摘要
目的构建绿色荧光蛋白/PAPa融合表达载体,并检则其在肝癌细胞SMMC7721细胞中的表达。方法采用PCR方法获得PAPa基因;将该基因片段克隆入带有绿色荧光蛋白(greenfluorescentprotein,CFP)报告基因的真核表达载体pEGPN1中,构建融合表达载体pEGFPPAPa;通过脂质体介导的方法将该载体转染到SMMC7721中,Westernblot检测PAPa的瞬时表达,荧光显微镜检测绿色荧光蛋白表达。结果pEGFPPAPa转染SMMC7721细胞后,在细胞中检测到PAPa基因片段,Westernblot可以检测到PAPa在SMMC7721细胞表达,用荧光显微镜观察到转染细胞中有绿色荧光蛋白表达。结论pEGFPPAPa在SMMC7721细胞中获得了表达,表达的融合蛋白具有PAPa和绿色光蛋白的双重活性,该载体的成功表达是研究PAPa抗肿瘤或抗病毒活民生的基础。
Objective To construct a PAP-a GFP fusion gene and express the gene in SMMC-7721 cells. Methods Amplify PAP-a gene by PCR and insert in into plasmid pEGFP-N1. Transfect SMMC-7721 with pEGFP-PAPa, detect the expressed PAP-a by Western-blot and fluorescence microscope. Results PAP-2 gene gene and PAP-a protein were detected in the transfected SMMC-7721 cells, meanwhile the expressed GFP was observed under fluorescence microscipe. Conclusion The PAP-a and GFP fusion gene was expressed in SMMC-7721 cells. The expressed fusion protein showed double activity of PAP-a and GFP.
出处
《胃肠病学和肝病学杂志》
CAS
2005年第4期363-365,共3页
Chinese Journal of Gastroenterology and Hepatology
基金
湖北省科技攻关计划(2002012)
