Milanesi G, Barbanti-Brodano G, Negrini M, Lee D, Corallini A, Caputo A, Grossi M P, Ricciardi R P
Mol Cell Biol. 1984 Aug;4(8):1551-60. doi: 10.1128/mcb.4.8.1551-1560.1984.
We describe a novel expression vector, pBK TK-1, that persists episomally in human cells that can be shuttled into bacteria. This vector includes sequences from BK virus (BKV), the thymidine kinase (TK) gene of herpes simplex virus type 1, and plasmid pML-1. TK+-transformed HeLa and 143 B cells contained predominantly full-length episomes. There were typically 20 to 40 (HeLa) and 75 to 120 143 B vector copies per cell, although some 143 B transformants contained hundreds. Low-molecular-weight DNA from TK+-transformed cells introduced into Escherichia coli were recovered as plasmids that were indistinguishable from the input vector. Removal of selective pressure had no apparent effect upon the episomal status of pBK TK-1 molecules in TK+-transformed cells. BKV T antigen may play a role in episomal replication of pBK TK-1 since this viral protein was expressed in TK+ transformants and since a plasmid that contained only the BKV origin of replication was highly amplified in BKV-transformed human cells that synthesize BKV T antigen.
我们描述了一种新型表达载体pBK TK-1，它能在可转入细菌的人细胞中以附加体形式持续存在。该载体包含来自BK病毒（BKV）、单纯疱疹病毒1型胸苷激酶（TK）基因以及质粒pML-1的序列。TK⁺转化的HeLa细胞和143 B细胞主要含有全长附加体。每个细胞通常有20至40个（HeLa细胞）和75至120个143 B载体拷贝，不过一些143 B转化体含有数百个拷贝。将TK⁺转化细胞的低分子量DNA导入大肠杆菌后，可作为与输入载体无法区分的质粒回收。去除选择压力对TK⁺转化细胞中pBK TK-1分子的附加体状态没有明显影响。BKV T抗原可能在pBK TK-1的附加体复制中起作用，因为这种病毒蛋白在TK⁺转化体中表达，并且因为仅包含BKV复制起点的质粒在合成BKV T抗原的BKV转化人细胞中高度扩增。
