Construction and initial application of HNF4 α enhanced 1.0-fold HBVreplication modelinvitro

Abstract

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

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.

References

1 Günther S,Li BC,Miska S,et al.Anovel method for efficient a Mplification of w hole hepatitis Bvirus geno mes permits rapid functional analysis and 4eveals deletion Mutants in im Munosuppressed patients.JVirol,1995,69:5437-5444.
2 Tang H,McLanchalan A. Transcriptional regulation of hepatitis Bvirus by nuclear horm one receptors is critical determinant of viral tropism. Proc Natl Acad Sci USA,2001,98:1841-1846.
3 Beasley RP,Lin CC,et al.Hepatocellular carcino ma and hepatitis Bvirus:a prospective study of men in Taiwan. Lancet,1981,2:1129-1133.
4 Sells MA,et al.Production of hepatitis Bvirus particlesin Hep G2 cells transfected with cloned hepatitis Bvirus DNA. Proc Natl Acad Sci USA,1987,84:1005-1009.
5 Weiss L,Kekule AS,et al.The HBV-producing cellline Hep G2-4 A5:Anew in vitro system for studying the regulation of HBVreplication and for screening anti-hepatitis Bvirus drugs. Virol,1996,216:214-218.
6 Yang HL,Westland C,et al.In vitro antiviral susceptiblity of full-length clinical hepatitis Bvirus isolates cloned with a novel expression vector. Antiviral Res,2004,61:27-36.
7 Long Y,Chen E,Liu C,et al.The correlation of hepatocyte nuclear factor 4 alpha and 3 beta with hepatitis Bvirus replication in the liver of chronic hepatitis Bpatients. JViral Hepat,2009,16:537-546.
8 Tang H,Raney AK,McLachlan A. Replication of the wild type and a natural hepatitis Bvirus nucleocapsid pro Moter variant is differentially regulated by nuclear horm one receptors in cell culture.JVirol,2001,75:8937-8948.
9 裴彦祯,韩涛,马骁艳,等. HBs Ag及HBVDNA定量水平在慢性乙型肝炎、肝硬化和肝癌患者中的变化.中华肝脏病杂志,2011,10:743.
10 Ning BF,Ding J,Yin C,et al.Hepatocyte nuclear factor 4 alpha suppresses the develop ment of hepatocellular carcino ma. Cancer Res,2010,70:7640-7651.