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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.

利用哺乳动物细胞重组DNA技术生产生物物质，无论是基础研究还是工业生产，都是最重要的步骤之一。与微生物细胞不同的是，质粒并没有被用作哺乳动物细胞的外壳。在这方面，我们试图开发一种新的载体系统，该系统具有较大的拷贝数，如果可能，其拷贝数可以通过温度控制。从猴病毒40(SV40)基因组中，利用大T抗原的温度敏感突变和细菌neo^lt；Gamma&gt；基因构建了哺乳动物细胞的复活载体。该质粒在39ﾟC以上复制受到抑制，在33ﾟC以下表现出旺盛的增殖能力。利用该载体可以非常有效地生产人促红细胞生成素。对于我们基于猿猴病毒40的新型Renaway类载体的工业应用，我们研究了这种载体的宿主范围特异性，特别是对人类衍生的细胞系，因为人类细胞作为HoT似乎对通过基因工程生产药物非常重要。在33ﾟC的允许温度下，失控型载体在HeLa细胞中有较高的复制率，并能有效地产生促红细胞生成素。我们还构建了啮齿动物细胞的上体型表达载体，通过定点突变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。将突变的多瘤细胞与选择标记连接，构建了复制受温度调控的新型啮齿动物细胞表达载体。