慢病毒介导的人Bax基因和人肝细胞生长因子基因在小鼠胚胎成纤维细胞中的共表达  

Co-expression of hBax and hHGF genes in NIH3T3 cells mediated by lentiviral vector

作　　者：李珂[1] 常青[2] 徐平[2] 高洪波[2] 

机构地区：[1]青岛阜外心血管病医院心脏中心,山东省266034 [2]青岛大学医学院附属医院心外科

出　　处：《中华临床医师杂志（电子版）》2012年第23期7547-7551,共5页Chinese Journal of Clinicians(Electronic Edition)

基　　金：山东省自然科学基金(2005zrb104001)

摘　　要：目的构建绿色荧光蛋白标记的人Bax基因(hBax)和人肝细胞生长因子基因(hHGF)双基因共表达的重组慢病毒。证实已包装成功的慢病毒能感染正常细胞并表达目的蛋白。方法通过重叠PCR技术构建attB1-K-hBAX/T2A/eGFP/P2A/hHGF-attB2基因片段,利用gateway technology构建慢病毒载体质粒pLV.EX2d.null-EF1A>hBAX/T2A/eGFP/P2A/hHGF和阴性对照质粒pLV.EX2d.null-EF1A>eGFP并测序,上述两种质粒分别与辅助质粒共转染293FT细胞包装病毒,荧光显微镜检测病毒滴度。实验分为实验组(感染了共表达Bax和HGF的慢病毒)、阴性对照组(仅感染了表达绿色荧光的慢病毒)和空白组(未作任何处理的正常NIH3T3细胞)三组,RT-PCR和Western blot检测染毒后各组细胞Bax和HGF的表达。结果经鉴定慢病毒载体质粒构建正确,荧光显微镜检测hBax和hHGF共表达慢病毒滴度为7.8×107TU/ml,仅表达绿色荧光蛋白的阴性病毒滴度为9×107TU/ml。将包装成功的慢病毒感染NIH3T3细胞48h后,RT-PCR和Western blot结果显示实验组与其他两组相比,Bax和HGF的表达明显上调(P<0.05)。结论标记增强型绿色荧光蛋白的表达hBax和hHGF双基因慢病毒构建成功并获得高滴度的病毒感染液。通过慢病毒感染途径成功将HGF和Bax基因导入NIH3T3细胞并表达蛋白,为进一步研究Bax和HGF双基因共表达的重组慢病毒对血管内皮细胞、平滑肌细胞和成纤维细胞的影响奠定了基础。Objective To construct enhanced green fluorecence protein(EGFP) labeled lentiviral vector carrying hBax and hHGF genes.To explore normal cell can be infected by the lentiviral vector and express interest proteins.Methods The attB1-K-hBAX /T2A /eGFP /P2A /hHGF-attB2 gene fragment was constructed by overlap PCR method.Gateway technology was used to construct the pLV.EX2d.null-EF1A > hBAX /T2A /eGFP /P2A /hHGF plasmid and pLV.EX2d.null-EF1A > eGFP plasmid.After sequencing,they were packaged through co-transfected into human embryonic kidney cell line-293T with helper plasmids.Then the virus titer was examined by fluorescence microscope.The cultured NIH3T3 cells were divided into 3 groups and treated with EGFP labeled lentiviral vector carrying hBax and hHGF genes(experimental group),lentiviral vector just carrying EGFP(negative control group) and nothing(blank group).The expressions of Bax and HGF were detected by RT-PCR and Western blot.Results The recombinant lentiviral transfer vector plasmids were constructed correctly:the titer of lentiviral-hBAXeGFP-hHGF was 7.8 × 10 7 TU /ml,and lentiviral-eGFP was 9 × 10 7 TU /ml.The expression levels of Bax and HGF in the experimental group were up-regulated significantly,which were much higher than those of the other two groups at 48 h after infection(P < 0.05).Conclusions The lentiviral vector was successfully constructed and efficient lentivirus particles with high titer were packaged.After the EGFP labeled lentiviral vectors carrying hBax and hHGF genes could successfully infected NIH3T3 cells,interest genes and proteins could be expressed successfully.This research provided the foundation for further study of the effects on vascular endothelial cells,smooth muscle cells,and fibrocytes of the EGFP labeled lentiviral vector carrying hBax and hHGF genes.

关 键 词：慢病毒属 肝细胞生长因子 BCL-2相关X蛋白质 冠状动脉旁路移植术 非体外循环 NIH3T3细胞 

分 类 号：Q78[生物学—分子生物学]