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

目的构建稳定过表达乙肝病毒(HBV) S基因Pre-S区突变(Pre-S1缺失突变、Pre-S2缺失突变)的HepG2细胞株,并观察该突变对HepG2细胞生物学行为的影响。方法以NCBI中HBV JX661479. 1的Pre-S/S片段序列信息为模板,设计并确定Pre-S1突变、Pre-S2突变的核苷酸序列,采用全基因合成法获得目的片段并定向导入带有FLAG标签的p LVX慢病毒质粒载体(p Lenti-CMV-3FLAG-PGK-Puro),PCR、双酶切、Sanger测序技术检测重组质粒中目的片段的准确性。将HepG2细胞随机分为5组,空白对照组不处理,p LVX-vector组、野生型组、Pre-S1突变组、Pre-S2突变组分别感染p LVX-vector、p LVX-Pre-S/S、p LVX-Pre-S1 mut/S、p LVX-Pre-S2 mut/S慢病毒液;利用嘌呤霉素筛选HepG2稳定株,采用Western blotting法鉴定目的蛋白,分别采用克隆形成、细胞划痕试验观察HepG2细胞稳定株增殖、迁移能力。结果构建的p LVX-Pre-S/S、p LVX-Pre-S1 mut/S、p LVX-Pre-S2 mut/S重组表达质粒PCR电泳产物与理论值相符,经EcoRⅠ和Bam HⅠ双酶切后电泳产物与理论值相符; Sanger测序结果显示,p LVXPre-S1 mut/S、p LVX-Pre-S2 mut/S突变型除突变序列外,其他序列与HBV S基因野生型序列完全相同。空白对照组HepG2细胞全部死亡,其余组HepG2细胞部分存活。在野生型组、Pre-S1突变组、Pre-S2突变组43 k D分子量位置检测到目的条带表达,而p LVX-vector组无法检测到相应条带。与其他组比较,Pre-S2突变组HepG2的第14天克隆形成数增多、细胞相对迁移距离增大(P均<0. 05)。结论成功构建了HBV Pre-S/S基因野生型及Pre-S突变型的HepG2细胞稳定株,HBV的S基因Pre-S2缺失突变导致肝癌HepG2细胞增殖及迁移能力增强。

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 pLVX lentiviral plasmid vector(pLenti-CMV-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 pLVX-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 pLVX-vector,pLVX-Pre-S/S,pLVX-Pre-S1 mut/S,and pLVX-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 pLVX-Pre-S/S,pLVX-Pre-S1 mut/S,and pLVX-Pre-S2 mut/S were conformed to the theoretical values through PCR electrophoresis analysis,and the electrophoretic products after digestion of EcoR I and BamH I were in accordance with the theoretical values.Sanger sequencing results showed that the mutations of pLVX-Pre-S1 mut/S and pLVX-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 kDa molecular weight position,but not in pLVX-vector group.Compared with the other groups,the number of clones and the relative migration distance on the 14th 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.