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首页> 外文期刊>Molecular and Cellular Biology >Construction of a helper-free recombinant adenovirus that expresses polyomavirus large T antigen.
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Construction of a helper-free recombinant adenovirus that expresses polyomavirus large T antigen.

机译：表达多瘤病毒大T抗原的无辅助重组腺病毒的构建。

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Adenovirus-polyomavirus recombinant viruses were constructed in vitro by inserting a hybrid transcription unit composed of the adenovirus type 2 major late promoter and the early coding region of polyomavirus into the adenovirus type 5 vector Ad5 delta E1/dl309. The vector lacks the E1a and E1b transcription units and contains a unique restriction endonuclease cleavage site in their place. The polyomavirus genomic insert contained a small deletion which precluded the synthesis of functional small and middle T antigen but allowed for the synthesis of large T antigen. One recombinant virus, Ad5PyR39, which contained the hybrid transcription unit in the opposite transcriptional orientation from the overall direction of late-gene transcription, was studied in detail. Ad5PyR39 replicated efficiently without a helper virus in human 293 cells and expressed hybrid mRNAs of the expected size and composition that were translated to yield large T antigen. The large T antigen synthesized in 293 cells was the same size as that produced in mouse 3T6 cells lytically infected with polyomavirus, and this protein bound efficiently and specifically to the large-T-antigen-binding sites in polyomavirus DNA. Moreover, the large T antigen encoded by the recombinant virus proved capable of catalyzing the replication in mouse 3T6 cells of a plasmid containing the polyomavirus origin for DNA replication. Comparison of the amount of large T antigen produced in 3T6 cells infected with polyomavirus with that in 293 cells infected with Ad5PyR39, under optimal conditions for each system, revealed at least a fivefold greater yield of the protein on a per cell basis in the latter system compared with the former. Ad5PyR39 should prove to be useful to isolate large quantities of functional polyomavirus large T antigen for structural and biochemical studies.
机译：通过将由2型腺病毒主要晚期启动子和多瘤病毒的早期编码区组成的杂交转录单元插入5型腺病毒载体Ad5 delta E1 / dl309中，体外构建了腺病毒-多瘤病毒重组病毒。该载体缺少E1a和E1b转录单位，并在其位置包含一个独特的限制性核酸内切酶切割位点。多瘤病毒基因组插入片段包含一个小的缺失，从而无法合成功能性的小T和中T抗原，但允许合成大T抗原。详细研究了一种重组病毒Ad5PyR39，它的杂种转录单位的转录方向与后基因转录的整体方向相反。 Ad5PyR39在没有辅助病毒的情况下在人的293细胞中有效复制，并表达了预期大小和组成的杂合mRNA，可翻译产生大T抗原。在293细胞中合成的大T抗原的大小与经溶瘤感染多瘤病毒的小鼠3T6细胞中产生的大小相同，并且该蛋白有效且特异性地与多瘤病毒DNA中的大T抗原结合位点结合。而且，由重组病毒编码的大T抗原被证明能够催化在小鼠3T6细胞中复制含有多瘤病毒来源的DNA复制的质粒。在每个系统的最佳条件下，比较感染多瘤病毒的3T6细胞和感染Ad5PyR39的293细胞中产生的大T抗原的量，发现在后者系统中，每个细胞的蛋白质产量至少高出五倍与前者相比。 Ad5PyR39应该被证明可用于分离大量功能性多瘤病毒大T抗原，用于结构和生化研究。

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