人前脑啡肽基因修饰人胚胎肾细胞的构建  

Construction of human embryonic kidney cells exhibiting human preproenkephalin gene expression

作　　者：白凤[1] 杨保仲[1] 薛朝霞[1] 潘喻飞[1] 黄平[1] 

机构地区：[1]山西医科大学第一临床医学院麻醉科,太原市030001

出　　处：《中华麻醉学杂志》2012年第6期673-675,共3页Chinese Journal of Anesthesiology

基　　金：山西省科技攻关项目（20090311058.2）

摘　　要：目的构建人前脑啡肽基因（hPPE）修饰的人胚胎肾细胞（HEK293细胞）。方法重组质粒peDNA3．1（＋）／hPPE经限制性内切酶HindHI和NotI进行双酶切获得hPPE基因，运用基因重组技术将hPPE基因与表达载体同源重组，转染293T细胞进行慢病毒包装、扩增、纯化，测定病毒滴度，再将重组的慢病毒载体转染HEK293细胞。用Westernblot法检测hPPE基因在HEK293细胞中的表达。结果重组慢病毒载体阳性克隆测序结果和基因库的hPPE基因序列完全一致。含有hPPE基因的慢病毒载体，滴度为2．07×10^8TU／ml。转染慢病毒载体后的HEK293细胞，在荧光显微镜下未见到GFP荧光。转染Ubc—GFP—L．V．空病毒载体的HEK293细胞，可见到较强的荧光。Westernblot法检测到经慢病毒载体转染后的HEK293细胞中hPPE基因表达呈阳性。结论成功构建了hPPE基因修饰的HEK293细胞，使hPPE基因可在HEK293细胞中稳定表达。Objective To construct human embryonic kidney cells （HEK293） modified with human pre- proenkephalin （hPPE） gene. Methods hPPE gene fragments were obtained from recombinant plasmid pcDNA3.1 （ ＋ ）/hPPE by using restriction endonuclease Hind m and Not I . Homologous recombination of lentivirus and hPPE gene was produced by using recombinant DNA technology. HEK293 cells were then transfected with the recombinant lentivirus vectors. The expression of hPPE gene in HEK293 cells was detected by Western blot. Re- sults The results of DNA sequencing indicated that the positive clone of recombinant lentivirus was completely consistent with sequencing of hPPE in Genebank. The titer of the concentrated virus was 2.07 × 10^8 TU/ml. GFP fluorescence was not seen in HEK293 cells transfected with the lentiviral vector under fluorescence microscope. A strong fluorescence was seen in HEK293 cells transfected with Ube-GFP-L. V. empty viral vector. Positive expression of hPPE was demonstrated in HEK293 cells transfected with lentiviral vector by Western blot. Conclusion HEK293 cells modified with hPPE gene were successfully constructed and the target gene hPPE was stably expressed in HEK293 cells.

关 键 词：脑啡肽类 细胞系 生物 基因修饰 基因疗法 

分 类 号：R96[医药卫生—药理学]