谷氨酸酰合成酶(GS)基因扩增选择系统的建立  

Development of the Glutamine Synthetase Gene Amplification System

作　　者：刘文军[1] 杨芙蓉[1] 阮力[1] 任贵方[1] 朱既明[1] 

机构地区：[1]中国预防医学科学院病毒研究所,北京100052

出　　处：《高技术通讯》1997年第2期44-49,共6页Chinese High Technology Letters

摘　　要：利用TR－PCR方法从CHOdhfr－细胞中克隆到五株GS基因。对三株GS基因进行了功能分析和序列分析。结果表明，895位核苷酸全部由G变为C，相应的氨基酸白Gly变为Arg，此处变异是CHOdhfr－细胞本身所固有的，不影响GS基因的功能。而399位核苷酸G到A的变异，直接影响到GS基因的功能。用只有895位核苷酸变异的GS基因作扩增选择基因构建表达CAT基因的质粒，转化细胞后，GS基因不仅自身具有扩增的能力，而且能够带动外源基因进行扩增。细胞内外源基因CAT拷贝数可达1000—2000拷贝／细胞，较已有报导的GS基因具有更高的扩增效率，基因扩增前后CAT基因的表达产量提高了10—20倍左右。Five clones of GS gene have been obtained from CHO dhfr-cells by RT-PCR method. Functional and sequence analysis data of three of these five clones of GS gene indicates that, in comparison with the published GS sequences data of CHO-K1 cells, all the three GS clones have a same mutated base pair located at 895 nucleotide, which leads to the change of an amino acid residue from Gly to Arg, suggesting that the mutation is an innate mutation of the CHO dhfr-cells. There is another mutated G to A base pair located at 399 nucleotide in the GS gene, which leads to the change of an amino acid residue from Met to lie. This mutation appears to influence the function of the GS protein. A functional GS gene which has only one mutated base pair located at 895 position as an amplifible selective marker has been used to constructed a plasmid to express CAT gene. Under the pressure of MSX, which is an inhibitor of the GS protein, the GS gene can be amplified itself and the foreign gene can be co-amplified with it. The copy number can reach 1000-2000 copies/cell. The GS gene that has been cloned yields more efficient amplification than the dhfr gene amplification system that has been employed.

关 键 词：基因扩增 GS基因 CHOdhfr-细胞 CAT基因 

分 类 号：Q75[生物学—分子生物学]