Development of runaway vector for mammalian cells and its application for production.

利用哺乳动物细胞重组DNA技术生产生物物质是基础研究或工业生产中最重要的步骤之一。与微生物细胞相比，质粒尚未被用作哺乳动物细胞的载体。在这方面，我们试图开发一种具有大拷贝数的新型矢量系统，如果可能的话，拷贝数可以由温度控制。以大T抗原温度敏感突变的猿猴病毒40（SV40）基因组和细菌neo^< γ >基因为载体，构建了哺乳动物细胞分离载体。在猿猴CV-1细胞中，该质粒在高于39ºC时复制受到抑制，而在低于33ºC时DNA增殖旺盛。利用该载体可有效地产生人促红细胞生成素。该载体在spppinner flask培养中工作得很好。基于猿猴病毒40的新型哺乳动物细胞载体的工业应用，我们研究了该载体的宿主范围特异性，特别是人类来源的细胞系，因为人类细胞作为热点似乎对基因工程药物的生产非常重要。失控型载体在33℃的允许温度下，在HeLa细胞中具有较高的复制率，并能有效地产生促红细胞生成素。质粒拷贝数与温度有关，但在KB细胞中相对较低。我们还建立了啮齿动物细胞的episomal型表达载体。利用定点诱变技术对克隆的野生型多瘤病毒基因组进行突变，引入温度依赖性复制。将突变的多瘤与选择标记连接，构建了复制受温度调控的鼠细胞表达载体。

Production of biological substances by recombinant DNA technology with mammalian cells is one of the most important procedures either for basic research or industrial production. In contrast to microbial cells, plasmids have not been used as a vestor for mammalian cells. In this regard, we tried to develop a novel vector system which has big copy number and, if possible, of which the copy number could be controlled by temperature.A renaway vector for mammalian cells was constructed from simian virus 40(SV40) genome with temperature sensitive mutation of large T antigen and bacterial neo^<gamma> gene. Replication of this plasmid was repressed above 39ﾟC and vigorous DNA propagation was observed below 33ﾟC in simian CV-1 cells. Human erythropoietin was very effectively produced by using this vector. This vector worked very well in spppinner flask cultures.For industrial applications of our novel renaway-type vector for mammalian cells based on simian virus 40, we studied host range specificity of this vestor, especially with human derived cell lines, since human cells as hots seem to be very important for the production of pharmaceuticals by genetic engineering. The runaway-type vector showed a high replication rate at a permissive temperature of 33ﾟC in HeLa cells and erythropoietin was produced effectively. The plasmid copy number was dependent on the temperature but was relatively low in KB cells.We also established an episomal type expression vector for rodent cells.Cloned wild type polyoma virus genome was mutated by site-directed mutagenesis to introduce the temperature-dependent replication. The mutated polyoma was ligated wiht selection marker, and new expression vector for rodent cells, of which replication was regulated by temperature, was constructed.