SV40载体在COS7细胞中的复制及外源基因的表达

为评价SV4 0载体 COS7稳定表达系统的可能应用价值 ,将含人组织型纤溶酶原激活剂 (t PA)编码区的SV4 0载体pctPA转染COS7细胞 ,并以G4 18选择培养 2 8d ,得到稳定的抗性细胞库 .Southern印迹和溶圈法分别分析该细胞库在随后的细胞传代中细胞内附加体拷贝数及t PA表达水平的变化 .在经选择培养 2 8d的COS7细胞库中 ,质粒pctPA以染色体外附加体的形式存在 (30 0拷贝 细胞 ) ,t PA的表达水平为 1 1μg d(10 6细胞 ) .在随后 3个月的动态观察中 ,随着细胞传代 ,该COS7细胞库tPA的表达水平虽逐渐递减 ,但在第 1个月内可保持在 1μg d(10 6细胞 )以上 .基于SV4 0载体 COS7稳定表达系统无需筛选克隆 ,在稳定的抗性细胞库形成后的 1个月内可能保持目的基因的高水平表达 ,因而较适合于需同时制备多种重组蛋白的实验 .

Replication and Exogenous Gene Expression of SV40 Vector in COS7 Cells

To evaluate the potential application for SV40 vectors/COS7 stable expression system, a stable, drug-resistant COS7 cell pool was formed by transfecting COS7 cells with SV40 vector pctPA containing an intact coding region of tissue-type plasminogen activator (t-PA), and then selecting the cells with G418 for 28 days. The changes of t-PA production and episomal copy number in the COS7 cell pool were analyzed by ring-dissolving and Southern blotting methods, respectively. In the drug-resistant COS7 cell pool, vector pctPA existed as extra-chromosomal episomes with a number of about 300 copies per cell, and the expression of t-PA was detected in the culture supernatant of the resistant COS7 cell pool with a level of about 1.1 μg/day (10+6 cells). In the subsequent observation for three-month, the yield of t-PA dropped gradually following cell passage, but stayed at a level higher than 1.0 μg/day (106 cells) in the first month. As there is no need to screen cell colonies and the expression of exogenous gene remains at relatively high level in the first month after formation of the cell pool, the SV40-COS7 stable expression system may be a good choice for preparing many recombinant proteins at the same time.