Inhibition of HBV targeted ribonuclease enhanced by introduction of linker

AIM: To construct human eosinophil-derived neurotoxin(hEDN) and HBV core protein (HBVc) eukaryotic fusion expression vector with a linker (Gly(4)Ser) (3) between them to optimize the molecule folding, which will be used to inhibit HBV replication in vitro. METHODS: Previously constructed pcDNA3.1(-)/TR was used as a template. Linker sequence was synthesized and annealed to form dslinker, and cloned into pcDNA3.1(-)/TR to produce plasmid pcDNA3.1(-)/HBc-linker. Then the hEDN fragment was PCR amplified and inserted into pcDNA3.1(-)/HBc-linker to form pcDNA3.1(-)/TNL in which the effector molecule and the target molecule were separated by a linker sequence. pcDNA3.1(-)/TNL expression was identified by indirect immunofluorescence staining. Radioimmunoassay was used to analyse anti-HBV activity of pcDNA3.1(-)/TNL. Meanwhile, metabolism of cells was evaluated by MTT colorimetry. RESULTS: hEDN and HBVc eukaryotic fusion expression vector with a linker (Gly(4)Ser)(3) between them was successfully constructed. pcDNA3.1(-)/TNL was expressed in HepG2.2.15 cells efficiently. A significant decrease of HBsAg concentration from pcDNA3.1(-)/TNL transfectant was observed compared to pcDNA3.1(-)/TR (P=0.036, P<0.05). MTT assay suggested that there were no significant differences between groups (P=0.08, P>0.05). CONCLUSION: Linker introduction enhances the inhibitory effect of HBV targeted ribonuclease significantly.     

Targeted ribonuclease can inhibit replication of hepatitis B virus

AIM: To study the effect of a novel targeted ribonuclease(TN), the fusion protein of HBVc and human eosinophilderived neurotoxin (hEDN), on the HBV replication in vitro.METHODS: The gene encoding the targeted ribonuclease was cloned into pcDNA3.1 (-) to form recombinant eukaryotic expression vector p/TN. Control plasmids, including p/hEDN,p/HBVc, and p/TNmut in which a Lys113→Arg mutation wasintroduced by sequential PCR to eliminate the ribonuclease activity of hEDN, were also constructed. Liposome-mediated transfection of 2.2.15 cells by p/TN, p/TNmut, p/hEDN, p/HBVc, and pcDNA3.1 (-), or mock transfection was performed. After that, RT-PCR was used to verify the transgene expression. Morphology of the transfected cells was observed and M‘IF assay was performed to detect the cytotoxicity of transgene expression. Concentration of HBsAg in the supernatant of the transfected cells was measured using solid-phase radioimmunoassay.RESULTS: Transgenes were successfully expressed in 2.2.15 cells. No obvious cytotoxic effect of transgene expressionon 2.2.15 cells was found. The HBsAg concentration in the p/TN transfected cells was reduced by 58 % compared with that of mock transfected cells. No such an effect was found in all other controls.CONCLUSION: The targeted ribonuclease can inhibit HBV replication in vitrowhile it has no cytotoxicity on host ceils.The targeted ribonuclease may be used as a novel antiviral agent for human HBV infection.     

VP22 fusion protein-based dominant negative mutant can inhibit hepatitis B virus replication

AIM: To investigate the inhibitory effect of VP22 fusion protein-based dominant negative (DN) mutant on Hepatitis Bvrus (HBV) replication. METHODS: Full-length or truncated fragment of VP22 was fused to C terminal of HBV core protein (HBc), and subcloned into pcDNA3.1 (-) vector, yielding eukaryotic expression plasmids of DN mutant. After transfection into HepG2.2.15 cells, the expression of DN mutant was identified by immunofluorescence staining. The inhibitory effect of DN mutant on HBV replication was indexed as the supernatant HBsAg concentration determined by RIA and HBV-DNA content by fluorescent quantification-PCR (FQ-PCR). Meanwhile, metabolism of HepG2.2.15 cells was evaluated by MTT colorimetry. RESULTS: VP22-based DN mutants and its truncated fragment were expressed in HepG2.2.15 cells, and had no toxic effect on host cells. DN mutants could inhibit HBV replication and the transduction ability of mutant-bearing protein had a stronger inhibitory effect on HBV replication. DN mutants with full length of VP22 had the strongest inhibitory effect on HBV replication, reducing the HBsAg concentration by 81.94%, and the HBV-DNA content by 72.30%. MTT assay suggested that there were no significant differences in cell metabolic activity between the groups. CONCLUSION: VP22-based DN mutant can inhibit HBV replication effectively.     

蛋白转导结构域介导乙型肝炎病毒聚合酶末端蛋白重链可变区抗体体外抑制病毒的复制

目的 蛋白转导结构域(TAT)介导HBV聚合酶末端蛋白(TP)重链可变区(VH)抗体,研究特异性TAT-VH抗体体外对HBV复制的影响.方法 将TAT-VH基因克隆入原核表达载体pET28a(+),在大肠埃希菌BL21(DE3)LysS内诱导融合蛋白表达并进行纯化.纯化的TAT-VH加入培养的HepG2.2.15细胞,间接免疫荧光法检测其导入HepG2.2.15细胞的效率,四甲基偶氮唑盐(MTT)法检测其对细胞生长代谢的影响,将TAT-VH加入培养的HepG2.2.15细胞,定量PCR法检测HBV DNA水平.数据行单因素方差分析和t检验.结果 成功制备了TAT-VH融合蛋白,间接免疫荧光及MTT证实TAT-VH可以跨膜导入HepG2.2.15细胞,且对细胞生长无影响;加入5 000 nmol/L TAT-VH的HepG2.2.15细胞培养上清液内HBV DNA为(1.211±0.132)lg拷贝/mL,对照组为(5.325±0.041)lg拷贝/mL(t=72.91,P＜0.05);细胞内分别为(3.521±0.411)和(8.532±0.132)Ig拷贝/mL(t=28.41,P＜0.05).结论 HBV聚合酶TP区特异性TAT-VH抗体在体外可抑制HBV复制,为应用细胞内抗体治疗HBV感染提供了良好的实验基础.     

Combination of small interfering RNA and lamivudine on inhibition of human B virus replication in HepG2.2.15 cells

AIM: To observe the inhibition of hepatitis B virus (HBV) replication and expression by combination of siRNA and lamivudine in HepG2.2.15 cells. METHODS: Recombinant plasmid psil-HBV was constructed and transfected into HepG2.2.15 cells. The transfected cells were cultured in lamivudine-containing medium (0.05 μmol/L) and harvested at 48, 72 and 96 h. The concentration of HBeAg and HBsAg was determined using ELISA. HBV DNA replication was examined by real- time PCR and the level of HBV mRNA was measured by RT-PCR. RESULTS: In HepG2.2.15 cells treated with combination of siRNA and lamivudine, the secretion of HBeAg and HBsAg into the supernatant was found to be inhibited by 91.80% and 82.40% (2.89 ± 0.48 vs 11.73 ± 0.38, P < 0.05; 4.59 ± 0.57 vs 16.25 ± 0.48, P < 0.05) at 96 h, respectively; the number of HBV DNA copies within culture medium was also significantly decreased at 96 h (1.04 ± 0.26 vs 8.35 ± 0.33, P < 0.05). Moreover, mRNA concentration in HepG2.2.15 cells treated with combination of siRNA and lamivudine was obviously lower compared to those treated either with siRNA or lamivudine (19.44 ± 0.17 vs 33.27 ± 0.21 or 79.9 ± 0.13, P < 0.05). CONCLUSION: Combination of siRNA and lamivudine is more effective in inhibiting HBV replication as compared to the single use of siRNA or lamivudine in HepG2.2.15 cells.     

RNA干扰抑制乙型肝炎病毒复制的实验研究

目的 以乙型肝炎病毒(HBV)核心区为靶位，构建表达小干扰RNA(siRNA)的质粒载体pSilencer3．1-Hlhygro，体外观察siRNA抗HBV的效果。方法 以HepG2 2．2．15细胞为靶细胞，利用脂质体Metafectene与表达siRNA的质粒载体pSilencer3．1-Hlhygro共转染，用定量聚合酶链反应检测细胞上清液中DNA，用逆转录聚合酶链反应检测HBV C-mRNA。结果 成功构建了表达siRNA的转录质粒载体，两条siRNA均可抑制HBV的复制，而且与siRNA浓度成正相关。结论 靶向HBV核心区的siRNA能抑制HBV的复制。     

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

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

青蒿琥酯对乙型肝炎病毒复制及肝癌细胞株HepG2.2.15凋亡的影响

目的 探讨青蒿琥酯在体外对乙型肝炎病毒复制及肝癌细胞株HepG2.2.15凋亡的影响.方法 将不同浓度青蒿琥酯作用于转染乙型肝炎病毒全基因组DNA的肝癌细胞株HepG2.2.15,收集48 h上清,采用酶联免疫吸附实验检测上清中乙型肝炎病毒表面抗原(HBsAg)和e抗原(HBeAg),采用荧光定量PCR法检测HBV-DNA,流式细胞术检测细胞凋亡.结果 青蒿琥酯对HBV复制具有抑制作用,随着浓度增加,对HBsAg和HBeAg的抑制率逐渐上升,细胞内HBV-DNA 复制水平下降;青蒿琥酯可诱导肝癌细胞早期凋亡及导致细胞死亡,随浓度增加,HepG2.2.15细胞早期凋亡率及死亡率均增加.结论 青蒿琥酯对HepG2.2.15细胞HBsAg和HBeAg的分泌及HBV-DNA复制具有抑制作用,并具有诱导HepG2.2.15细胞凋亡的作用.     

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.     

商陆抗病毒蛋白不同结构域的抗乙型肝炎病毒活性

目的 比较全长和不同片段缺失型商陆抗病毒蛋白(PAP)基因真核表达质粒体外抗HBV作用及其细胞毒性作用.方法 将全长和不同片段缺失型PAP基因真核表达质粒用脂质体转染HepG2.2.15细胞,收获生长良好的HepG2.2.15细胞,转染前1 d,接种于24孔培养细胞板,培养20 h后,待细胞密度达到40%～50%时进行转染.细胞随机分为4组:pXF3H组,转染空质粒pXF3H作为对照;pXF3H-PAP_(12)组,转染全长PAP的真核表达质粒pXF3H-PAP_(12);pXF3H-PAP_(14)组,转染C端缺失25个氨基酸的PAP的真核表达质粒pXF3H-PAP_(14);pXF3H-PAP3_(34)组,转染既缺失N端69个氨基酸又缺失C端25个氨基酸的PAP的真核表达质粒pXF3H-PAP_(34).转染的质粒剂量为每孔1.0μg,终浓度为2.0μg/ml,质粒DNA(μg)和脂质体(μl)的比例为1:2.5,转染72 h后收集细胞及培养上清液.酶联免疫吸附法检测培养上清液HBsAg和HBeAg,荧光定量PCR检测HBV DNA水平,四甲基偶氮唑盐比色法检测各质粒对转染细胞的毒性作用.应用SPSS12.0软件包处理数据,两样本均数的比较采用t检验,率的比较采用χ~2检验.结果 对HBsAg、HBeAg、HBVDNA的抑制率,pXF3H-PAP_(14)组分别为56.3%、75.8%和61.7%,pXF3H-PAP_(12)组分别为61.4%、84.2%和63.2%,两组间差异无统计学意义.但pXF3H-PAP_(14)组细胞毒性 (抑制率为10.2%)明显低于pXF3H-PAP_(12)组(抑制率为27.1%),χ~2=7.7,P<0.01.pXF3H-PAP_(34)组无细胞毒性,但其抗HBV作用也丧失,对HBsAg、HBeAg、HBV DNA的抑制率分别为7.8%、11.0%、20.5%.结论 PAP的C端25个氨基酸与细胞毒性相关,与抗HBV活性无关;PAP的N端69个氨基酸与抗HBV活性相关.     

Inhibition of the replication of the DNA polymerase M550V mutation variant of human hepatitis B virus by adefovir, tenofovir, L-FMAU, DAPD, penciclovir and lobucavir

Several nucleoside analogues (penciclovir, lobucavir, dioxalane guanine [DXG], 1-β-2,6-diaminopurine dioxalane [DAPD], L-FMAU, lamivudine) and acyclic nucleoside phosphonate analogues (adefovir, tenofovir) that are in clinical use, in clinical trials or under preclinical development for the treatment of hepatitis B virus (HBV) infections, were evaluated for their inhibitory effect on the replication of a la- mivudine-resistant HBV variant containing the methionine → valine substitution (M550V) in the polymerase nucleoside-binding domain. The antiviral activity was determined in the tetracycline-responsive HepAD38 and HepAD79 cells, which are stably transfected with either a cDNA copy of the wild-type pregenomic RNA or with cDNA containing the M550V mutation. As expected, lamivudine was much less (≈ 200-fold) effective at inhibiting replication of the M550V mutant virus than the wild-type virus. In contrast, adefovir, tenofovir, lobucavir, L-FMAU, DXG and DAPD proved almost equally effective against both viruses. A second objective of this study was to directly compare the antiviral potency of the anti-HBV agents in HepG2 2.2.15 cells (which are routinely used for anti-HBV drug-screening purposes) with that in HepAD38 cells. HepAD38 cells produce much larger quantities of HBV than HepG2 2.2.15 cells, and thus allow drug screening in a multiwell plate format. All compounds were found to be almost equally effective at inhibiting HBV replication in HepAD38 cells (as in HepG2 2.2.15 cells), except for penciclovir, which was clearly less effective in HepAD38 cells.     

La蛋白与乙型肝炎病毒mRNA稳定性和蛋白质表达的相关性

目的研究La蛋白与乙型肝炎病毒（HBV）mRNA稳定性和蛋白质表达之间的相关性。方法设计并合成La蛋白mRNA特异性小干扰RNA（siRNA），转染HepG2．2．15细胞，继续培养3d后分别裂解转染和未转染siRNA的细胞l抽提蛋白质，Westernblot检测La蛋白含量变化；于转染后的第1、2、3、5、7、9天分别收集细胞培养上清液，实时荧光定量聚合酶链反应技术检测上清液中HBVDNA的变化情况，电化学发光分析技术检测乙型肝炎表面抗原，乙型肝炎e抗原含量变化情况。结果La蛋白在转染siRNA的HepG2．2．15细胞中表达明显低于未转染siRNA的细胞；转染siRNA的细胞分泌到上清液中乙型肝炎表面抗原、乙型肝炎e抗原和HBVDNA的含量也明显低于未转染siRNA的细胞。结论在HepG2．2．15细胞内，La蛋白与HBVmRNA稳定性和蛋白质表达具有功能相关性。     

A novel HBV antisense RNA gene delivery system targeting hepatocellular carcinoma

AIM: To construct a novel HBV antisense RNA delivery system targeting hapatocellular carcinoma and study its inhibitory effect in vitro and in vivo.METHODS: GE7,a 16-peptide specific to EGFR, and HA20,a homologue of N-terminus of haemagglutinin of influenza viral envelope protein, were synthesized and conjugated with polylysin. The above conjugates were organized into the pEBAF-as-preS2, a hepatocarcinoma specific HBV antisense expression vector, to construct a novel HBV antisense RNA delivery system, named AFP-enhancing 4-element complex. Hepatocelluar carcinoma HepG2.2.15 cells was used to assay the in vitro inhibition of the complex on HBV. Expression of HBV antigen was assayed by ELISA.BALB/c nude mice bearing HepG2.2.15 cells were injected with AFP-enhancing 4-element complex. The expression of HBV antisense RNA was examined by RT-PCR and the size of tumor in nude mice were measured.RESULTS: The AFP-enhancing 4-element complex was constructed and DNA was completely trapped at the slot with no DNA migration when the ratio of polypeptide to plasmid was 1:1.The expression of HBsAg and HBeAg of HepG2.2.15 cells was greatly decreased after being transfected by AFP-enhancing 4-element complex. The inhibitory rates were 33.4 % and 58.5 % respectively. RT-PCR showed HBV antisense RNA expressed specifically in liver tumor cells of tumor-bearing nude mice. After 4 injections of AFP-enhancing 4-element complex containing 0.2 μg DNA, the diameter of the tumor was 0.995 cm&#177;0.35,which was significantly smaller than that of the control groups (2.215 cm&#177;025, P&lt;0.05).CONCLUSION: AFP-enhancing 4-element complex could deliver HBV antisense RNA targeting on hepatocarcinoma and inhibit both HBV and liver tumor cells in vitro and in vivo.     

维甲酸诱导基因-I诱导干扰素的产生在乙型肝炎病毒感染清除中的作用

目的探讨维甲酸诱导基因-I(RIG-I)在诱导HBV感染IFN产生中的影响,以期对阐明乙型肝炎慢性化机制,寻找新的治疗靶点提供新的思路。方法以RIG-I表达载体flagRIG-l-ful1转染培养6孔板中的HepG2、HepG2.2.15,24h后用水疱性口炎病毒(VSV)感染细胞,然后在0、8、16和24h收集细胞及培养上清液,采用quantitive RT-PCR、Western印迹检测RIG-I、MDA5、IPS-1、ISG54等基因的表达,ELISA检测培养上清液中分泌的IFN-β水平。结果 HepG2.2.15细胞在VSV感染后IFN-β分泌水平(11.18±1.34)pg/mL明显低于HepG2细胞(275.50±22.97)pg/mL(P<0.01);通过质粒flagRIG-I-ful1转染高表达RIG-I后,HepG2.2.15分泌IFN-β的能力恢复至(548.78±57.99)pg/mL与HepG2细胞(532.10±39.34)pg/mL接近的水平(P=0.7013)。结论 HepG2.2.15细胞感染VSV后IFN产生障碍,高表达RIG-I后IFN表达水平得到恢复,提示RIG-I功能缺陷导致抗病毒免疫应答降低,RIG-I可能在HBV感染清除中起重要作用。     

PTD-HBcAg融合蛋白诱导特异性CTL在HepG2.2.15细胞抑制HBV复制的研究

目的探讨PTD-HBcAg融合蛋白诱导小鼠体内特异性CTL并抑制HepG2.2.15细胞HBV复制的作用。方法 PTD-HBcAg、HBcAg和阴性对照分别与等体积的弗氏佐剂乳化后皮下免疫小鼠;第14d,分离脾淋巴细胞并分别用PTD-HBcAg、HBcAg、PTD和PBS加强刺激后收集上清,检测细胞因子IFN-γ、IL-12、IL-4和IL-10;刺激后的淋巴细胞作为效应细胞与HepG2.2.15细胞共培养,检测,效应细胞对HBsAg、HBVDNA的抑制作用及对HepG2.2.15细胞、HepG2细胞的杀伤效果。结果 PTD-HBcAg组分泌的IFN-γ、IL-12、IL-4和IL-10与HBcAg组和阴性对照组比较差异有统计学意义(P0.05);PTD-HBcAg融合蛋白组较HBcAg组和阴性对照组有更明显的病毒抑制作用(P0.05);PTD-HBcAg组对HepG2.2.15细胞的杀伤率明显高于HBcAg组和阴性对照组(P0.05)。结论 PTD-HBcAg可诱导HBV特异性CTL,能有效抑制HepG2.2.15细胞HBV的复制。