摘要
目的构建ADH1B慢病毒表达载体并建立ADH1B稳定过表达的人肝癌细胞株HepG2.2.15。方法根据ADH1B基因序列设计引物,PCR扩增并连接于GV492载体质粒交换转化DH5α感受态,选取阳性克隆转化子进行测序鉴定。采用三质粒系统包装ADH1B慢病毒载体转染293T细胞收集上清,测定滴度。感染人肝癌细胞HepG2.2.15,嘌呤霉素筛选得到过表达ADH1B的稳定细胞株,设立空白组、阴性对照组和过表达组,通过荧光显微镜观察各组GFP表达情况,并用Western blot及RT-qPCR从蛋白、mRNA水平对ADH1B基因表达进行检测。结果通过PCR、酶切鉴定及基因测序成功构建ADH1B过表达慢病毒载体,检测病毒滴度为2×109TU·mL-1。利用此病毒悬液感染HepG2.2.15细胞,成功筛选到ADH1B稳定表达的HepG2.2.15细胞株。Western blot及RT-qPCR结果显示:重组慢病毒载体LV-ADH1B能够有效感染HepG2.2.15细胞,与空白组和阴性对照组细胞相比,在过表达组细胞ADH1B m RNA和蛋白中表达量均升高。结论成功构建ADH1B基因的重组慢病毒载体LV-ADH1B,并筛选出稳定过表达ADH1B基因的人肝癌细胞株HepG2.2.15。
Objective To construct a lentiviral vector for ADH1 B and establish a human hepatocellular carcinoma cell line HepG2.2.15 with stable expression of ADH1 B. Methods The primers were designed based on the ADH1 B gene sequence,PCR amplification was connected to the GV492 vector plasmid exchange transformation into the DH5α regeneration receptor,and the positive clone transform was selected for sequencing identification. Supernatant was collected from 293 T cells transfected with the ADH1 B lentivirus vector and the titer was measured. In human liver cancer cells infected with HepG2.2.15,puromycin was screened to obtain stable cell lines overexpressing ADH1 B. The blank group,negative control group and overexpression group were setted up. GFP expression of three groups were observed by fluorescence microscopy,and ADH1 B gene expression was detected by Western blot and RT-qPCR from protein and m RNA levels.Results ADH1 B overexpression lentiviral vector was successfully constructed by PCR,enzyme digestion and gene sequencing. The titer of detection was 2×10^9 TU·m L^-1. HepG2.2.15 cells were infected with this virus suspension,and HepG2.2.15 cell line stably expressing ADH1 B was successfully screened. The results of Western blotting and RT-qPCR showed that the recombinant lentiviral vector LV-ADH1 B could effectively infect HepG2.2.15 cells. Compared with HepG2.2.15 blank group and negative control group cells,ADH1 B m RNA and protein levels were expressed in the overexpression group. The amount has increased significantly.Conclusion The recombinant lentiviral vector LV-ADH1 B of ADH1 B gene was successfully constructed and the human hepatocellular carcinoma cell line HepG2.2.15 stably over-expressing ADH1 B gene was screened to provide an in vitro cell model for the further study of the mechanism of ADH1 B in hepatocellular carcinoma.
作者
冯薛烟
丁向春
雒夏
董辉
赵志军
马丽娜
海龙
胡彦超
FENG Xueyan;DING Xiangchun;LUO Xia;DONG Hui;ZHAO Zhijun;MA Lina;HAI Long;HU Yanchao(Ningxia Medical University,Yinchuan 750004;Department of Infectious Diseases,the General Hospital of Ningxia Medical University,Yinchuan 750004;Laboratory of Pathogenic Microorganisms,the General Hospital of Ningxia Medical University,Yinchuan 750004)
出处
《宁夏医科大学学报》
2018年第8期869-874,共6页
Journal of Ningxia Medical University
基金
国家自然科学基金地区项目(81760363)
宁夏自然基金重点项目(2018AAC02014)
