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

哺乳动物细胞失控载体的开发及其生产应用。

基本信息

批准号：
07555256
负责人：
IIJIMA Shinji
金额：
$ 10.24万
依托单位：
Nagoya University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1996
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07555256/
关键词：
runaway-plasmid episome mammalian cells genetic engineering erythropoietin

项目摘要

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.

通过哺乳动物细胞的重组DNA技术生产生物物质是基础研究或工业生产中最重要的程序之一。与微生物细胞相比，质粒尚未被用作哺乳动物细胞的载体。开发一种新的大拷贝数载体系统，如果可能的话，其拷贝数可以通过温度控制。以猿猴病毒40（SV40）基因组为基础，构建了哺乳动物细胞的renaway载体，该载体具有温度敏感突变大 T 抗原和细菌 neo^<gamma> 基因的复制在 39°C 以上受到抑制，而在 33°C 以下，使用该载体可以非常有效地产生人促红细胞生成素。该载体在 spppinner 烧瓶培养中效果非常好。为了我们基于猿猴病毒 40 的哺乳动物细胞新型 renaway 型载体的工业应用，我们研究了宿主范围特异性这种载体，特别是人类来源的细胞系，因为人类细胞作为热源似乎对于通过基因工程生产药物非常重要，失控型载体在 HeLa 的允许温度 33°C 下表现出高复制率。细胞和促红细胞生成素的有效产生取决于温度，但在 KB 细胞中相对较低。我们还建立了啮齿动物细胞的附加型表达载体。克隆的野生型多瘤病毒基因组。通过定点诱变引入温度依赖性复制，将突变的多瘤细胞与选择标记连接，构建了复制受温度调节的啮齿动物细胞的新表达载体。