preference to small t. One of the latter monoclonal antibodies also immunoprecipitated large T but not small t of simian virus 40, suggesting that the site recognized by this antibody may be functionally important. None of the monoclonal antibodies yielded an immunoprecipitate active in phosphorylating middle T in vitro.
多瘤病毒小t抗原从基因工程大肠杆菌中纯化出来，并用作生产多克隆和单克隆抗体的免疫原。通过用与16S rRNA 3'端具有更高互补度的化学合成序列替换先前发表的质粒中编码核糖体结合位点的序列，构建了一系列用于增加多瘤病毒T抗原或T抗原-β-半乳糖苷酶融合蛋白表达的新质粒。用合成序列的质粒表达融合蛋白的细胞在诱导3小时后所含融合蛋白比对照细胞多5至10倍。对携带新质粒的细胞进行脉冲标记显示，诱导后T抗原大量合成：小t占总合成量的10%；Py-1387T中T（中T的截短突变体）占15%；中T可能占1%至5%。在考马斯亮蓝染色的十二烷基硫酸钠-聚丙烯酰胺凝胶上分析总细胞蛋白时，小t和Py-1387T中T而非野生型中T表现为次要条带。一种从超声破碎细菌的沉淀中纯化细菌小t的简单、快速方法，每升细菌培养物可产生1至2毫克小t，纯度为80%至90%。用纯化的小t制备的高效价多克隆兔抗血清可识别所有三种T抗原，适用于中T的免疫亲和纯化。针对细菌小t制备的小鼠单克隆抗体有四类，可免疫沉淀所有三种多瘤病毒T抗原、仅小t和中T、主要是小t，或优先于小t的中T和大T。后一种单克隆抗体之一还可免疫沉淀猿猴病毒40的大T而非小t，表明该抗体识别的位点可能具有功能重要性。这些单克隆抗体均未产生在体外使中T磷酸化的免疫沉淀活性。
