摘要
将猪白介素4（Porcine interleukin4，PIL-4）基因亚克隆到真核表达载体pEGFP～N1中，构建重组表达质粒pEGFP—PIL-4，利用脂质体法转染CHO-K1细胞，采用荧光显微镜实时观察、RT—PCR和Western-blot分别检测CHO细胞转录表达目的分子的情况，通过MTT法检测所表达蛋白的生物学活性。结果在将重组质粒转染CHO-K1细胞中24、48h后的均观察到绿色荧光；经G418筛选14～20d后转染的细胞形成了阳性细胞集落，用RT-PCR扩增出约339bp的目的基因片段；经Western—blot检测到约为39000的特异蛋白分子条带，并证明在转染重组质粒的细胞中表达了高生物活性的重组蛋白。
The IL 4 gene of porcine was subcloned into the eukaryotic expression vector pEGFP-N1 to construct the recombinant plasmid pEGFP PIL-4, which was transfccted into the CHO-K1 cells by the method of using Lipofectin,The expression of PIL-4 was determined by fluoroscopy, RT-PCR and Western-blot. After 24 hours and 48 hours,the Green fluorescence can be observed under fluorescence microscope and a 339 bp cDNA was detected by RT-PCR after screening 14 days with G418,39 000 protein was detected by Western-blot. The results indicate that fusion protein of PoIL 4 and EGFP was successfully expressed in CHO-K1 cells. And expressed protein had obviously biological activity.
出处
《中国兽医学报》
CAS
CSCD
北大核心
2008年第12期1452-1455,1467,共5页
Chinese Journal of Veterinary Science
基金
国家"863"计划资助项目(2006AA10A203)
兰州市科技攻关项目(05-1-47)
