山东医药2019，Vol.59Issue(2)：36-39,4.DOI:10.3969/j.issn.1002-266X.2019.02.009
乙肝病毒S基因Pre-S区突变的人肝癌细胞HepG2稳定株构建及其生物学行为变化
Construction of a stable strain of hepatitis B virus S gene mutation in Pre-S region of human hepatocellular carcinoma cell line HepG2 and its changes in biological behavior
摘要
Abstract
Objective To construct the HepG2 cell lines stably overexpressing hepatitis B virus ( HBV) S gene Pre-S region mutation ( Pre-S1 deletion mutation and Pre-S2 deletion mutation) , and to observe the effect of this mutation on the biological behavior of HepG2 cells. Methods The sequence of Pre-S/S fragment of HBV virus was determined based on JX661479. 1 sequence in NCBI, the deletion fragment of Pre-S1 and Pre-S2 mutation was designed subsequently. The target fragment was obtained by whole-genome synthesis method and inserted into the p LVX lentiviral plasmid vector ( p LentiCMV-3FLAG-PGK-Puro) with FLAG tag. We detected the accuracy of the target fragment in recombinant plasmid by PCR, double digestion and Sanger sequencing. HepG2 cells were randomly divided into 5 groups: the blank control group, the p LVX-vector group, wild type group, Pre-S1 mutation group, and Pre-S2 mutation group, and the cells in the latter four groups were transfected with p LVX-vector, p LVX-Pre-S/S, p LVX-Pre-S1 mut/S, and p LVX-Pre-S2 mut/S lentiviral solution, respectively. The HepG2 stable strain was screened by puromycin, and the expression of the target protein in HepG2 cells was identified by Western blotting. Colony Formation and scratch test were used to detect the impact of hepatitis B virus Pre-S mutation on the proliferation and migration abilities of HepG2 cells, respectively. Results The constructed recombinant expression plasmids of p LVX-Pre-S/S, p LVX-Pre-S1 mut/S, and p LVX-Pre-S2 mut/S were conformed to the theoretical values through PCR electrophoresis analysis, and the electrophoretic products after digestion of EcoR I and Bam H I were in accordance with the theoretical values. Sanger sequencing results showed that the mutations of p LVX-PreS1 mut/S and p LVX-Pre-S2 mut/S were identical to those of wild type of HBV S except mutation sequence. In the blank control group, all HepG2 cells died, and in the other groups, partial HepG2 cells survived. The expression of the target band was detected in the wild type group, Pre-S1 mutant group and Pre-S2 mutant group at 43 k Da molecular weight position, but not in p LVX-vector group. Compared with the other groups, the number of clones and the relative migration distance on the 14 th day of HepG2 cells in the Pre-S2 mutant group increased ( all P < 0. 05) . Conclusions The stable strain of HepG2 cells with wild and mutant HBV Pre-S/S genes is successfully constructed. The deletion of HBV S protein Pre-S2 results in the enhancement of proliferation and migration of hepatocellular carcinoma HepG2 cells关键词
乙肝病毒/S基因Pre-S突变/肝癌/HepG2细胞/细胞增殖/细胞迁移Key words
hepatitis B virus/S gene Pre-S mutant/hepatocellular carcinoma/HepG2 cells/cell proliferation/cell migration分类
医药卫生引用本文复制引用
郑羽飘,钱宝鑫,覃琴,骆莹,朱争艳,高英堂,王凤梅..乙肝病毒S基因Pre-S区突变的人肝癌细胞HepG2稳定株构建及其生物学行为变化[J].山东医药,2019,59(2):36-39,4.基金项目
艾滋病和病毒性肝炎等重大传染病防治国家科技重大专项(2018ZX10732202_004) （2018ZX10732202_004）
天津市慢性病防治科技重大专项(17ZXMFSY00170) （17ZXMFSY00170）
天津市科技攻关项目(16KG151). （16KG151）
