新型CHO/dhfr^-细胞定点整合和表达系统的建立

Construction of targeted integration and high expression system in CHO/dhfr- cell line

目的 :建立CHO/dhfr-细胞的定点整合和表达系统。方法 :利用常规分子生物学方法 ,构建了一个新的高效筛选表达载体 ,该载体含有一个由FRT(flprecombinationtarget)位点、报告基因 (LacZ)及筛选基因 (zeocin)三片段构成的融合基因 ,而且该基因的表达受一个弱化的SV4 0启动子的控制。借助于随机整合及高效筛选 ,实现FRT位点在CHO/dhfr-细胞基因组中转录活跃区的整合 ,然后利用该位点介导的特异性重组实现了目的基因 (单链抗体 尿激酶融合基因 )的定点整合和有效表达 ,而且随后通过DHFR/MTX系统的加压扩增 ,以提高单链抗体 尿激酶融合基因在宿主细胞中的表达水平。结果与结论 :获得了能够实现外源基因在基因组中定点整合和有效表达的CHO/dhfr-细胞系 ,并利用该细胞系实现了单链抗体 尿激酶融合基因的高表达 ,表达量达到 5 μg/ (10 6细胞·2 4h)。

Objective:To construct targeted integration and high expression system in CHO/dhfr- cell line.Methods:By using routine methods of molecular biology,a new expression vector that could express a three-segment fusion gene of FRT site, reporter gene(lacZ) and selection gene(zeocin) was firstly constructed,and the fusion gene was under the control of an attenuated SV40 promotor.By using the constructed vector,the CHO/dhfr- cell line that had an integrated FRT site in transcriptionally active region was identified after a series of assays.Then,by taking advantage of the flp-FRT reconbination system,the interesting gene(IIn-UK fusion gene ) was integrated into the FRT site in the genome of CHO/dhfr- cell and the expression level was increased by amplification with MTX.Results and Conclusion:Targeted integration and high expression system in CHO/dhfr- cell line was constructed,and the expression level of IIn-UK fusion gene reached 5 μg/10 6cells per 24 h by using constructed targeted integration and high expression system.