Tumstatin-EGFP真核表达载体构建及稳定转染CHO细胞系的建立  

Construction of tumstatin-EGFP eukaryotic expression vector and establishment of CHO cell line

作　　者：姚丽娟[1] 罗以勤[1] 孔建新[1] 赵亮[1] 常珍[1] 濮跃晨[1] 马筱玲[1] 

机构地区：[1]安徽医科大学附属省立医院,安徽合肥230001

出　　处：《山东医药》2008年第34期27-30,共4页Shandong Medical Journal

基　　金：国家自然科学基金项目(30672437)。

摘　　要：目的构建重组tumstatin-增强型绿色荧光蛋白(EGFP)的分泌型真核表达载体质粒,建立稳定转染的中国仓鼠卵巢细胞系(CHO-K1)。方法利用重叠多聚酶链反应(PCR)技术从pGEM-T/STL质粒和pEGFP-C2质粒中扩增出STL-EGFP,用DNA重组技术将片段定向插入pIRESneo3质粒中,经酶切和序列验证正确后,用lipo-fectamine 2000转染CHO-K1细胞,通过G418筛选建立稳定转染的CHO细胞系,用逆转录-PCR、Western blot检测tumstatin-EGFP的表达,用倒置荧光显微镜观察绿色荧光蛋白的表达。结果重组真核表达载体正确构建并在CHO细胞获得稳定表达,tumstatin-EGFP基因整合入细胞基因组DNA,培养液上清有融合蛋白tumstatin-EGFP的分泌,绿色荧光蛋白的表达高达95%以上。结论成功构建了质粒PIRESneo3-STL-EGFP,获得稳定表达tumstatin-EGFP的细胞系。Objective To construct the eukaryotic expression plasmid of recombinant tumstatin-EGFP, establish CHO- K1 cell line. Methods STL-EGFP was obtatined from pGEM-T/STL and PEGFP-C2 by overlap PCR technology and was inserted into pIRESneo3. After identified by restriction endonuclease and sequence analysis, the recombinant expression plasmid was transfected with lipofectamine 2000 to CHO cells and established CHO-STL-EGFP cell line by screening of antibiotic G418. tumstatin-EGFP expression was detected by RT-PCR and Western blot , EGFP was detected by inverted fluorescent microscope. Results The recombinant eukaryotic expression plasmid were identified correct and the obtained CHO cell line stably expressed STL-EGFP. Tumstatin-EGFP has intergrated into CHO cell genome, the supematant of CHO-STL--EGFP culture medium secreting fusion protein of tumstatin-EGFP, and inverted fluorescence microscope showed that about 95% of the cells had positive fluorescent signals. Conclusions We have successfully constructed a recombinant expression plasmid of PIRESneo3-STL- EGFP and have established a CHO cell line stably expressing tumstatin-EGFP which may help to further studies of tumstatin or its fusion protein.

关 键 词：肿瘤抑素 增强型绿色荧光蛋白 融合蛋白 表达载体 

分 类 号：R780.2[医药卫生—口腔医学] R392.11[医药卫生—临床医学]