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[1]冯薛烟,丁向春,雒 夏,等.过表达ADH1B基因HepG2.2.15细胞株的建立[J].宁夏医科大学学报,2018,(08):869-874.[doi:10.16050/j.cnki.issn1674-6309.2018.08.001]
 FENG Xueyan,DING Xiangchun,LUO Xia,et al.Establishment of Stable HepG2.2.15 Cell Strain Overexpressing ADH1B Gene[J].Ningxia Medical University,2018,(08):869-874.[doi:10.16050/j.cnki.issn1674-6309.2018.08.001]
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过表达ADH1B基因HepG2.2.15细胞株的建立(PDF)
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《宁夏医科大学学报》[ISSN:1005-8486/CN:64-1029/R]

卷:
期数:
2018年08期
页码:
869-874
栏目:
论著
出版日期:
2018-08-30

文章信息/Info

Title:
Establishment of Stable HepG2.2.15 Cell Strain Overexpressing ADH1B Gene
文章编号:
1674-6309(2018)08-0869-06
作者:
冯薛烟1 丁向春2 雒 夏12 董 辉3 赵志军3 马丽娜2 海 龙2 胡彦超2
(1. 宁夏医科大学,银川 750004; 2. 宁夏医科大学总医院感染疾病科,银川 750004; 3. 宁夏医科大学总医院病原微生物实验室,银川 750004)
Author(s):
FENG Xueyan1 DING Xiangchun2 LUO Xia12 DONG Hui3 ZHAO Zhijun3 MA Lina2 HAI Long2 HU Yanchao2
(1. Ningxia Medical University, Yinchuan 750004; 2. Department of Infectious Diseases, the General Hospital of Ningxia Medical University, Yinchuan 750004; 3. Laboratory of Pathogenic Microorganisms,the General Hospital of Ningxia Medical University,Yi
关键词:
ADH1B基因慢病毒载体HepG2.2.15细胞肝细胞肝癌
Keywords:
ADH1B gene lentiviral vector HepG2.2.15 cell hepatocellular carcinoma
分类号:
R735.7
DOI:
10.16050/j.cnki.issn1674-6309.2018.08.001
文献标志码:
A
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 mRNA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。
Abstract:
Objective To construct a lentiviral vector for ADH1B and establish a human hepatocellular carcinoma cell line HepG2.2.15 with stable expression of ADH1B. Methods The primers were designed based on the ADH1B 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 293T cells transfected with the ADH1B 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 ADH1B. The blank group,negative control group and overexpression group were setted up. GFP expression of three groups were observed by fluorescence microscopy, and ADH1B gene expression was detected by Western blot and RT-qPCR from protein and mRNA levels. Results ADH1B overexpression lentiviral vector was successfully constructed by PCR, enzyme digestion and gene sequencing. The titer of detection was 2×109 TU·mL-1. HepG2.2.15 cells were infected with this virus suspension, and HepG2.2.15 cell line stably expressing ADH1B was successfully screened. The results of Western blotting and RT-qPCR showed that the recombinant lentiviral vector LV-ADH1B could effectively infect HepG2.2.15 cells. Compared with HepG2.2.15 blank group and negative control group cells,ADH1B mRNA and protein levels were expressed in the overexpression group. The amount has increased significantly. Conclusion The recombinant lentiviral vector LV-ADH1B of ADH1B gene was successfully constructed and the human hepatocellular carcinoma cell line HepG2.2.15 stably over-expressing ADH1B gene was screened to provide an in vitro cell model for the further study of the mechanism of ADH1B in hepatocellular carcinoma.

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备注/Memo

备注/Memo:
收稿日期:2018-04-22
基金项目:国家自然科学基金地区项目(81760363);宁夏自然基金重点项目(2018AAC02014)
作者简介:冯薛烟(1993-),女,在读硕士研究生,研究方向:病毒性肝炎相关疾病的基础与临床研究。
通信作者:丁向春(1972-),男,博士,副教授,主任医师,博士研究生导师,研究方向:病毒性肝炎相关疾病的基础与临床研究。 E-mail:13619511768@163.com
更新日期/Last Update: 2018-08-30