Davidson D, Hassell J A
J Virol. 1987 Apr;61(4):1226-39. doi: 10.1128/JVI.61.4.1226-1239.1987.
To overproduce biologically active polyomavirus middle T antigen, we used an adenovirus vector and human 293 cells as hosts. Two helper-independent recombinant adenoviruses were isolated that contain a hybrid transcription unit, in differing orientations, at a site in the adenovirus genome from which the E1a and most of the E1b transcription units have been deleted. The hybrid transcription unit consists of the adenovirus type 2 major late promoter and tripartite leader and a cDNA segment capable of encoding polyomavirus middle T antigen and accompanying 3' RNA-processing signals. Both recombinant viruses were stable and replicated to high titers in human 293 cells. The polyomavirus sequences were expressed, predominantly at late times after infection of 293 cells, to yield mRNAs that encoded middle T antigen. One of the recombinant viruses also expressed a middle T antigen-related protein in 293 cells. The latter was translated from one of several novel mRNA species that resulted from aberrant splicing and incomplete RNA processing of precursor RNA transcripts. Comparison of the amount of middle T antigen produced in 3T6 cells infected with polyomavirus with that in 293 cells infected with either of the recombinant adenoviruses, under optimal conditions for each system, revealed at least a 10-fold greater yield of the protein on a per-cell basis in the latter system than in the former. The recombinant-virus-encoded middle T antigen was biologically active, as evidenced by its ability to associate with and serve as a substrate for human pp60c-src. The functionality of the middle T antigen was further confirmed by demonstrating that both recombinant viruses efficiently transformed Rat-1 cells. These recombinant viruses will be useful to overproduce middle T antigen and to introduce the polyomavirus oncogene into a wide variety of mammalian cells.
为了过量生产具有生物活性的多瘤病毒中T抗原，我们使用腺病毒载体和人293细胞作为宿主。分离出两种辅助非依赖型重组腺病毒，它们在腺病毒基因组的一个位点含有一个杂交转录单元，其方向不同，该位点的E1a和大部分E1b转录单元已被删除。杂交转录单元由2型腺病毒主要晚期启动子和三联体前导序列以及一个能够编码多瘤病毒中T抗原和伴随的3'RNA加工信号的cDNA片段组成。两种重组病毒都很稳定，并且在人293细胞中能复制到高滴度。多瘤病毒序列在感染293细胞后的晚期主要表达，产生编码中T抗原的mRNA。其中一种重组病毒在293细胞中还表达了一种与中T抗原相关的蛋白质。后者是由几种新的mRNA种类之一翻译而来，这些mRNA是由前体RNA转录本的异常剪接和不完全RNA加工产生的。在每个系统的最佳条件下，比较感染多瘤病毒的3T6细胞中产生的中T抗原量与感染任何一种重组腺病毒的293细胞中产生的中T抗原量，发现后一系统中每细胞产生的该蛋白产量比前一系统至少高10倍。重组病毒编码的中T抗原具有生物活性，这可通过其与人类pp60c-src结合并作为其底物的能力来证明。通过证明两种重组病毒都能有效地转化Rat-1细胞，进一步证实了中T抗原的功能。这些重组病毒将有助于过量生产中T抗原，并将多瘤病毒癌基因导入多种哺乳动物细胞。
