EXPRESSION OF PAPOVAVIRUS TUMOR ANTIGENS

需要更方便和多功能的克隆载体， 外源基因在真核细胞中的表达促使我们开发了 一种病毒载体系统，可以容纳和表达大范围的 外源基因序列 我们的系统设计允许特定的选择 表达高水平外源基因产物的重组病毒 在病毒转录启动子的控制下。 过去三 多年来，我们已经制定了一系列表达高水平的策略， 的SV40肿瘤抗原。 我们成功 在通过构建SV40 A基因mRNA获得过量生产中， 重组病毒，其中A基因在转录下， 腺病毒主要晚期启动子的控制。 然而，我们发现， TmRNA的有效翻译似乎需要特异性前导序列 在一些实施方案中，T抗原mRNA包含待附接至T抗原mRNA的5'端的序列。 这一发现 让我们假设在我们宿主中翻译的有效起始 载体系统需要特定的顺式作用序列元件， 通常存在于腺病毒晚期mRNA的5 ′末端。 在此，我们建议 识别晚期转录因子前导序列中的翻译控制信号， 病毒mRNAs，并探讨功能之间的关系， 三部分前导序列和mRNA的选择性翻译， 病毒感染的宿主细胞。 我们打算进一步探索我们的能力， 将外源DNA定位在腺病毒基因组内， 重组 除了构建和分离重组 病毒，我们还将应用一系列技术来表征 表达的基因的基因组、转录物和蛋白质结构不同， 腺病毒重组体。 最后，我们建议推广表达式 通过构建含有SV40辅助细胞的克隆载体， 功能基因与其他外源基因融合， 其他基因的表达。 特别是，我们计划建造一系列 设计用于表达多瘤肿瘤抗原的重组载体 病毒 我们对多瘤T抗原的生化特性的兴趣 源于研究表明，虽然多瘤病毒A基因编码 在结构和功能上与SV40 T类似的多肽 抗原之间存在显著差异， 影响T抗原介导的转录的调节序列 以及多瘤病毒和SV40中的病毒DNA合成。

The need for more convenient and versatile cloning vectors for the expression of foreign genes in eukaryotic cells has prompted us to develop a viral vector system that can accomodate and express a large range of foreign gene sequences. Our system is designed to allow specific selection of recombinant viruses that express high levels of foreign gene products under the control of viral transcription promoters. In the past three years, we have developed a number of strategies for expressing high levels of the SV40 tumor antigen in a mammalian viral vector system. We succeeded in obtaining overproduction of the SV40 A gene mRNA by constructing recombinant viruses in which the A gene was under the transcriptional control of the adenovirus major late promoter. However, we discovered that efficient translation of T mRNA appeared to require specific leader sequences to be attached to the 5' end of the T antigen mRNA. This finding let us to hypothesize that efficient initiation of translation in our host vector system requires specific cis-acting sequence elements that are commonly found at the 5' ends of adenovirus late mRNAs. Here we propose to identify the translational control signals in the leader sequences of late viral mRNAs and to explore the relationship between the function of the tripartite leader sequence and the selective translation of mRNAs in virus-infected host cells. We intend to further explore our ability to position foreign DNA within the adnovirus genome by homologous recombination. In addition to constructing and isolating recombinant viruses, we will also apply a battery of techniques to characterize the genome, transcript and protein structures of the genes expressed various adenovirus recombinants. Finally, we propose to generalize the expression system by constructing cloning vectors that contain the SV40 helper function gene fused to other foreign genes in order to select for expression of other genes. In particular, we plan to construct a series of recombinant vectors designed to express the tumor antigen of polyoma virus. Our interest in the biochemical properties of polyoma T antigen stems from studies which indicate that although the polyoma A gene encodes a polypeptide that is analogous in structure and function to the SV40 T antigen, there are significant differences between the arrangement of regulatory sequences that influence the T antigen mediated transcription and viral DNA synthesis in polyoma and SV40.