POLYOMAVIRUS SMALL T-ANTIGEN - OVERPRODUCTION IN BACTERIA, PURIFICATION, AND UTILIZATION FOR MONOCLONAL AND POLYCLONAL ANTIBODY-PRODUCTION
POLYOMAVIRUS SMALL T-ANTIGEN - OVERPRODUCTION IN BACTERIA, PURIFICATION, AND UTILIZATION FOR MONOCLONAL AND POLYCLONAL ANTIBODY-PRODUCTION
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DOI：
10.1128/jvi.60.3.1075-1084.1986
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发表时间：
1986-12-01
影响因子：
5.4
通讯作者：
ROBERTS, TM
中科院分区：
文献类型：
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作者：
PALLAS, DC;SCHLEY, C;ROBERTS, TM
多瘤病毒小 t 抗原从基因工程大肠杆菌中纯化，用作生产多克隆和单克隆抗体的免疫原。通过用与16S rRNA的3''端具有更高程度的互补性的化学合成序列替换先前公开的质粒的核糖体结合位点的编码序列，构建了用于增加多瘤病毒T抗原或T抗原-β-半乳糖苷酶融合蛋白的表达的新系列质粒。诱导3小时后，从具有合成序列的质粒表达融合蛋白的细胞所含的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 抗原的合成水平很高：小 t 占总合成的 10%； Py-138T 中间 T 为 15%，中间 T 的截短突变体；中 T 可能为 1% 至 5%。在考马斯蓝染色的十二烷基硫酸钠-聚丙烯酰胺凝胶上分析时，小 t 和 Py-138T 中 T（而非野生型 T）被视为总细胞蛋白中的次要条带。通过简单、快速的程序从超声处理的细菌沉淀中纯化小细菌，每升细菌培养物可产生 1 至 2 毫克小细菌，同质性为 80% 至 90%。高滴度多克隆兔抗血清和针对纯化小 t 的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 产生的小鼠单克隆抗体分为四类，免疫沉淀 (i) 所有三种多瘤病毒 T 抗原，(ii) 仅小 t 和中 T，(iii) 主要是小 t，或 (iv) 中 T 和大 T 优先于小 t。后者的一种单克隆抗体也能免疫沉淀猿猴病毒 40 的大 T，但不能免疫沉淀小 t，这表明该抗体识别的位点可能具有重要的功能。所有单克隆抗体均未产生体外磷酸化中 T 的免疫沉淀活性。
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 polymavirus 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 complementary 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-138T middle T, a truncated mutant of middle T; and probably 1 to 5% for middle T. Small t and Py-138T middle T, but not wild-type 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 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 and 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 (i) all three polyomavirus T antigens, (ii) small t and middle T only, (iii) primarily small t, or (iv) 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.