低密度cDNA Macroarray对干扰素α抗病毒蛋白的筛选

目的 基于低密度cDNA Macoarray技术筛选出差异表达的干扰素(IFN)α抗病毒基因,以探讨IFN α抗病毒蛋白的表达与HBV复制的关系. 方法 以一定浓度的IFN α处理肝胚瘤细胞株HepG2和HepG2.2.15细胞6h,用cDNA Macroarray分析比较两细胞株IFN α抗病毒基因表达谱,并筛选出差异表达的IFNα抗病毒基因.将表达HBV核心蛋白(HBc)的质粒pHBc-EGFP转染HepG2细胞,RT-PCR法分析HBc对IFN α抗病毒基因表达的影响.将表达抗黏病毒A蛋白(MxA)的表达质粒pcDNA3.1-Flag-MxA转染HepG2.2.15,以酶联免疫吸附试验、Dot blot、Southern blot等方法分别检测HepG2.2.15细胞表达释放的HBsAg与HBeAg、细胞外HBV DNA和细胞内HBV DNA复制中间体(松弛环状DNA、双股线性DNA),以判断HBV复制情况.两组间数据比较采用t检验,组间不同时间点数据比较采用单因素方差分析.结果 cDNA Macroarray分析显示HepG2和HepG2.2.15细胞的抗病毒基因表达谱具有差异性:IFNa抗病毒基因中干扰素诱导跨膜蛋白(IFITM)1、IFITM2、IFITM3、RING4等在HepG2.2.15细胞的表达被部分抑制,而重要的抗病毒蛋白MxA表达被完全抑制.HBc转染组细胞中MxA mRNA表达的相对水平为0.31±0.05,低于空白对照组的0.74±0.04,差异有统计学意义,P＜0.05.MxA蛋白转染HepG2.2.15细胞48、72 h后,MxA转染组细胞上清液中HBsAg的S/CO值分别为1.42+0.21和1.58±0.18,HBeAg的S/CO值为1.44±0.14和2.28±0.24,而空白对照组细胞上清液中HBsAg的S/CO值为1.92±0.19和2.79±0.25,HBeAg的S/CO值为2.31±0.46和3.37±0.29,两组细胞上清液中HBV抗原的S/CO值差异均有统计学意义,P值均＜0.05.细胞外HBV DNA、胞内HBV复制中间体DNA均无明显变化.结论 HBV及其抗原成分的复制和表达影响着IFNα抗病毒蛋白的表达;HBV通过抑制IFN α抗病毒蛋白的表达而发挥拮抗IFNα的抗病毒活性.     

Hepatitis B virus upregulates host expression of α-1,2-mannosidases via the PPARα pathway

AIM To assess the effects of hepatitis B virus(HBV) on the expression of host α-1,2-mannosidases and determine the underlying mechanisms.METHODS We measured the expression levels of MAN1A1, MAN1A2, MAN1B1, and MAN1C1 in cell lines HepG 2.2.15, HepN 10, HepA D38 and Hep G2 by Western blot. Viral antigens(HBs Ag and HBe Ag) in the culture medium were measured using the chemiluminescence method. HBV DNA quantification assays were performed using a commercial real-time PCR kit. Protein levels of human liver tissue α-1,2-mannosidases were also evaluated by Western blot. Plasmids containing seven individual viral genes of HBV(PTT22-HBx, PTT22-HBs, PTT22-pre S2, PTT22-pre S1, PTT22-HBc, PTT22-HBe, and PTT22-HBp) or control plasmids(PTT22-vector) were transfected into Hep G2 cells. MK886(PPARα) and GW9662(PPARγ) inhibitors were used to explore the effects of HBV on α-1,2-mannosidase expression after the PPARα and PPARγ pathways were blocked.RESULTS We showed that the expression of α-1,2-mannosidases was higher in stably transfected HBV cells than in controls. The expression levels of α-1,2-mannosidase were higher in AD38 cells than those in ND10 cells, which were in turn greater than those in G2.2.15 cells, and positively correlated with the expression of HBsA gin all the cell lines. Levels of α-1,2-mannosidase in nontumorous liver tissues of HBV-related HCC patients were also higher than in the tissues from non-HBVrelated HCC patients. Moreover, transfecting Hep G2 cells with a component of the HBV viral envelope also increased the expression of α-1,2-mannosidases. However, this envelope protein component could not induce MAN1C1 expression in the presence of a PPARα inhibitor, MK886. We also found that MK886 did not affect the expression of MAN1C1 in AD38 cells without tetracycline in the culture medium. This phenomenon was not observed in the case of GW9662.CONCLUSION Our results indicate that HBV increases the expression of α-mannosidases both in vitro and in vivo via activation of the PPARα pathway by its envelope protein.     

Apoptosis and its pathway in X gene-transfected HepG2 cells

AIM: To investigate the effect of hepatitis B virus (HBV) X gene on apoptosis and expressions of apoptosis factors in X gene-transfected HepG2 cells. METHODS: The HBV X gene eukaryon expression vector pcDNA3-X was transiently transfected into HepG2 cells by lipid-media transfection. Untransfected HepG2 and HepG2 transfected with pcDNA3 were used as controls. Expression of HBx in HepG2 was identified by RT-PCR. MTT and TUNEL were employed to measure proliferation and apoptosis of cells in-three groups. Semi-quantified RT-PCR was used to evaluate the expression levels of Fas/FasL, Bax/Bcl-xL, and c-myc in each group. RESULTS: HBV X gene was transfected into HepG2 cells successfully. RT-PCR showed that HBx was only expressed in HepG2/pcDNA3-X cells, but not expressed in HepG2 and HepG2/pcDNA3 cells. Analyzed by MTT, cell proliferation capacity was obviously lower in HepG2/pcDNA3-X cells (0.08910±0.003164) than in HepG2(0.14410±0.004927) and HepG2/pcDNA3 cells (0.12150±0.007159) (P<0.05 and P<0.01). Analyzed by TUNEL, cell apoptosis was much more in HepG2/pcDNA3-X cells (980/2 000) than HepG2 (420/2 000), HepG2/pcDNA3 cells (520/2 000) (P<0.05 and P<0.01). Evaluated by semi-quantified RT-PCR, the expression level of Fas/FasL was significantly higher in HepG2 cells transfected with HBx than in HepG2 and HepG2/ pcDNA3 cells (P<0.05 and P<0.01). Bax/Bcl-xL expression level was also elevated in HepG2/pcDNA3-X cells (P<0.05 and P<0.01). Expression of c-myc was markedly higher in HepG2/pcDNA3-X cells than in HepG2 and HepG2/pcDNA3 cells (P<0.05 and P<0.01). CONCLUSION: HBV X gene can impair cell proliferation capacity, improve cell apoptosis, and upregulate expression of apoptosis factors. The intervention of HBV X gene on the expression of apoptosis factors may be a possible mechanism responsible for the change in cell apoptosis and proliferation.     

Inhibition of hepatitis B virus replication by APOBEC3G in vitro and in vivo

AIM: To investigate the effect of APOBEC3G mediated antiviral activity against hepatitis B virus (HBV) in cell cultures and replication competent HBV vector-based mouse model. METHODS: The mammalian hepatoma cells Huh7 and HepG2 were cotransfected with various amounts of CMV-driven expression vector encoding APOBEC3G and replication competent 1.3 fold over-length HBV. Levels of HBsAg and HBeAg in the media of the transfected cells were determined by ELISA. The expression of HBcAg in transfected cells was detected by western blot. HBV DNA and RNA from intracellular core particles were examined by Northern and Southern blot analyses. To assess activity of the APOBEC3G in vivo, an HBV vector-based model was used in which APOBEC3G and the HBV vector were co-delivered via high-volume tail vein injection. Levels of HBsAg and HBV DNA in the sera of mice as well as HBV core-associated RNA in the liver of mice were determined by ELISA and quantitative PCR analysis respectively. RESULTS: There was a dose dependent decrease in the levels of intracellular core-associated HBV DNA and extracellular production of HBsAg and HBeAg. The levels of intracellular core-associated viral RNA also decreased, but the expression of HBcAg in transfected cells showed almost no change. Consistent with In vitro results, levels of HBsAg in the sera of mice were dramatically decreased. More than 1.5 log 10 decrease in levels of serum HBV DNA and liver HBV RNA were observed in the APOBEC3G-treated groups compared with the control groups. CONCLUSION: These findings indicate that APOBEC3G could suppress HBV replication and antigen expression both in vivo and in vitro, promising an advance in treatment of HBV infection.     

Quantifying anti-HBV effect of targeted ribonuclease by real-time fluorescent PCR

AIM:To quantify the inhibition of HBV replication by targetedribonuclease by using real-time fluorescent PCR.METHODS:Targeted ribonuclease was introduced into 2.2.15cells by liposome-mediated transfection or HIV-TAT mediatedprotein transduction.Forty-eight hours after the transfectionand 24 h after the transduction,supernatants of 2.2.15 cellswere collected and HBV DNA in the supernatants wasquantified by real-time fluorescent PCR with a commercial kit.RESULTS:HBV DNA concentrations in the supernatants of2.2.15 cells transfected or transducted with targetedribonuclease were 4.9±2.4×10~8 copies/L and 8.3±4.0×10~8copies/L,respectively.Compared with controls,transfectionor transduction of targeted ribonuclease reduced HBV DNAconcentration in the supematants of 2.2.15 cells by 90.4%and 90.1%,respectively(P<0.05).CONCLUSION:Targeted ribonuclease can inhibit HBVreplication in 2.2.15 cells.     

Construction, expression and characterization of human interferon α2b-（G4S）n-thymosin α1 fusion proteins in Pichia pastoris

AIM: Interferon α2b (IFNα2b) and thymosin α1 (Tα1) exhibit synergic effects in the treatment of hepatitis B and hepatitis C when used together. For developing a fusion protein drug, fusion proteins of IFNc(2b and To(1 linked by different lengths of (G4S)n (n=1-3) were constructed and expressed in Pichia pastoris.METHODS: Using PCR and molecular clone techniques,the fusion genes of IFNα2b-(G4S)n-Tα1(n=1-3) were constructed and subcloned into the eukaryotic expression vector pPIC9. After transformation of these plasmids into P. pastoris, the expressed fusion proteins IFNα2b-(G4S) n-Tα1(n=1-3) were obtained. These proteins were purified through diethylaminoethyl (DEAE) affinity chromatography and Superdex^TM 75 gel filtration and analyzed by SDSPAGE and Western blot. Antiviral and E-rosette assays were used to investigate the bioactivities of these fusion proteins.RESULTS: DNA sequencing confirmed that the fusion genes of IFNα2b-(G4S)n-Tα1(n=1-3) were correctly cloned to the pPIC9 vector. The recombinant IFNα2b-(G4S)n-Tα1(n=1-3) fusion proteins expressed in P. pastoris were purified with DEAE and Superdex^TM 75 gel filtration chromatography. The fusion proteins could be observed on sodium dodecylsulfate-polyacrylamide gel electrophoresis with molecular weight (MW) of 23.2, 22.9, and 22.6ku, respectively, and reacted to the IFNα2b monoclonal antibody and Tα1 polyclonal antibody. The purified fusion proteins exhibit antiviral activity and can enhance the percentage of E-rosette-forming-cell in E-rosette assay.CONCLUSION: The recombinant IFNα2b-(G4S)n-Tα1(n=1-3) fusion proteins were successfully expressed in P. pastoris. Purified fusion proteins exhibit both antiviral activity of IFNα2b and immunomodulatory activity of Tα1 in vitro. These results will be the basis for further evaluation of the fusion proteins‘ function in vivo.     

Construction and expression of eukaryotic plasmids containing lamivudine-resistant or wild-type strains of Hepatitis B Virus genotype C

AIM： To construct eukaryotic expression plasmids of full-length Hepatitis B Virus （HBV） genotype C genome, which contain lamivudine-resistant mutants （YIDD, YVDD） or wild-type strain （YMDD）, and to observe the expression of HBV DNA and antigens [hepatitis B surface antigen （HBsAg） and hepatitis B e antigen （HBeAg）] of the recombinant plasmids in HepG2 cells. METHODS： Three HBV full-length genomes were amplified from the plasmids pMD18T-HBV/YIDD, pMD18T-HBV/YVDD and pMD18T-HBV/YMDD, using PCR. Three recombinant plasmids were generated by inserting each of the PCR products into the eukaryotic expression vector pcDNA3.1 （＋）, between the EcoRI and HindⅢ sites. After being characterized by restriction endonuclease digestion, and DNA sequence analysis, the recombinant plasmids were transfected into HepG2 cells. At 48 and 72 h post-transfection, the levels of intracellular viral DNA replication were detected by real-time PCR, and the expression of HBsAg and HBeAg in the cell culture supernatant was determined by ELISA.
RESULTS： Restriction endonuclease digestion and DNA sequence analysis confirmed that the three recombinant plasmids were correctly constructed. After transfecting the plasmids into HepG2 cells, high levels of intracellular viral DNA replication were observed, and HBsAg and HBeAg were secreted into the cell culture supernatant.
CONCLUSION： Eukaryotic expression plasmids pcDNA3.1 （＋）-HBV/YIDD, pcDNA3.1 （＋）-HBV/YVDD or pcDNA3.1 （＋）-HBV/YMDD, which contained HBV genotype C full-length genome, were successfully constructed. After transfection into HepG2 cells, the recombinant plasmids efficiently expressed HBV DNA, HBsAg and HBeAg. Our results provide an experimental basis for the further study of HBV lamivudine-resistant mutants.     

Transfection of apoptosis related gene Fas ligand in human hepatocellular carcinoma cells and its significance in apoptosis

AIM: To evaluate the expression of apoptosis related gene Fas ligand (FasL) in human hepatocellular carcinoma (HCC) cells HepG2 and its significance in apoptosis. METHODS: Levels of soluble Fas ligand (sFasL) in a group of patients with hepatitis B virus (HBV)-induced chronic hepatitis, HBV-positive liver cirrhosis and HCC were evaluated. In a further study, the recombinant eukaryotic expression plasmid pcDNA3.1hisB-FasL was transfected into HCC cells HepG2 by lipofection, and then soluble FasL was examined in the supernatant of culture cells by EIA, FasL expression in HepG2 cells was detected by immuohistochemistry. After being stained by annexin V and propidium iodine, cells were passed through a flow cytometer and examined by a fluorescence microscope and a laser scanning microscope. RESULTS: The sFasL levels were significantly lower in patients with HCC when compared to the patients with hepatitis or liver cirrhosis. In comparison with untransfected cells, the soluble FasL could be detected in the supernatant of transfected cells. FasL was expressed on the membranes and cytoplasm of transfected cells. The apoptotic cell rate was 36.30% in transfected cells, and was 11.53% in untransfected cells. Moreover, the different stage of apoptotic cells could be distinguished by annexin V and propidium iodine staining. CONCLUSION: Fas ligand is an apoptotic pathway of HCC cells.     

Construction, expression and characterization of human interferon α2b-(G4S)n-thymosin α1 fusion proteins in Pichia pastoris

AIM: Interferon α2b (IFNα2b) and thymosin α1 (Tα1) exhibit synergic effects in the treatment of hepatitis B and hepatitis C when used together. For developing a fusion protein drug, fusion proteins of IFNα2b and Ta1 linked by different lengths of (G4S)n(n = 1-3) were constructed and expressed in Pichia pastoris. METHODS: Using PCR and molecular clone techniques, the fusion genes of IFNα2b-(G4S)n-Tα1 (n = 1-3) were constructed and subcloned into the eukaryotic expression vector pPIC9. After transformation of these plasmids into P. pastoris, the expressed fusion proteins IFNα2b-(G4S) n-Tα1 (n = 1-3) were obtained. These proteins were purified through diethylaminoethyl (DEAE) affinity chromatography and Superdex?75 gel filtration and analyzed by SDS-PAGE and Western blot. Antiviral and E-rosette assays were used to investigate the bioactivities of these fusion proteins. RESULTS: DNA sequencing confirmed that the fusion genes of IFNa2b-(G4S)n-Tα1 (n= 1-3) were correctly cloned to the pPIC9 vector. The recombinant IFNα2b-(G4S)n-Tα1 (n = 1-3) fusion proteins expressed in P. pastoris were purified with DEAE and Superdex?75 gel filtration chromatography. The fusion proteins could be observed on sodium dodecylsulfate-polyacrylamide gel electrophoresis with molecular weight (MW) of 23.2, 22.9, and 22.6 ku, respectively, and reacted to the IFNa2b monoclonal antibody and Tal polyclonal antibody. The purified fusion proteins exhibit antiviral activity and can enhance the percentage of E-rosette-forming-cell in E-rosette assay. CONCLUSION: The recombinant IFNa2b-(G4S)n-Tα1 (n = 1-3) fusion proteins were successfully expressed in P. pastoris. Purified fusion proteins exhibit both antiviral activity of IFNa2b and immunomodulatory activity of Tal in vitro. These results will be the basis for further evaluation of the fusion proteins' function in vivo.     

Interferon-α response in chronic hepatitis B-transfected HepG2.2.15 cells is partially restored by lamivudine treatment

INTRODUCTION Hepatitis B (HBV) is a hepatotropic DNA virus capable of causing both acute and chronic hepatitis in humans. It is estimated that over 350 million people are chronically infected with HBV worldwide. Currently approved thera- peutic strategies for treatment of HBV include interferon- alpha (IFN-a), the nucleoside analogue lamivudine and the nucleotide analogue adefovir[1,2]. However, only a minority of patients treated with IFN-a has a long-term sustained response with …     

乙型肝炎病毒复制调控元件对HBV DNA疫苗诱导的免疫应答

目的研究乙型肝炎病毒（HBV）复制调控元件增强子Ⅰ（ENHⅠ）及前S2（Pre-S2）抗原基因对HBV DNA疫苗诱导的免疫应答的影响。方法采用常规聚合酶链反应（PCR）从HBV adr亚型全基因DNA序列中分别扩增HBsAg、PreS2-HBsAg、HBsAg-ENHI和PreS2-HBsAg-ENHⅠ基因片段，重组到VR1012载体中，构建4种HBV DNA疫苗，转染HepG2细胞并免疫Balb／C小鼠。通过细胞免疫化学、酶联免疫分析（ELISA）、酶联免疫斑点试验（ELISPOT）等方法检测其在HepG2细胞内的表达及小鼠的体液及细胞免疫应答。结果转染的HepG2细胞表达相应的目的蛋白．ENHⅠ及Pre-S2抗原基因均可增强HBV DNA疫苗转染HepG2细胞表达HBsAg；免疫接种小鼠后第2周产生抗-HBs及HBsAg特异性细胞毒T淋巴细胞（CTL），Pre—S2抗原基因可增强HBV DNA疫苗免疫Balb／C小鼠诱导的抗-HBs及HBsAg特异性CTL的产生，ENHⅠ基因对免疫应答无影响。结论ENHI及Pre—s2抗原基因均可增强HBVDNA疫苗转染HepG2细胞表达HBsAg．Pre-S2抗原基因可增强HBVDNA疫苗免疫Balb／C小鼠引起的免疫应答。     

Interferon-α response in chronic hepatitis B-transfected HepG2.2.15 cells is partially restored by lamivudine treatment

AIM: To characterize the IFN-response and its modulation by the antiviral compound lamivudine in HBVtransfected HepG2.2.15 cells.METHODS: HepG2.2.15 and HepG2 cells were stimulated with various concentrations of IFN-α2a in the presence or absence of lamivudine. Then, total RNA was extracted and analysed by customised cDNA arrays and northern blot for interferon-inducible genes (ISGs). In addition, cellular proteins were extracted for EMSA and western blot. HBV replication was assessed by southern blot or ELISAs for HBsAg and HBeAg.RESULTS: Two genes (MxA, Cig5) with completely abolished and 4 genes (IFITM1, -2, -3, and 6-16)with partially reduced IFN-responses were identified in HepG2.2.15 cells. In 2 genes (IFITM1, 6-16), the response to IFN-α could be restored by treatment with lamivudine. This effect could not be explained by a direct modulation of the Jak/Stat signalling pathway since EMSA and western blot experiments revealed no suppression of Stat1 activation and ISGF3 formation after stimulation with IFN-α in HepG2.2.15 compared to HepG2 cells.CONCLUSION: These results are consistent with the assumption that chronic hepatitis B may specifically modulate the cellular response to IFN by a selective blockage of some ISGs. Antiviral treatment with lamivudine may partially restore ISG expression by reducing HBV gene expression and replication.     

Interferon-α response in chronic hepatitis B-transfected HepG2.2.15 cells is partially restored by lamivudine treatment

AIM: To characterize the IFN-response and its modulation by the antiviral compound lamivudine in HBV-transfected HepG2.2.15 cells. METHODS: HepG2.2.15 and HepG2 cells were stimulated with various concentrations of IFN-alpha2a in the presence or absence of lamivudine. Then, total RNA was extracted and analysed by customised cDNA arrays and northern blot for interferon-inducible genes (ISGs). In addition, cellular proteins were extracted for EMSA and western blot. HBV replication was assessed by southern blot or ELISAs for HBsAg and HBeAg. RESULTS: Two genes (MxA, Cig5) with completely abolished and 4 genes (IFITM1, -2, -3, and 6-16) with partially reduced IFN-responses were identified in HepG2.2.15 cells. In 2 genes (IFITM1, 6-16), the response to IFN-alpha could be restored by treatment with lamivudine. This effect could not be explained by a direct modulation of the Jak/Stat signalling pathway since EMSA and western blot experiments revealed no suppression of Stat1 activation and ISGF3 formation after stimulation with IFN-alpha in HepG2.2.15 compared to HepG2 cells. CONCLUSION: These results are consistent with the assumption that chronic hepatitis B may specifically modulate the cellular response to IFN by a selective blockage of some ISGs. Antiviral treatment with lamivudine may partially restore ISG expression by reducing HBV gene expression and replication.     

HCV核心蛋白与截短型HBV表面抗原中蛋白的协同反式激活功能

目的 探讨HCV核心蛋白与截短型HBV表面抗原中蛋白的协同反式激活作用。方法 构建表达HCV核心蛋白的重组质粒pcDNA3.1(-)-core和表达截短型HBV表面抗原中蛋白的重组质粒pcDNA3.1(-)-Mt；转染HepG2细胞，从转录和翻译水平鉴定病毒基因的瞬时表达；与报告质粒pSV-lacZ共转染HepG2细胞，检测β－半乳糖苷酶（β-gal)表达活性，酶的活性反映了表达的肝炎病毒蛋白对SV40病毒早期启动子（增强子）功能的影响。结果 构建成HCV核心蛋白及截短型HBV表面抗原中蛋白的重组表达载体pcDNA3.1(-)-core、pcDNA3.1(-)-Mt；在HepG2细胞均能瞬时表达相应的病毒蛋白；单独的共转染实验中pcDNA3.1(-)-core、pcDNA3.1(-)-Mt组的β-gal的表达分别是对照的4．6和3．2倍，两种质粒共同转染时酶的表达是对照组的8．4倍；表达质粒对β－gal表达的激活作用呈剂量依赖性。结论 HepG2细胞中表达的HCV核心蛋白和截短型HBV表面抗原中蛋白均具有反式激活SV40早期启动子（增强子）的功能，并且两种蛋白的反式激活功能具有协同特性。本实验有助于解释HCV、HBV感染，尤其是共同感染的致病（癌）机制。     

MxA inhibits hepatitis B virus replication by interaction with hepatitis B core antigen

Human MxA, an interferon-inducible cytoplasmic dynamin-like GTPase, possesses antiviral activity against multiple RNA viruses. Recently, MxA has also been demonstrated to have activity against the hepatitis B virus (HBV), a well-known DNA virus responsible for acute and chronic liver disease in humans. We investigated the molecular mechanism for the anti-HBV activity of MxA. Our results demonstrated that in HepG2.2.15 cells, MxA GTPase independently suppressed the production of hepatitis B surface antigen and HBV DNA without changing the level of hepatitis B core antigen (HBcAg) and the distribution of HBV mRNA. MxA significantly reduced the level of the encapsidated pregenomic RNA. Through its central interactive domain, MxA interacted with HBcAg, causing accumulation of the proteins in perinuclear compartments. MxA-HBcAg interaction significantly affected the dynamics of HBcAg by immobilizing HBcAg in the perinuclear structures. CONCLUSION: MxA displays antiviral activity against HBV involving a mechanism of MxA-HBcAg interaction that may interfere with core particle formation.