肝细胞核因子4α增强型1.0倍HBV复制子体外复制模型建立及初步应用

摘要/Abstract

摘要： 目的建立一种肝细胞核因子4α（HNF4α）增强型1.0倍HBV复制子体外复制模型。方法从临床获得的急性乙型肝炎患者血清中扩增并克隆HBV全长DNA;手术获得肝组织中提取总RNA,扩增并克隆HNF4α基因cDNA。Bsp QI/ScaI双酶切回收HBV全长DNA,HNF4α基因cDNA定向连接到真核表达载体pcDNA3.1（+）中,构建pcDNA3.1-4α表达载体。将HBV全长DNA1μg/孔,按比例共转染pcDNA3.1-4α（0,0.5μg,1μg）于Hep G2细胞,96 h后检测细胞上清中的HBs Ag、HBeAg和HBVDNA;以0.5μg/1μGpcDNA3.1-4α与HBV全长DNA共转染Hep G2细胞,加入不同浓度LAM（0,100μMol/L）和ETV（0,10μMol/L）,评价建立的体外复制模型。结果 1％TAE琼脂糖凝胶电泳鉴定HBV全长DNA和HNF4α cDNAPCR产物,条带长度为3.2 kb和1.5 kb;目的片段克隆后测序鉴定正确。胶回收HBV全长DNA克隆质粒酶切目的片段,构建的pcDNA3.1-4α表达载体酶切鉴定正确后,测序鉴定。不同浓度比例的pcDNA3.1-4α与HBV全长DNA转染Hep G2细胞后,0μg/1μg组合：HBeAg阴性,HBs Ag阴性,上清HBVDNA载量为10³;0.5μg/1μg组合：HBeAg阴性,HBs AGA值从0.1升高到1.99,上清HBVDNA载量从1×10³IU/ML升高到4 ×10⁴IU/ML;1μg/1μg组合：HBeAg阴性,HBs AGA值从0.1升高到2.4,上清HBVDNA载量从1×10³IU/ML升高到1×10⁵IU/ML;LAM从0~100μMol/L,细胞上清HBVDNA降低了97.6％;ETV从0~10μMol/L,上清HBVDNA降低了99.0％。结论 HNF4α增强型1.0倍HBV复制子体外复制模型,可以体外评价临床常用抗病毒药物,为后续HBV研究提供新思路。

关键词: HNF4α, 乙型肝炎, 体外复制, 抗乙肝药物

Abstract: Objective To construct a hepatocyte nuclear factor 4 alpha（HNF4 α）enhanced 1.0-fold hepatitis Bvirus （HBV）replication Model. Methods Afull-length HBVDNAfro Mseru Mof acute hepatitis B（AHB）patients was isolated for am plification and cloning,and total RNAof liver tissue fro Msurgery was extracted for HNF4 α gene cDNAam plifying and cloning. Bsp QI/ScaIwas used to digest HBVDNAplasmid and recover HBVDNAproduction. HNF4 α gene cDNAwas directionally connected to the eukaryotic expression vector pcDNA3.1（+）for construction of pcDNA3.1-4 α expression vector. Hep G2 cells were transfected with full-length HBVDNA（1μg/well）,according to proportion of transfection pcDNA3.1-4 α（0μg,0.5μg,1μg）. After 96 hours,HBs Ag,HBe AGlevels and HBVDNAload in cell supernatant were detected. Hep G2 cells were transfected with 0.5μg/1μGpcDNA3.1-4 α and full-length HBVDNA,which were added with different concentrations of LAM（0μMol/L,100μMol/L）and ETV（0μMol/L,10μMol/L）to evaluate the replication Modelin vitro. Results The 1％TAEagarose gel detection showed that the lengths of the strips of full-length HBVDNAand HNF4 α cDNAPCRproduct were 3.2 kb and 1.5 kb,respectively,of which cloning target frag ments were sequenced and identified. The full-length HBVDNAwas recycled fro Mgel for cloning digested target frag ments. The constructed pcDNA3.1-4 α expression vector was sequenced and identified. Hep G2 cells were transfected with different ratios of pcDNA3.1-4 α and full-length HBVDNA.In 0μg/1μGgroup,HBe AGand HBs AGwere negative with a HBVDNAload of 10³in supernatant;in 0.5μg/1μGgroup,HBe AGwas negative and HBs AGODrose fro M0.1 to 1.99 with an increasing HBVDNAload fro M1×10³to 4×10⁴;in 1μg/1μGgroup,HBe AGwas negative and HBs AGODbook=29,ebook=33rose fro M0.1 to 2.4 with an increasing HBVDNAload fro M1×10³to 1×10⁵. HBVDNAload in supernatant was reduced by 97.6％as lamivudine increasing fro M0μMol/Lto 100μMol/L,and was also reduced by 99.0％as entecavir increasing fro M0μMol/Lto 10μMol/L. Conclusion HNF4 α enhanced 1.0-fold HBVreplication Model is constructed successfully,which could be used to evaluate anti-HBVagents in vitro and provide new insights for HBVstudy.

Key words: HNF4 alpha, Hepatitis Bvirus, Replication in vitro, Antiviral HBV

丁宁, 张明香. 肝细胞核因子4α增强型1.0倍HBV复制子体外复制模型建立及初步应用[J]. 肝脏, 2016, 21(1): 28-33.

DING Ning, ZHANG Ming-xiang. Construction and initial application of HNF4 α enhanced 1.0-fold HBVreplication modelinvitro[J]. Chinese Hepatolgy, 2016, 21(1): 28-33.

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