IFN-αinhibits HBV transcription and replication in cell culture and in humanized mice by targeting
Research article IFN-α inhibits HBV transcription and replication in cell culture and in humanized mice by targeting the epigenetic regulation of the nuclear cccDNA minichromosome
Laura Belloni,1,2,3 Lena Allweiss,4 Francesca Guerrieri,1,3,5 Natalia Pediconi,1,3,5 Tassilo Volz,4 Teresa Pollicino,6 Joerg Petersen,7 Giovanni Raimondo,6 Maura Dandri,4 and Massimo Levrero1,2,3,5 1EAL Inserm U785, Sapienza University of Rome, Rome, Italy. 2Laboratory of Gene Expression, Fondazione A. Cesalpino, Rome, Italy.
3Department of Internal Medicine, University La Sapienza, Rome, Italy. 4Department of Internal Medicine, University Medical Hospital Hamburg-Eppendorf, Hamburg, Germany. 5Rome Oncogenomic Center, Istituto Regina Elena, Rome, Italy. 6Department of Internal Medicine, University of Messina, Messina, Italy.
7IFI Institute for Interdisciplinary Medicine, Asklepios Clinic St. Georg, Hamburg, Germany.
HBV infection remains a leading cause of death worldwide. IFN-α inhibits viral replication in vitro and in vivo, and pegylated IFN-α is a commonly administered treatment for individuals infected with HBV. The HBV genome contains a typical IFN-stimulated response element (ISRE), but the molecular mechanisms by which IFN-α suppresses HBV replication have not been established in relevant experimental systems. Here, we show that IFN-α inhibits HBV replication by decreasing the transcription of pregenomic RNA (pgRNA) and sub-genomic RNA from the HBV covalently closed circular DNA (cccDNA) minichromosome, both in cultured cells in which HBV is replicating and in mice whose livers have been repopulated with human hepatocytes and infected with HBV. Administration of IFN-α resulted in cccDNA-bound histone hypoacetylation as well as active recruitment to the cccDNA of transcriptional corepressors. IFN-α treatment also reduced binding of the STAT1 and STAT2 transcription factors to active cccDNA. The inhibitory activity of IFN-α was linked to the IRSE, as IRSE-mutant HBV transcribed less pgRNA and could not be repressed by IFN-α treatment. Our results identify a molecular mechanism whereby IFN-α mediates epigenetic repression of HBV cccDNA tran-scriptional activity, which may assist in the development of novel effective therapeutics.
Introduction
Hepatitis?B?Virus?(HBV)?infection?remains?a?major?health?prob-lem?worldwide?despite?the?availability?of?a?highly?effective?preven-tive?vaccine.?HBV?is?a?noncytopathic?hepatotropic?DNA?virus?that?belongs?to?the?family?Hepadnaviridae,?whose?members?share?a?distinctive?strategy?for?replication.?HBV?replication?occurs?in?the?cytoplasm?within?viral?capsids?(core?particles),?where?a?genome-sized?RNA?replicative?intermediate,?termed?the?pregenome?(pgRNA),?is?converted?by?the?virally?encoded?RNA-dependent?and?DNA-dependent?reverse?transcriptase/polymerase?into?a?specific?open?circular?(OC)?duplex?DNA?(1).?Transcription?in?the?nucleus?of?the?pgRNA?from?the?covalently?closed?circular?DNA?(cccDNA)?is?the?critical?step?for?genome?amplification?and?ultimately?deter-mines?the?rate?of?HBV?replication?(2).?The?cccDNA,?which?also?serves?as?the?template?for?the?transcription?of?all?viral?messen-ger?RNAs,?is?organized?into?a?minichromosome?in?the?nuclei?of?infected?hepatocytes?by?histone?and?nonhistone?proteins,?and?its?function?is?regulated,?similarly?to?cellular?chromatin,?by?the?activ-ity?of?nuclear?transcription?factors,?transcriptional?coactivators?and?corepressors,?and?chromatin-modifying?enzymes?(2–4). Current?antiviral?therapies?involve?the?use?of?nucleoside?analogs?and?pegylated?IFN-α?(5).?IFN-α,?a?type?I?IFN,?engages?the?IFN-α/β?receptor?complex?to?activate?the?intracellular?Jak/Stat?signaling?pathway,?which?modulates?the?transcription?of?a?diverse?set?of?target?genes,?referred?to?as?IFN-stimulated?genes?(ISGs)?(6).?ISG?modulation?results?in?an?antiviral?response?in?target?cells?aimed?at?limiting?both?viral?replication?and?spreading.?IFN-α?has?been?reported?to?inhibit?HBV?replication?through?a?variety?of?mecha-nisms,?including?a?block?of?RNA-containing?core?particle?forma-tion,?an?accelerated?decay?of?replication-competent?core?particles,?and?degradation?of?the?pgRNA?(7–9).
An?IFN-stimulated?response?element?(ISRE)?has?been?identified?in?the?enhancer?1/X?gene?promoter?region?of?the?HBV?genome?(10),?and?IFN-α?has?been?shown?to?suppress?viral?gene?expression?(11,?12).?Subsequent?studies?failed?to?establish?the?role?of?IFN-α,?the?HBV?ISRE,?and?the?STAT?proteins?on?HBV?transcription?(13).?However,?most?studies?were?conducted?in?vitro?or?in?a?nonrepli-cative?context.?We?sought?to?employ?relevant?in?vitro?replicative?models?and?in?vivo?infection?systems?to?investigate?whether?IFN-α?targets?cccDNA?transcription?to?inhibit?viral?replication?and?attempted?to?define?the?molecular?mechanisms?of?IFN-α?repres-sion.?To?this?aim,?we?made?use?first?of?a?plasmid-free?HBV?trans-fection?cell-based?replication?assay?relying?on?the?generation?of?transcriptionally?active?nuclear?cccDNA?to?replicate?HBV?(3,?14).?Second,?SCID/beige?mice?transgenic?for?the?urokinase?plasmino-gen?activator?(uPA)?under?control?of?the?albumin?promoter?were?used?to?repopulate?mouse?livers?with?human?hepatocytes?derived?from?a?single?liver?donor?(15,?16).?This?model?minimizes?the?impact?of?host?variation?factors?and?allows?the?investigation?of?in?vivo?interactions?occurring?between?HBV?and?human?hepatocytes,?the?natural?target?cell?of?infection?and?replication.?Our?results?indicate?that?IFN-α?suppresses?HBV?replication?by?targeting?the?epigenetic?control?of?cccDNA?function?and?transcription.
Conflict of interest:?The?authors?have?declared?that?no?conflict?of?interest?exists. Citation for this article:?J Clin Invest.?2012;122(2):529–537.?doi:10.1172/JCI58847.
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Results
IFN-α inhibits cccDNA transcription and HBV replication in HCC cells .?Class?I?IFNs?inhibit?HBV?replication?in?a?variety?of?plasmid-based?replication?assays?in?HCC?cell?lines?and?nonhuman?pri-mary?hepatocytes?(7,?17–20).?We?examined?the?impact?of?IFN-α?treatment?on?cccDNA?transcription?and?HBV?replication?in?a?cccDNA-driven?replication?system?(3,?4).?Equivalent?numbers?of?HepG2?cells?were?transfected?with?linear?WT?HBV?(genotype?A)?genomes?and?exposed?to?IFN-α?(1000?U/ml).?We?confirmed?that?in?IFN-α–treated?HepG2?cells,?STAT1?and?STAT2?are?rapidly?phosphorylated?(Supplemental?Figure?1;?supplemental?material?available?online?with?this?article;?doi:10.1172/JCI58847DS1)?and?translocate?to?the?nucleus?to?bind?the?promoter?regions?of?ISGs?to?stimulate?their?transcription?(data?not?shown?and?Supplemental?Figure?2).?Cells?were?harvested?at?the?indicated?times?after?trans-fection?(Figure?1,?A?and?B),?cytoplasmic?viral?core?particles?were?isolated,?and?capsid-associated?HBV?DNA?was?quantified?by?real-time?PCR?and?analyzed?by?hybridization?with?a?32P-labeled?full-length?HBV?DNA?probe.?As?shown?in?Figure?1,?A?and?B,?the?level?of?capsid-associated?HBV?DNA?and?cccDNA?molecules?per?cell?was?highest?at?24?and?48?hours?after?transfection?and?decreased?in?the?following?days.?IFN-α?administration?performed?shortly?after?transfection?strongly?suppressed?steady-state?amounts?of?capsid-associated?HBV?DNA?compared?with?the?viral?loads?determined?in?untreated?cells,?although?the?number?of?cccDNA?copies?per?cell?was?not?modified?by?IFN-α?at?24?and?48?hours?of?treatment?(Figure?1B).?Notably,?Northern?blot?analysis?revealed?that?IFN-α?treatment?also?lowered?the?steady-state?levels?of?HBV?transcripts?in?HBV-replicating?cells.?Both?3.5?kb?pgRNA?and?2.4–2.1?mRNA?spe-cies?(preS/S?RNA)?levels?were?significantly?reduced?in?IFN-α–treat-ed?cells?(Figure?1C).?Quantification?of?pgRNA?levels?by?real-time?PCR?showed?a?60%?to?70%?decrease?after?24?and?48?hours?of?IFN-α?treatment?(Figure?1D),?without?affecting?levels?of?cccDNA?copies?per?cell?(Figure?1B).?These?results?confirm?that?IFN-α?inhibits?HBV?replication?in?our?system?and?indicate?that?IFN-α?lowers?steady-state?levels?of?HBV?transcripts.
Several?types?of?evidence?support?the?notion?that?cccDNA?tran-scription?is?controlled?by?epigenetic?modifications?of?cccDNA-bound?histones.?Indeed,?HBV?replication?and?cccDNA?transcrip-tion?are?modulated?by?substances?that?interfere?with?the?activity?of?known?chromatin-modifying?enzymes,?such?as?class?I/II?histone?deacetylases?(3).?Moreover,?using?the?cccDNA?ChIP?assay,?which?couples?a?classical?ChIP?step?with?a?cccDNA-specific?real-time?PCR?to?selectively?detect?the?immunoprecipitation?of?cccDNA-bound?histones?and?nonhistone?proteins,?we?showed?that?cccDNA?tran-scriptional?activity?and?HBV?replication?are?controlled,?both?in?cell?culture?systems?and?in?vivo?in?the?liver?of?chronic?HBV?carriers,?by?the?acetylation?status?of?cccDNA-bound?H3?and?H4?histones?(2,?3). We?found?that?48?hours?after?transfection,?cccDNA-bound?H4?histones?were?significantly?hypoacetylated?in?IFN-α–treated?cells,?thus?linking?the?repression?of?HBV?transcription?by?IFN-α?with?cccDNA-bound?histone?hypoacetylation?(Figure?1D).
The?regulatory?protein?HBx?is?recruited?onto?the?cccDNA?minichromosome?in?HBV-replicating?cells?(4)?and?is?required?for?both?cccDNA?transcription?and?HBV?replication?in?the?context?of?a?natural?HBV?infection?(21).?In?the?absence?of?HBx,?cccDNA-bound?histones?are?hypoacetylated,?and?the?cccDNA?transcribes?
significantly?less?pgRNA?(4).?We?sought?to?assess?whether?the?
Figure 1
IFN-α inhibits HBV replication and cccDNA transcription in HCC cells. (A ) Left panel: HepG2 cells were transfected with monomeric linear full-length WT HBV adw (genotype A) genomes. HBV core particles were isolated from untreated and IFN-α–treated cells at the indicated time points after transfection. Results are expressed as number of HBV DNA copies per transfected cell. Right panel: Southern blot hybridization. OC, open circular duplex HBV DNA; DS, double-stranded HBV DNA replicative intermediates; SS, single-stranded HBV DNA replicative intermediates. (B ) Left panel: cccDNA levels (copies per transfected cell) were analyzed by qPCR with selective cccDNA primers and β-globin primers (DNA sample normalization). Right panel: Southern blot analysis. (C ) Left panel: pgRNA levels were analyzed by qPCR using pgRNA selective prim-ers and GAPDH primers (equal loading of each RNA sample). Right panel: Northern blot analysis. pgRNA, HBV pregenomic RNA. (D ) Cross-linked chromatin was immunoprecipitated with a relevant control IgG or specific anti-AcH4 antibody and analyzed by qPCR with HBV cccDNA selective primers. Results are expressed as fold induction relative to the untreated cells using the comparative Ct method. (E ) HepG2 cells were transfected with monomeric linear full-length WT or HBx mutant HBV genomes (4). Core particles HBV-DNA (left panel) and pgRNA (right panel) results are expressed as in Figure 1, A and C, respectively. All histograms show mean values from 3 independent experiments; bars indicate SD. P values were determined using Student’s t test. *P < 0.05, **P < 0.01, ***P < 0.001.
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repression?of?cccDNA?transcriptional?activity?by?IFN-α?is?medi-ated?by?or?requires?HBx.?As?shown?in?Figure?1E,?IFN-α?inhibited?capsid-associated?HBV?DNA?and?pgRNA?levels?to?a?similar?extent?in?HepG2?cells?replicating?WT?or?HBx?mutant?viruses,?indicating?that?the?repression?of?cccDNA?transcriptional?activity?by?IFN-α?is?HBx?independent.
IFN-α inhibits cccDNA transcription and HBV replication in chimeric uPA/SCID mice .?The?lack?of?efficient?HBV?infection?models?has?greatly?limited?the?study?of?the?antiviral?mechanisms?induced?by?IFN-α?in?HBV-infected?hepatocytes?in?vivo.?To?determine?the?impact?of?IFN?treatment?on?intrahepatic?viral?activity,?chimeric?uPA/SCID?mice?were?repopulated?with?primary?human?hepatocytes?and?infected?with?HBV?in?vivo?(15).?10?HBV-infected?mice?display-ing?stable?median?viremia?levels?of?2?×?108?HBV?DNA?copies/ml?(range,?2?×?107?to?6?×?108)?received?either?daily?injections?of?human?IFN-α?(n =?5)?or?saline?(n =?5).?Comparative?analysis?of?viremia?changes?performed?in?single?mice?after?5?days?of?IFN?treatment?showed?a?significant?reduction?in?viremia?(range,?0.4?to?2.5?log;?median,?0.8?log)?compared?with?baseline?in?all?animals?analyzed?(Figure?2A).?Similarly?to?what?we?observed?in?HBV-replicating?HCC?cell?lines,?IFN-α?treatment?reduced?both?the?levels?of?pgRNA?and?subgenomic?RNAs?(preS/S?RNA)?in?all?animals?analyzed,?as?shown?by?Northern?blot?(Figure?2B)?and?real-time?RT-PCR?analysis?(Figure?2C)?performed?on?liver?tissues?obtained?8?hours?after?the?last?IFN?application,?whereas?no?changes?in?the?intrahepatic?cccDNA?load?were?observed?(Figure?2D).?Notably,?the?reduction?of?HBV?RNA?transcripts?was?accompanied?by?a?sharp?reduction?of?cccDNA-bound?histone?H4?acetylation?(Figure?2E).?These?data?indicate?that?IFN-α?exhibits?a?direct?antiviral?effect?in?infected?primary?human?hepatocytes?by?inducing?the?hypoacetylation?of?cccDNA-bound?histones?and?decreasing?the?levels?of?HBV?transcripts?in?vivo?with-out?any?measurable?effect?on?the?size?of?the?nuclear?cccDNA?pool.
The HBV ISRE mediates STAT1 and STAT2 recruitment on the cccD-NA and IFN-α transcriptional repression .?Next,?we?sought?to?inves-tigate?whether?the?ISRE?element?present?on?the?HBV?cccDNA?is?functional?in?vivo?and?might?be?therefore?involved?in?the?HBV?transcriptional?repression?observed?after?IFN-α?treatment.?As?shown?in?Figure?3A,?STAT1,?STAT2,?and?their?phosphorylated?forms?all?bind?to?the?cccDNA?in?unstimulated?cells.?After?IFN-α?treatment,?both?cccDNA-bound?phospho-STAT1?and?phospho-STAT2?and,?to?a?lesser?extent,?STAT1?and?STAT2,?recruitment,?are?greatly?reduced?(Figure?3B).?These?results?indicate?that?the?HBV?ISRE?is?functional?in?the?context?of?HBV?replication?and?that?STAT?binding?to?the?cccDNA?changes?upon?IFN-α?treat-ment.?Although?we?do?not?have?formal?evidence,?these?results?also?suggest?that,?in?response?to?IFN-α,?there?is?a?dynamic?change?in?the?quality?of?the?complexes?bound?to?the?HBV?ISRE?with?loss?of?phosphorylated?STAT2?and?STAT1?and?a?decrease?of?unphos-phorylated?STAT?proteins.?In?this?respect,?it?is?noteworthy?that?chronic?HBV?infection?of?human?hepatocytes?transplanted?in?uPA/SCID?mice?impairs?the?induction?of?IFN-α?target?genes?by?inhibiting?nuclear?translocation?of?STAT1?(16).?The?presence?of?STAT1?and?STAT2?complexes?on?the?cccDNA?before?IFN-α?treatment?apparently?defies?the?classical?paradigm?of?the?STATs?as?latent?transcription?factors?in?the?cytoplasm,?entering?the?nucleus?to?induce?gene?expression?only?in?response?to?cytokine?stimulation?to?bind?DNA?and?activate?their?transcriptional?pro-gram,?but?it?is?not?unprecedented.?Using?a?ChIP-Seq?approach,?it?has?been?shown?that?more?than?85%?of?STAT1-binding?peaks?overlap?before?and?after?IFN-γ?stimulation?(22).?Recently,?using?a?ChIP-on-chip?approach?on?a?large?subset?of?IFN-α?direct?target?genes,?we?could?show?an?occupancy?of?ISRE?sites?by?STAT2?in?about?half?of?the?promoters?we?studied?in?unstimulated?HCC?
cells?lines?and?primary?human?hepatocytes?(23).
Figure 2
IFN-α inhibits cccDNA transcription and HBV replication in chimeric uPA/SCID mice. Uninfected and chronically HBV infected (median, 2 × 108 serum HBV-DNA copies/ml serum) chimeric uPA/SCID mice transplanted with thawed human hepatocytes received daily injections of human IFN-α (1,300 IU/g body weight) for 5 days or saline. (A ) HBV viremia (log HBV-DNA copies/ml serum) were determined in individual animals (n = 5) at baseline, shortly before IFN treatment, and 8 hours after the last adminis-tration of IFN-α. (B ) Representative Northern blot analysis from one IFN-α– treated mouse and one untreated mouse. (C ) Steady-state levels of pre-genomic RNA (left panel) and preS/S RNAs (right panel). Intrahepatic preS/S RNAs were determined by subtracting pgRNA amounts from total HBV RNAs (pgRNA + preS/S RNA) estimated in the same RNA prepara-tion as described in Methods, and values were normalized using human-specific GAPDH primers. (D ) Intrahepatic cccDNA loads in IFN-α–treated (n = 5) and control (saline) HBV-infected mice (n = 5). Real-time qPCR analysis was performed using selective cccDNA primers, and β-globin primers were used to normalize cccDNA copies (median + SD) per human hepatocyte (expressed as human genome equivalents) determined in chi-meric livers. (E ) Cross-linked chromatin from liver samples of chronically HBV-infected untreated and IFN-α–treated (48 hours) uPA chimeric mice was immunoprecipitated with a relevant control IgG or specific anti-AcH4 antibody. Immunoprecipitated chromatin was analyzed by qPCR as in Figure 1D. All histograms show mean values from 3 independent experi-ments; bars indicate SD.
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To?confirm?the?role?of?STAT1?and?STAT2?in?mediating?the?effect?of?IFN-α?on?HBV?replication,?we?generated?a?replication-compe-tent?HBV?genome?bearing?ISRE-inactivating?mutations?(IRESmt)?(ref.?13?and?Figure?4A).?As?expected,?cccDNA?molecules?harbor-ing?an?ISREmt?do?not?recruit?STAT2?(Figure?4B).?We?found?that?ISREmt?HBV?transcribes?less?pgRNA?(Figure?4C)?and?accordingly?replicates?less?efficiently?(Figure?4D).?Importantly,?pgRNA?tran-scription?from?the?ISREmt?HBV?cccDNA?is?not?repressed?by?IFN-α?treatment?(Figure?4C),?supporting?the?notion?that?the?ISRE?indeed?mediates?IFN-α?transcriptional?repression?of?cccDNA?transcrip-tion.?On?the?other?hand,?the?observed?reduction?of?pgRNA?pro-duction?in?untreated?ISREmt?HBV–replicating?cells?(Figure?4C)?would?suggest?that?the?binding?of?STATs,?and?possibly?of?other?transcription?factors/coactivators,?to?the?WT?HBV?ISRE?contrib-utes,?in?the?absence?of?exogenous?IFN-α,?to?cccDNA?transcrip-tion?and?replication.?Furthermore,?the?replicative?activity?of?the?ISREmt?HBV?displays?a?reduced?but?still?significant?repression?by?IFN-α?(Figure?4D)?that?likely?reflects?the?contribution?of?the?tran-scription-independent?effects?mediated?by?IFN-α?(7).
IFN-α affects the epigenetic control of the HBV cccDNA minichromo-some .?To?gain?further?mechanistic?insights?in?the?epigenetic?reg-ulation?of?the?HBV?minichromosome?function?by?IFN-α?and?to?investigate?how?active?cccDNA-bound?STAT?complexes?are?con-verted?into?repressive?ones,?we?performed?additional?cccDNA?ChIP?experiments.?HBV-transfected?HepG2?cells?were?exposed?to?IFN-α?for?48?hours,?and?chromatin?was?immunoprecipitated?using?antibodies?that?specifically?recognize?the?histone?deacety-lases?HDAC1?and?hSirt1,?the?polycomb?protein?enhancer?of?zeste?homolog?2?(Ezh2),?and?the?transcription?factors?YY1?(yin?yang?1).?HDAC1?and?hSIRT1?were?previously?shown?to?bind?the?cccDNA,?and?their?recruitment?and?activity?correlated?with?decreased?viral?replication?in?cell?culture?and,?in?the?case?of?HDAC1,?in?the?liver?of?HBV?chronic?hepatitis?patients?(3,?4).?YY1?is?a?ubiquitous?zinc-finger?transcription?factor?of?the?Polycomb?Group?(PcG)?protein?family?that,?depending?on?the?binding?context,?can?activate?or?repress?the?transcription?of?many?viral?and?cellular?genes?(24).?YY1-binding?sites?have?been?located?upstream?of?DR1?in?the?HBV?genome?(25).?To?exert?its?repressive?function,?YY1?interacts?with?different?HDACs?(26)?and?with?Ezh2,?a?PcG?chromatin-modify-ing?enzyme?with?histone?lysine?methyltransferase?(HKMT)?activity?(27).?As?shown?in?Figure?5A,?48?hours?after?transfection,?when?the?cccDNA-bound?histones?H4?are?significantly?hypoacetylated?in?IFN-α–treated?cells?(Figure?1D),?the?recruitment?of?HDAC1,?Sirt1,?Ezh2,?and?YY1?is?increased.?It?is?noteworthy?that?Ezh2?occupancy?on?the?cccDNA?is?still?conserved?48?hours?after?IFN-α?treatment?is?stopped?(Figure?5B).?Accordingly,?pgRNA?transcription?(Figure?
5B)?and?cytoplasmic?HBV?core?particle?production?(Figure?5B)?are?
both?persistently?repressed?after?IFN-α?release.?These?results?indi-cate?that?IFN-α?induces?a?persistent?recruitment?of?several?core-pressors?and?components?of?the?polycomb?repressive?complex?2?(PRC2)?that?target?histone?tail?acetylation?and?methylation,?thus?providing?a?molecular?mechanism?for?long-term?off-treatment?IFN-α?repression?of?HBV?transcription?(Figure?6).Finally,?we?studied?the?impact?of?the?ISRE?mutation?on?cccDNA?
chromatin?status?in?response?to?IFN-α.?We?found?that?the?ISREmt?
HBV?cccDNA?does?not?recruit,?as?expected,?STAT2?proteins?and?
displays,?according?to?its?reduced?transcription/replication?capac-ity,?a?reduced?acetylation?of?cccDNA-bound?H4?histones?that?is?not?further?modified?by?IFN-α?(Figure?5C).?We?also?show?that?the?ISREmt?HBV?cccDNA?fails?to?recruit?HDAC1?in?response?to?IFN-α?
(Figure?5D),?thus?further?linking?the?presence?of?a?functional?ISRE?with?the?epigenetic?changes?of?the?cccDNA?chromatin?that?char-acterize?the?transcription?repression?response?to?IFN-α.?Interest-ingly,?the?ISREmt?cccDNA?displays?an?increased?hSirt1?binding?in?the?absence?of?IFN-α?stimulation?and?further?recruits?hSirt1?in?response?to?IFN-α?stimulation.?These?observations?suggest?that?the?presence?of?hSirt1?on?the?ISREmt?cccDNA?might?contribute?to?its?reduced?transcriptional?activity?in?unstimulated?cells,?whereas?its?further?recruitment?after?IFN?treatment?might?mediate?in?part?the?IFN-α?repression?of?the?ISREmt?HBV?cccDNA.Discussion
Together,?our?results?identify?a?new?molecular?mechanism?by?which?IFN-α?inhibits?HBV?replication.?We?show?that?IFN-α?inhibits?ccc-DNA-driven?transcription?of?genomic?and?subgenomic?RNAs,?both?in?HBV-replicating?cells?in?culture?and?in?HBV-infected?chimeric?uPA/SCID?mice?repopulated?with?primary?human?hepatocytes.?By?generating?a?replication-competent?HBV?genome?carrying?ISRE-inactivating?mutations,?we?also?linked?IFN-α?inhibitory?activity?with?the?presence?of?a?functional?ISRE.?ISREmt?HBV?transcribes?less?pgRNA,?replicates?less?efficiently,?and,?more?importantly,?can-not?be?repressed?by?IFN-α.?Both?STAT1?and?STAT2?transcription?factors?and?their?phosphorylated?“active”?forms?are?recruited?on?the?cccDNA,?and?their?binding?is?reduced?by?IFN-α?treatment.?Since,?under?the?same?experimental?conditions,?several?cellular?ISGs?were?activated?(Supplemental?Figure?1),?one?possible?mecha-nism?to?explain?IFN-α–repressive?activity?on?cccDNA?transcrip-tion?might?be?an?IFN-α–induced?redistribution?of?active?STAT1/STAT2?complexes?from?the?viral?minichromosome?to?cellular?target?genes.?However,?we?also?found?that,?in?response?to?IFN-α, cccDNA-bound?histones?become?hypoacetylated?and?both?com-ponents?of?the?transcriptional?repressor?complex?PRC2,?namely?YY1?and?Ezh2,?and?the?histone?deacetylases?HDAC1?and?hSirt1?are?actively?recruited?on?the?cccDNA.?These?latter?observations?suggest?an?active?mechanism?for?IFN-α?repression?of?HBV?transcription,?
?
Figure 3
STAT1 and STAT2 transcription factors are recruited on the cccDNA.
(A ) Cross-linked chromatin from HepG2 cells transfected with mono-meric linear full-length HBV DNA was immunoprecipitated with a rel-evant control IgG or specific anti-STAT1, anti–phospho-STAT1, anti-STAT2, and anti–phospho-STAT2 antibodies. Immunoprecipitated chromatin samples were analyzed by real-time PCR with either HBV cccDNA selective primers (upper panel) or primers specific for the cyclin A2 coding region as a negative control (lower panel). (B ) Chro-matin was prepared from untreated and IFN-α–treated HepG2 cells
transfected with WT HBV genomes. Immunoprecipitated chromatin was
analyzed and results expressed as in Figure 1D. All histograms show mean values from 3 independent experiments; bars indicate SD.
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where?changes?of?cccDNA-bound?transcriptional?regulators?translate?into?changes?in?the?epigenetic?control?of?cccDNA?mini-chromosome?function,?rather?than?a?simple?“passive”?relocation?of?ISFG3?complexes.?It?will?be?important?in?the?future?to?define?the?precise?nature?of?the?signals?that?IFN-α?provides?in?order?to?not?only?modify?the?composition?and?the?function?of?STAT1-?and?STAT2-containing?complexes,?but?also?the?organization?of?other?cccDNA-bound?transcription?factors?and?regulators.?In?this?respect,?it?has?been?recently?reported?that?inflammatory?signaling?involving?the?induction?of?the?TNF-α?receptor/p38?MAPK?cascade?results?in?Ezh2?phosphorylation,?increased?binding?to?YY1,?and?transcriptional?repression?of?target?genes?(28).
Virion?productivity,?being?defined?as?the?ratio?between?the?number?of?intrahepatic?relaxed?circular?HBV?DNA?and?cccDNA?molecules,?depends?on?pgRNA?synthesis?and?thus?also?reflects cccDNA?transcription.?From?a?more?translational?point?of?view,?our?data?also?provide?a?molecular?frame?explaining?previous?stud-ies?reporting?a?very?strong?HBV?suppression,?with?a?99%?inhibi-tion?of?intrahepatic?virion?productivity,?in?patients?who?had?been?treated?with?the?combination?of?IFN-α?and?the?nucleotide?ana-log?adefovir?dipivoxil?for?1?year?(29).?The?observation?that?during?the?following?2?years,?when?patients?were?shifted?to?ADV?mono-therapy,?the?suppression?of?viral?productivity?was?reduced?to?76%,?although?the?clinical?benefit?was?maintained?in?most?patients,?suggests?that?IFN-α?may?have?direct?antiviral?effects?in?vivo?that?are?qualitatively?different?from?those?of?HBV?DNA?polymerase?
inhibitors.?Indeed,?PEG–IFN-α?results?in?the?highest?rate?of?off-treatment?sustained?virological?response?after?a?1-year?course?of?therapy?(5).?This?sustained?virological?suppression,?achieved?in?30%–35%?of?HBeAg-positive?patients?and?20%–25%?of?HBeAg-negative?patients,?is?commonly?thought?to?reflect?the?transition,?induced?by?IFN-α?treatment,?to?the?“immune-control”?phase?that?characterizes?the?inactive?HBsAg?carrier?state.?Our?results?indicate?that?IFN-α?induces?a?persistent?condition?of?“active?epigenetic?con-trol”?of?HBV?cccDNA?minichromosome,?involving?all?HBV?tran-scripts,?that?may?contribute?to?the?persistent,?yet?reversible,?“off-therapy”?inhibition?of?HBV?replication?(Figure?6).?Understanding IFN-α–induced?mechanisms?acting?both?at?posttranscriptional?(7,?12)?and?transcriptional?levels?to?suppress?HBV?replication?and?how?these?IFN-α?direct?activities?are?linked?with?other?signals?from?both?adaptive?and?innate?immune?responses?may?assist?in?the?development?of?more?effective?therapeutic?approaches.Methods
Cell cultures and IFN-α treatments .?HepG2?hepatoma?cells?were?maintained?in?DMEM?supplemented?with?10%?FBS?(Gibco;?Invitrogen),?1%?penicil-lin/streptomycin,?and?1%?glutamine?(Sigma-Aldrich).?IFN-α?was?used?at?a?final?concentration?of?1000?IU/ml?and?was?added?directly?to?the?cul-ture?medium?every?24?hours,?starting?4?hours?after?transfection,?unless?otherwise?indicated.
Transient transfection of full-length HBV DNA genomes .?Monomeric?linear?full-length?(14)?WT?and?ISREmt?HBV?genomes?were?released?from?the?pCR.HBV.A.EcoRI?(4)?and?the?pCR.ISREmt.A.EcoRI?plasmids?using?EcoRI-PvuI?(New?England?Biolabs)?The?pCR.ISREmt.A.EcoRI?has?been?generated?by?site-directed?mutagenesis?(QuikChange?Site-Directed?Mutagenesis?Kit;?Stratagene?Inc.)?from?the?pCR.HBV.A.EcoRI?plasmid.?The?oligonucleotides?used?for?mutagenesis?were?as?follows:?sense,?5′-ATA-CAAGCTAAACAGGCCTTTACCTTCTCGCCAACTTACAAG-3′;?anti-sense,?5′-CTTGTAAGTTGGGCAGAAGGTAAAGGCCTGTTTAGC-3′.?These?nucleotide?substitutions?in?the?ISRE?sequence?have?been?shown?to?abolish?ISGF3?binding?in?EMSA?assays?(13),?but?do?not?alter?the?HBV?polymerase?polypeptide?sequence.?Linear?HBV?monomers?were?transfect-ed?into?HepG2?human?hepatoma?cells?using?the?Mirrus?Bio?transIT-LT1?(Mir?2300A).?Briefly,?HepG2?cells?were?seeded?at?a?density?of?2–3?million?cells?in?100-mm-diameter?Petri?dishes?and?transfected?24?hours?later?with?1?μg?to?2?μg?of?digested?HBV?DNA.?Unless?specified?otherwise,?culture?medium?was?changed?1?day?after?transfection?and?cells?were?harvested?at?the?indicated?times.?All?transfection?included?0.5?μg?of?GFP?expression?vector?to?assess?transfection?efficiency?(HepG2?cells,?range?28%–32%).?To?exclude?nonhomologous?recombination?events?at?the?level?of?the?2?ends?of?the?transfected?linear?HBV?DNA?and?the?possible?generation?of?circular?HBV?DNA?molecules?carrying?sequence?modifications?at?the?recombina-tion?site,?the?HBV?region?spanning?the?predicted?ends?of?the?linear?dsDNA?was?amplified?and?sequenced?(3).
Purification and quantification of core particles associated with HBV DNA from HBV-replicating cells .?To?purify?HBV?DNA?from?intracellular?core?particles,?transfected?cells?were?washed?once?with?ice-cold?PBS?and?lysed?in?50?mmol?Tris-HCl,?pH?7.4,?1?mmol?EDTA,?and?1%?NP-40?(lysis?buffer?A).?Nuclei?were?pelleted?by?centrifugation?for?1?minute?at?10,000?g .?The?supernatant?was?adjusted?to?100?mmol?MgCl 2?and?treated?with?100?mg/ml?of?DNase?I?for?30?minutes?at?37°C.?The?reaction?was?stopped?by?adding?EDTA?to?a?final?con-centration?of?25?mmol.?Protein?was?digested?with?0.5?mg/ml?proteinase?K and?1%?SDS?for?2?hours?at?50°C.?Nucleic?acids?were?purified?by?phenol-chloroform?(1:1)?extraction?and?ethanol?precipitation?adding?glycogen?and?examined?by?Southern?blot?analysis?(3).?HBV?DNA?was?quantified?by?real-time?PCR?in?a?Light?Cycler?instrument?(Roche)?using?the?following?
?
Figure 4
HBV ISRE mediates IFN-α transcriptional repression. (A ) Sequence of
the HBV enhancer 1/X gene promoter around the HBV ISRE. ISRE muta-tions are shown. The nucleotide substitutions do not alter the HBV poly-merase polypeptide sequence. (B ) Chromatin prepared from untreated and IFN-α–treated HepG2 cells transfected with WT or ISREmt HBV genomes was immunoprecipitated with a relevant control IgG or specific anti-STAT2 antibodies. Immunoprecipitated chromatin was analyzed by qPCR and results expressed as in Figure 1D. (C ) mRNAs were pre-pared from untreated and IFN-α–treated HepG2 cells transfected with WT and ISREmt HBV genomes, and HBV pregenomic RNA was quanti-fied by qPCR using specific primers. GAPDH amplification was used to normalize for equal loading of each RNA sample. (D ) Cytoplasmic HBV core particles were isolated from untreated and IFN-α–treated HepG2 cells 48 hours after transfection with monomeric linear full-length WT or ISREmt genomes. Results are expressed as in Figure 1A. Results are shown as mean values from 3 independent experiments; bars indicate SD. P values were determined using Student’s t test. **P < 0.01.
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primers?and?probes:?forward,?5′-CTCGTGGTGGACTTCTCTC-3′,?and?reverse?5′-CAGCAGGATGAAGAGGAA-3′.?We?also?used?specific?FRET?hybridization?probes:?5′-CACTCACCAACCTCCTGTCCTCCAA-FL-3′,?Red640,?5′-TGTCCTGGTTATCGCTGGATGTGTCT-3′.?Amplifications?were?performed?as?follows:?95°C?for?5?minutes,?followed?by?45?cycles?at?95°C?for?10?seconds,?58°C?for?10?seconds,?and?72°C?for?20?seconds. HBV cccDNA quantification.?HepG2?cells?were?collected?at?the?indicated?times?after?transfection,?resuspended?in?lysis?buffer?A?(see?above),?and?incu-bated?10?minutes?at?4°C.?Lysates?were?centrifuged?1?minute?at?10,000?g; pelleted?nuclei?were?resuspended?in?lysis?buffer?B?(10?mM?Tris-HCL, 10?mM?EDTA,?150?mM?NaCl,?0.5%?SDS,?and?0.5?mg/ml?protein?K)?and?incubated?overnight?at?37°C.?Nucleic?acids?were?purified?by?phenol-chlo-roform?(1:1)?extraction?and?ethanol?precipitation.?500?ng?aliquots?of?each?extracted?DNA?were?treated?for?45?minutes?at?37°C?with?10?U?plasmid?safe?DNase?I?(Epicentre?Inc.).?DNase?was?inactivated?by?incubating?the?reactions?for?30?minutes?at?70°C.?Real-time?PCR?experiments?were?per-formed?in?a?Light-Cycler?(Roche?Diagnostics)?using?a?20?l?reaction?volume?containing?20?ng?of?DNA,?3?mmol/l?MgCl2,?0.5?mmol/l?of?forward?and?reverse?primers,?0.2?mmol/l?of?3′-fluorescein–labeled?(FL-labeled)?probe,?and?0.4?mmol/l?of?5′-Red640–labeled?(R640-labeled)?probe.?Forward?and?reverse?primers?were?as?follows:?5′-CTCCCCGTCTGTGCCTTCT-3′?(NCCC1?nt?1548–1566)?and?5′-GCCCCAAAGCCACCCAAG-3′?(CCCAS2?nt?1903–1886),?respectively.?Hybridization?probes?were?5′-GTTCACG-GTGGTCTCCATGCAACGT-FL-3′?and?5′-R640-AGGTGAAGCGAAGT-GCACACGGACC-3′,?respectively.?Amplification?was?performed?as?follows:?95°C?for?10?minutes,?then?45?cycles?of?95°C?for?10?seconds,?62°C?for?10?seconds,?and?72°C?for?20?seconds.?The?efficacy?of?DNase?treatment?in?the?elimination?of?OC?and?SS?forms?of?HBV?DNA?prior?to?PCR?was?confirmed?by?the?abrogation?of?the?PCR?amplification?of?HBV?DNA?extracted?from?cytoplasmic?viral?particles?by?the?nonselective?HBV oligonucleotide?primers?targeting?the?HBs?ORF?(3).?Globin?amplifica-tion?was?performed?using?the?Light-Cycler?Globin?Control?Kit?(Roche?Diagnostics).?Serial?dilutions?of?a?plasmid?containing?a?monomeric?geno-type?A?HBV?insert?(Clonit?Srl.)?were?used?as?quantification?standards.?Southern?blot?analysis?was?performed?following?standard?procedures?as?previously?described?(3).
HBV RNAs and cellular mRNA analysis.?Total?RNA?was?extracted?from?HepG2?cells?48?hours?and?96?hours?after?transfection?with?1?mg?of?linear?monomeric?HBV?DNA?using?the?TRI zol?reagent?(Invitrogen)?as?recom-mended?by?the?manufacturer.?The?RNA?samples?were?treated?with?RQ1?RNase-Free?DNase?(Promega)?for?30?minutes?at?37°C?and?stored?until?used.?RNA?quality?and?quantity?were?monitored?by?ethidium?bromide?staining?and?by?UV?absorbance.?For?pgRNA?analysis,?2?mg?of?DNase-treated?RNA?was?reverse?transcribed?and?amplified?by?the?ThermoScript?RT-PCR?Sys-tem?(Invitrogen).?Then?2?μl?of?each?cDNA?was?quantified?by?real-time?PCR?analysis?(Light?Cycler;?Roche?Diagnostics)?using?the?following?pgRNA-specific?primers?and?probes:?forward?primer,?5′-GCCTTAGAGTCTCCT-GAGCA-3′,?reverse?primer,?5′-GAGGGAGTTCTTCTTCTAGG-3′,?FRET?hybridization?probes,?5′–AGTGTGGATTCGCACTCCTCCAGC-FL-3′,?and?Red640-5′ATAGACCACCAAATGCCCCTATCTTATCAAC-3′.?The h-GAPDH?housekeeping?gene?Light?Cycler?Set?(Roche?Diagnostics)?was?used?to?normalize?the?RNA?samples.
For?Northern?blot?analysis,?25?μg?of?total?RNA?per?sample?was?sepa-rated?on?a?1%?formaldehyde-agarose?gel?and?blotted?onto?Zeta-Probe?GT?membranes?(Bio-Rad?Laboratories).?Radioactive?probes?were?prepared?by?random?priming?protocol,?using?either?full-length?HBV?DNA?or?18S?cDNA?Figure 5
IFN-α modulates the epigenetic control of cccDNA func-
tion by affecting the recruitment of chromatin-modifying
enzymes. (A) Cross-linked chromatin from untreated and
IFN-α–treated HepG2 cells transfected with WT HBV
genomes was immunoprecipitated with a relevant control
IgG or anti-HDAC1, anti-YY1, anti-hSirt1 and anti-EzH2
antibodies and analyzed as in Figure 1D. (B) HepG2 cells,
transfected as in A, were either left untreated (96nt), or
treated for 96 hours after transfection with IFN-α (96t), or
treated with IFN-α for 48 hours and then left untreated for
48 hours (48t + 48nt). Left panel: cross-linked chromatin
immunoprecipitated with a relevant control IgG or anti-Ezh2
antibody was analyzed as in Figure 1D. pgRNA (middle
panel) and cytoplasmic core particles HBV-DNA (right panel)
were quantified by real-time qPCR. Results are expressed
as in Figure 1. (C and D) Chromatin was prepared from
untreated and IFN-α–treated HepG2 cells transfected with
WT or ISREmt HBV genomes and immunoprecipitated with
a relevant control IgG or anti-AcH4 (B) or anti-HDAC1 (C)
antibodies. Immunoprecipitated chromatin was analyzed by
qPCR and results expressed as in Figure 1D. All histograms
show the mean from 3 independent experiments; bars indi-
cate SD. P values were determined using Student’s t test.
*P < 0.05; **P < 0.01; ***P
< 0.001.
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templates?and?32P?α-dCTP?(Amersham).?After?hybridization,?the?membrane?was?washed?and?exposed?to?X-Omat?film?(Kodak?Inc.)?at?–80°C.
For?the?analysis?of?cellular?ISG?expression?by?TaqMan?Low-Density?Arrays?(TLDAs)?(Applied?Biosystems),?total?RNAs?were?reverse?transcribed?using?the?Random?Primers?Superscript?Kit?(Invitrogen),?and?200?ng?of?each?complementary?DNA?was?loaded?in?double?on?a?95-ISG?customized?TLDA?with?18S?RNA?used?as?a?control.?TLDAs?were?run?on?an?AB?7900HT?ther-mocycler,?and?real-time?PCR?data?were?collected?and?analyzed?using?the?SDS?2.2?program?(Applied?Biosystems).
Generation of human chimeric mice, HBV infection experiments, and IFN treatment .?Alb?uPA?transgenic?mice?(Jackson?Laboratories)?crossed?with?SCID/beige?mice?(Taconic?Farms)?were?housed?and?maintained?under?specific?pathogen–free?conditions?in?accordance?with?institutional?guide-lines?under?approved?protocols.?The?presence?of?the?uPA?transgene?and?the?maintenance?of?the?SCID?phenotype?were?determined?as?reported?(15).?Three-?to?four-week-old?homozygous?uPA?SCID/beige?mice?were?anesthetized?with?isoflurane?and?injected?intrasplenically?with?1?×?106?cryopreserved?and?thawed?viable?human?hepatocytes?(30)?isolated?from?1?liver?specimen?obtained?from?a?reduced?size?liver?transplant.?Informed?
consent?was?obtained?from?donors,?and?all?procedures?were?approved?by?the?ethical?committee?of?the?city?and?state?of?Hamburg?according?to?the?principles?of?the?Declaration?of?Helsinki.?Human?hepatocyte?repopula-tion?levels?were?determined?by?measuring?HSA?concentrations?in?mouse?serum?with?the?Human?Albumin?ELISA?Quantitation?Kit?(Bethyl?Labo-ratories,?Biomol?GmbH)?as?recommended?by?manufacturers.?Human?chi-meric?animals?displaying?HSA?concentrations?of?1?or?more?mg/ml?were?used?for?the?study.?Ten?to?twelve?weeks?after?mice?received?a?single?perito-neal?injection?of?HBV-positive?mouse-derived?serum?(2?×?107?HBV?DNA?copies),?stably?infected?animals?were?treated?with?IFN-α?(1300?IU/g?body?weight,?daily,?in?200?μl),?while?control?animals?received?saline.?Liver?speci-mens?removed?at?sacrifice?were?snap-frozen?in?liquid?nitrogen?for?further?histological?and?molecular?analyses.?All?animal?experiments?were?con-ducted?in?accordance?with?the?European?Communities?Council?Directive?(86/EEC),?and?were?approved?by?the?city?of?Hamburg,?Germany.
Virological measurements in uPA chimeric mice .?After?homogenization?of?the?mouse?liver?specimens,?DNA?and?RNA?were?extracted?in?paral-lel?using?the?Master?Pure?DNA?Purification?Kit?(Epicentre;?Biozym)?and?RNeasy?RNA?Purification?Kit?(QIAGEN).?Human?
hepatocyte?
Figure 6
Schematic representation of cccDNA chromatin changes in response to IFN-α treatment. cccDNA-bound histone acetylation status and the recruitment of chromatin-modifying enzymes onto the viral minichromosome change in relation to viral replication and IFN-α treatment. The draw-ings reflect what is observed in an in vitro replication system. The translation into the clinical scenario as it is observed in patients (lower boxes) is inferred, and it awaits to be confirmed by ex vivo experiments. In the context of high HBV replication and in the absence of IFN-α treatment, cccDNA-bound histones are hyperacetylated, cccDNA-associated chromatin is in an open configuration, pgRNA is actively transcribed, and HBV replication is unrestricted (left drawing). The clinical correlate is an active HBV carrier with high HBV viremia, reflecting high levels of intrahepatic viral replication and liver disease progression. In response to IFN-α, HDACs (HDAC1 and Sirt1) substitute HAT enzymes (p300, CBP, and P/CAF) on the cccDNA, and a PRC2-repressor complex is recruited. This leads to histone deacetylation/methylation at specific lysine residues, a “closed” chromatin configuration, and a striking reduction of pgRNA transcription, HBsAg synthesis, and HBV replication (middle drawing). In the clinical setting, this would translate into a rapid serum HBsAg decline and viral suppression (inactive carrier) with disease remission. When treatment is stopped, the chromatin changes imposed by IFN-α tend to persist (right drawing) resulting in the “off-therapy” maintenance of the virological sup-pression and clinical improvement (achieved in 30%–35% of HBeAg-positive patients and 20%–25% of HBeAg-negative patients). Darker forms in the middle and right drawings indicate components of the PRC2 complex whose recruitment has been directly investigated in this paper.
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genome?equivalents?and?intrahepatic?viral?loads?were?determined?per?ng?genomic?DNA?isolated?from?chimeric?livers.?After?treating?purified?DNA?with?20?U?plasmid-safe?DNase?I?(Epicentre;?Biozym)?to?enrich?the?cccDNA?fraction,?intrahepatic?cccDNA?amounts?were?determined?using cccDNA-specific?primers?and?FRET?probes?as?reported?above.?Cloned?HBV?DNA?was?used?to?establish?a?standard?curve?for?quantification.?cccDNA?copies?were?normalized?for?human?hepatocyte?contents?using?the?β-globin?Quantification?Kit?(Roche?Applied?Science)?(15,?29).?Viral?RNA?was?reverse?transcribed?using?oligo-dT?primers?and?the?Transcrip-tor?Kit?(Roche?Applied?Science)?and?quantified?by?using?primers?spe-cific?for?pgRNA?and?total?HBV-RNA?(tot)?sequences.?Steady-state?levels?of?intrahepatic?preS/S?RNAs?were?determined?by?subtracting?pgRNA?amounts?from?total?HBV?RNAs?(pgRNA?+?preS/S?RNA)?estimated?in?the?same?RNA?preparation?as?reported?(31),?and?values?were?normalized?using?human-specific?GAPDH?primers?(QuantiTect?Primer?Assay;?QIA-GEN)?(15).?Northern?blot?analysis?was?performed?as?described?above. ChIP assays.?48?hours?after?transfection?with?linear?HBV?monomers,?HepG2?cells?were?resuspended?in?1?ml?of?ChIP?lysis?buffer?(50?mM?Tris?HCL,?pH?8,?0.5%?NP40,?1?mM?EDTA,?and?100?mM?NaCL)?and?incubated?10?minutes?at?4°C.?The?lysate?was?centrifuged?at?10,000?g?for?2?minutes?to?pellet?the?nuclei.?The?supernatant?was?removed,?and?the?nuclei?were?fixed?in?1%?formaldehyde?for?30?minutes?at?4°C.?Iso-lated?cross-linked?nuclei?were?extracted?with?a?20?mM?Tris,?pH?8,?3?mM?MgCl2,?20?mM?KCl?buffer?containing?protease?inhibitors,?pelleted?by?microcentrifugation,?and?lysed?by?incubation?in?SDS?lysis?buffer?(1%?sodium?dodecyl?sulfate,?10?mM?EDTA,?50?mM?Tris-chloride,?pH?8.1)?containing?protease?inhibitors.?The?resulting?chromatin?solution?was?sonicated?for?5?pulses?of?45?seconds?at?80%?power?to?generate?300-?to?1000-bp?DNA?fragments?using?a?Bioruptor?Sonicator?(Diagenode?Inc).?After?microcentrifugation,?the?supernatant?was?diluted?1:10?in?a?dilution?buffer?(0.01%?sodium?dodecyl?sulfate,?1.1%?Triton?X-100,?1.2?mM?EDTA,?16.7?mM?Tris-chloride,?pH?8.1,?167?mM?NaCl,?con-taining?protease?inhibitors),?precleared?with?blocked?Protein?G?Plus?(Pierce),?and?divided?into?aliquots.?The?chromatin?was?then?subjected?to?immunoprecipitation?for?14–16?hours?at?4°C?using?antibodies?spe-cific?to?H4?(ab1791;?Abcam),?AcH4?(06-866,?Upstate;?rabbit?polyclonal?IgG?recognizing?histone?H4,?which?is?tetra-acetylated?at?lysines?6,?9,?13,?and?17),?Sirt1?(sc15404/;?Santa?Cruz?Biotechnology?Inc.),?HDAC1?(no.?06-720;?Upstate),?STAT1?(sc476;?Santa?Cruz?Biotechnology?Inc.),?STAT2?(sc476;?Santa?Cruz?Biotechnology?Inc.),?phospho-STAT1?(9171;?Cell?Signaling),?phospho-STAT2?(07224;?Upstate),?YY1?(sc1703;?Santa?Cruz?Biotechnology?Inc.),?and?EZH2?(3147;?Cell?Signaling).?Immuno-precipitations?with?nonspecific?immunoglobulins?(Santa?Cruz?Biotech-nology?Inc.)?were?included?in?each?experiment?as?a?negative?control.?After?the?reverse?cross-linking,?immunoprecipitated?chromatin?was?purified?by?phenol/chloroform?(1:1)?extraction?and?ethanol?precipitation?and?analyzed?by?either?PCR?amplification?using?specific?primers?for?the?HBV?cccDNA?(NCC1?and?CCCAS)?or?real-time?PCR?using?the?same?cccDNA?primers?and?specific?probes?(FL?and?Red).
ChIP?assays?of?liver?specimens?of?chronically?HBV-infected?uPA?chi-meric?mice?were?performed?with?the?following?modifications:?liver?tis-sues?were?homogenized?in?500?μl?lysis?buffer?(5?mM?PIPES,?85?mM?KCl,?0.5%?NP40)?and?incubated?at?4°C?for?10?minutes.?After?microcentrifu-gation,?the?pelleted?nuclei?were?fixed?in?1%?formaldehyde?for?15?min-utes?at?4°C.?After?sonication?and?dilution,?the?chromatin?was?subjected?to?immunoprecipitation?for?14–16?hours?at?4°C?using?antibodies?spe-cific?to?acetylated?H4?(06-866;?Upstate)?or?nonspecific?immunoglobu-lins?(BD?Biosciences).?Immunoprecipitated?DNA?was?extracted?using?the?Master?Pure?DNA?Purification?Kit?(Epicentre;?Biozym).?After?treat-ment?with?20?U?plasmid-safe?DNAase?I?(Epicentre;?Biozym),?DNA?was?analyzed?by?real-time?PCR?using?cccDNA-specific?primers?and?FRET?probes?as?reported?above.
Immunoblotting.?Cells?were?lysed?in?RIPA?buffer?(50?mM?Tris,?pH?7.6,?1%?NP-40,?140?mM?NaCl,?0.1?%?SDS),?and?the?insoluble?pellet?was?dis-carded?after?centrifugation.?Protein?concentration?was?determined?by?the?BCA?protein?assay?reagent?(Bio-Rad).?Protein?lysates?were?trans-ferred?to?a?nitrocellulose?membrane?and?incubated?with?STAT1?(sc346;?Santa?Cruz?Biotechnology?Inc.)?and?STAT2?(sc476;?Santa?Cruz?Biotech-nology?Inc.)?antibodies.
Statistics.?P?values?were?determined?using?the?2-tailed?Student’s?t?test.?The?Wilcoxon?rank-sum?test?was?used?for?nonparametric?pair-wise?compari-sons.?P?
Acknowledgments
M.?Levrero?and?G.?Raimondo?are?supported?by?grants?from?Associ-azione?Italiana?per?la?Ricerca?sul?Cancro?and?PRIN-MIUR?(Progetti?di?Ricerca?di?Interesse?Nazionale?del?Ministero?dell’Istruzione,?dell’Università?e?della?Ricerca).?M.?Levrero?and?J.?Petersen?were?also?supported?by?the?Vigilance?Network?for?the?management?of?antivi-ral?drug?resistance?(Virgil).?M.?Dandri?is?supported?by?the?Deutsche?Forschungsgemeinschaft?(SFB?481)?and?receives?research?support?from?Roche?Palo?Alto?LLC?(grant?number?09-631).
Received?for?publication?May?3,?2011,?and?accepted?in?revised?form?November?9,?2011.
Address?correspondence?to:?Maura?Dandri,?Department?of?Internal?Medicine,?University?Medical?Hospital?Hamburg-Eppendorf,?Mar-tinistrasse?52,?20246?Hamburg,?Germany.?Phone:?49.40.741052949;?Fax:?49.40.741057232;?E-mail:?m.dandri@uke.de.?Or?to:?Massimo?Levrero,?Department?of?Internal?Medicine,?DMISM,?Laboratory?of?Gene?Expression,?Sapienza?University,?Viale?del?Policlinico?155,?00161?Rome,?Italy.?Phone:?39.06.49970892;?Fax:?39.06.4452388;?E-mail: massimo.levrero@uniroma1.it.
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原核&真核表达载体构建
原核、真核表达载体构建 真核表达载体和原核表达载体的区别：主要是因为原核和真核表达系统所需的表达元件不同。 比如说启动子，终止子在两种表达系统中是不一样的。 带有真核表达元件的是真核载体，能在真核生物内表达； 带有原核表达元件的是原核载体，能在原核生物内表达。两者都具有的为穿梭载体。 ㈠原核表达载体指：能携带插入的外源核酸序列进入原核细胞中进行复制的载体。 原核表达载体调控原件 1.启动子 启动子是DNA链上一段能与RNA聚合酶结合并起始RNA合成的序列，它是基因表达不可缺少的重要调控序列。没有启动子，基因就不能转录。由于细菌RNA聚合酶不能识别真核基因的启动子，因此原核表达载体所用的启动子必须是原核启动子。原核启动子是由两段彼此分开且又高度保守的核苷酸序列组成，对mRNA的合成极为重要。在转录起始点上游5～10 bp处，有一段由6～8个碱基组成，富含A和T的区域，称为Pribnow 盒，又名TATA 盒或－10区。来源不同的启动子，Pribnow 盒的碱基顺序稍有变化。在距转录起始位点上游35 bp 处，有一段由10 bp组成的区域，称为-35区。转录时大肠杆菌RNA聚合酶识别并结合启动子。-35区与RNA聚合酶s亚基结合，-10区与RNA聚合酶的核心酶结合，在转录起始位点附近DNA被解旋形成单链，RNA聚合酶使第一和第二核苷酸形成磷酸二酯键，以后在RNA聚合酶作用下向前推进，形成新生的RNA 链。原核表达系统中通常使用的可调控的启动子有Lac(乳糖启动子)、Trp(色氨酸启动子)、Tac(乳糖和色氨酸的杂合启动子) 、lPL (l噬菌体的左向启动子)、T7噬菌体启动子等。 2. SD序列 1974年Shine和Dalgarno首先发现，在mRNA上有核糖体的结合位点，它们是起始密码子AUG和一段位于AUG上游3～10 bp处的由3～9 bp组成的序列。这段序列富含嘌呤核苷酸，刚好与16S rRNA 3￠末端的富含嘧啶的序列互补，是核糖体RNA的识别与结合位点。以后将此序列命名为Shine-Dalgarno序列，简称SD序列。它与起始密码子AUG之间的距离是影响mRNA转录、翻译成蛋白的重要因素之一，某些蛋白质与SD序列结合也会影响mRNA与核糖体的结合，从而影响蛋白质的翻译。另外，真核基因的第二个密码子必须紧接在ATG 之后，才能产生一个完整的蛋白质。 3．终止子 在一个基因的3￠末端或是一个操纵子的3'末端往往有特定的核苷酸序列，且具有终止转录功能，这一序列称之为转录终止子，简称终止子(terminator)。转录终止过程包括：RNA聚合酶停在DNA模板上不再前进，RNA的延伸也停止在终止信号上，完成转录的RNA从RNA聚合酶上释放出来。对RNA聚合酶起
重组HER2真核表达载体的构建及其稳定转染EMT6细胞株的筛选
重组HER2真核表达载体的构建及其稳定转 染EMT6细胞株的筛选 作者：徐腾飞, 张文卿, 于红, 李丹 【摘要】目的: 构建人表皮生长因子受体（HER2）胞外区（1 896 bp）基因的真核表达质粒（pcDNA6/v5his HER2）, 转染小鼠乳腺癌细胞（EMT6）, 获得其稳定表达细胞株(EMT6/ HER2)。方法: 用PCR方法从含HER2全长基因的pcDNA3.1HER2质粒上扩增HER2胞外区基因序列; 经酶切、连接构建pcDNA6/v5his HER2; 转化大肠杆菌DH5α, 筛选阳性克隆, 对其进行酶切及测序鉴定; 以PEI法将pcDNA6/v5his HER2导入EMT6小鼠乳腺癌细胞, 经杀稻瘟菌素(Blasticidin)筛选1～2周, 获得抗性克隆EMT6/HER2; 用RT PCR检测EMT6/HER2中HER2 mRNA, 免疫组化法检测其HER2蛋白的表达。结果: PCR产物与预期片段大小一致; pcDNA6/v5his HER2经酶切、琼脂糖凝胶电泳后, 可见与PCR产物大小相同的片段; DNA测序结果显示, pcDNA6/v5his HER2 中HER2 基因序列无误, 读码框正确; 用RT PCR可在EMT6/HER2中检测到HER2 mRNA, 免疫组化法证实, EMT6/HER2中有HER2的阳性信号。结论: 成功地构建了HER2胞外区真核表达载体, 获得稳定表达HER2基因的小鼠乳腺癌EMT6细胞株, 为进一步研究HER2基因过表达与乳腺癌发生的关系及其基因治疗奠定基础。
【关键词】HER2; 真核表达; 转染 ［Abstract］AIM: To construct an eukaryotic vector encoding extracellular domain of human epidermal growth factor receptors (HER2), pcDNA6/v5his HER2, and to screen HER2 positive clones from mouse breast cancer cell line EMT6. METHODS: The extracellular domain of HER2 was amplified from pcDNA3.1HER2 by PCR. pcDNA6/v5 his HER2 was prepared by inserting the fragment into the plasmid pcDNA6/v5his. Then the recombinant vector was identified by restriction enzyme and sequencing. Next, pcDNA6/v5his HER2 was transfected into the EMT6 cell line and the positive clones (EMT6/HER2) were screened with blasticidin. Finally, the expression of HER2 in EMT6/HER2 was detected by RT PCR and immunohistochemistry. RESULTS: The fragment of HER2 was amplified and pcDNA6/v5his HER2 was prepared successfully. No errors were found both in the sequence and ORF of the acquired fragment. The expected fragment of HER2 (1896 bp) was amplified from EMT6/HER2 by RT PCR and positive signals of HER2 were detected in EMT6/HER2 by immunohistochemistry. CONCLUSION: An eukaryotic plasmid encoding HER2 (pcDNA6/v5 his HER2) has been constructed and a cell line expressing HER2 stably has been prepared successfully.
小鼠γ干扰素IFN-γ试剂盒使用方法
小鼠γ干扰素(IFN-γ)试剂盒使用方法 检测范围：96T 30ng/L -800 ng/L 使用目的： 本试剂盒用于测定小鼠血清、血浆及相关液体样本中γ干扰素(IFN-γ)含量。 实验原理 本试剂盒应用双抗体夹心法测定标本中小鼠γ干扰素(IFN-γ)水平。用纯化的小鼠γ干扰素(IFN-γ)抗体包被微孔板，制成固相抗体，往包被单抗的微孔中依次加入γ干扰素(IFN-γ)，再与HRP标记的γ干扰素(IFN-γ)抗体结合，形成抗体-抗原-酶标抗体复合物，经过彻底洗涤后加底物TMB显色。TMB在HRP酶的催化下转化成蓝色，并在酸的作用下转化成最终的黄色。颜色的深浅和样品中的γ干扰素(IFN-γ)呈正相关。用酶标仪在450nm波长下测定吸光度（OD值），通过标准曲线计算样品中小鼠γ干扰素(IFN-γ)浓度。 试剂盒组成 1 30倍浓缩洗涤液20ml×1瓶7 终止液6ml×1瓶 2 酶标试剂6ml×1瓶8 标准品（1600ng/L）0.5ml×1瓶 3 酶标包被板12孔×8条9 标准品稀释液 1.5ml×1瓶 4 样品稀释液6ml×1瓶10 说明书1份 5 显色剂A液6ml×1瓶11 封板膜2张 6 显色剂B液6ml×1/瓶12 密封袋1个 标本要求 1．标本采集后尽早进行提取，提取按相关文献进行，提取后应尽快进行实验。若不能马上进行试验，可将标本放于-20℃保存，但应避免反复冻融 2．不能检测含NaN3的样品，因NaN3抑制辣根过氧化物酶的（HRP）活性。 操作步骤 1.标准品的稀释：本试剂盒提供原倍标准品一支，用户可按照下列图表在小试管中进行稀 释。 800 ng/L 5号标准品150μl的原倍标准品加入150μl标准品稀释液 400 ng/L 4号标准品150μl的5号标准品加入150μl标准品稀释液 200 ng/L 3号标准品150μl的4号标准品加入150μl标准品稀释液 100 ng/L 2号标准品150μl的3号标准品加入150μl标准品稀释液 50 ng/L 1号标准品150μl的2号标准品加入150μl标准品稀释液 2.加样：分别设空白孔（空白对照孔不加样品及酶标试剂，其余各步操作相同）、标准孔、 待测样品孔。在酶标包被板上标准品准确加样50μl，待测样品孔中先加样品稀释液40μl，然后再加待测样品10μl（样品最终稀释度为5倍）。加样将样品加于酶标板孔底部，尽量不触及孔壁，轻轻晃动混匀。 3.温育：用封板膜封板后置37℃温育30分钟。
构建目的基因的真核表达载体实战操作
构建目的基因的真核表达载体实战操作 最后更新：2010-5-16 阅读次数：451 【字体：小中大】 目的：构建目的基因的真核表达载体 一.PCR扩增得到目的片段 1.引物的设计与合成： 这里有很多血与泪的教训。我前后一共送过九个载体测序，其中居然有五个是引物出错。最后一次挑的两个克隆都是上游引物出错（此上游引物40个碱基），后来和这家引物公司交涉，他们拿走了我的板子，一次送了五个克隆测序，三个是上游引物出错，两个是正确的克隆，60％的出错率。 现在想想，有几点教训： (1)尽量避免长引物的合成。越长意味着出错的几率越大，哪家公司都这样 (2)选择一家质量可靠的公司，千万别在这省钱，引物再贵也花不了多少钱，可一轮构建下来，一测序发现引物错了，不知白扔了多少钱不说，那种心情实在是难以收拾 （3）一旦发现引物有问题，及时换公司，我就让他们拿回去又是纯化又是重新合成过，结果还是错，时间咱耽误不起 2.Pfu酶扩增： （1）扩增效率低的问题： 往往taq酶扩的很好，一换成pfu就是扩不出来。解决办法：a延长延伸时间(我1.2kb用3分15秒才扩出来)，并适当增加循环数;b多试几家的pfu，总有一家能扩出来;c多设计几对引物试试，总有一对能扩出来，因为pfu有外切活性，可否适当的延长引物的3’互补端；d构建中间载体：一旦用某一对引物能扩出来，把此目的片段连接t－easy中间载体，测序正确后，即可以此中间载体做pcr扩增的模板。实践证明以此中间载体为模板的pfu扩增效率远远高于以cDNA为模板。 （2）pfu保真性的问题： 即使是pfu，也会出错。现在各家公司都有号称高保真的pfu酶出售，价格不一。虽然都是pfu，但质量不不见得一样，在都能扩增出来的情况下，选择信誉好的公司的pfu。我用的是一家小公司的pfu，很便宜，100u才80块钱，但我1.2kb的片段却频频出错。所以万不可在酶上省那百十块钱，最后花了大钱不说，还浪费了时间精力。 3．cDNA模板的问题： 在pfu摸条件这一步往往要耗费大量cDNA模板，因此在开始要准备足够的cDNA。100ul，200ul都不为过。新提取的RNA证明很好的话，就再多逆转录几管，我的RNA在－80℃放了两个月再逆转录就扩增不出目的片段了。当然这其中可能有很多原因，酶的问题，RNA 的降解等等，但是当你做PCR做到很烦的时候，发现cDNA模板不够了，又重新养细胞提RNA是一件非常痛苦的事情。 二．酶切的问题： 用于构建的酶切一定要非常充分！ 新买回来的酶要分装，用于“酶切后回收”和“酶切鉴定”的酶要分开。因为用于“酶切鉴定”时，只要能切出来就行了，而用于“酶切后回收再做连接”的酶一定要保证切的充分，才能提高连接效率，更重要的是减少筛选时的麻烦！我有一段时间连挑了三十多个克隆都是空载体，当然一方面怪我没有做载体自连的阴性对照，及时发现这一问题，另一方面酶反复从冰箱拿出来活性下降酶切效率降低，导致大量的载体自身连接。我觉得内切酶这东西还是挺娇气的，所以还是建议大家新酶分装，用于“酶切鉴定”的可稍放松，用于“酶切后回收”的酶一定要尽量减少冻融次数，并尽量减少在室温放置的时间。 三．PCR产物直接酶切：
人γ干扰素IFN-γ试剂盒使用方法
人γ干扰素(IFN-γ)试剂盒使用方法 检测范围：96T 10 ng/L -400 ng/L 使用目的： 本试剂盒用于测定人血清、血浆及相关液体样本中γ干扰素(IFN-γ)含量。 实验原理 本试剂盒应用双抗体夹心法测定标本中人γ干扰素(IFN-γ)水平。用纯化的人γ干扰素(IFN-γ)抗体包被微孔板，制成固相抗体，往包被单抗的微孔中依次加入γ干扰素(IFN-γ)，再与HRP标记的γ干扰素(IFN-γ)抗体结合，形成抗体-抗原-酶标抗体复合物，经过彻底洗涤后加底物TMB显色。TMB在HRP酶的催化下转化成蓝色，并在酸的作用下转化成最终的黄色。颜色的深浅和样品中的γ干扰素(IFN-γ)呈正相关。用酶标仪在450nm波长下测定吸光度（OD值），通过标准曲线计算样品中人γ干扰素(IFN-γ)浓度。 试剂盒组成 1．标本采集后尽早进行提取，提取按相关文献进行，提取后应尽快进行实验。若不能马上进行试验，可将标本放于-20℃保存，但应避免反复冻融 2．不能检测含NaN3的样品，因NaN3抑制辣根过氧化物酶的（HRP）活性。 操作步骤 1.标准品的稀释：本试剂盒提供原倍标准品一支，用户可按照下列图表在小试管中进行稀 2.加样：分别设空白孔（空白对照孔不加样品及酶标试剂，其余各步操作相同）、标准孔、 待测样品孔。在酶标包被板上标准品准确加样50μl，待测样品孔中先加样品稀释液40μl，然后再加待测样品10μl（样品最终稀释度为5倍）。加样将样品加于酶标板孔底部，尽量不触及孔壁，轻轻晃动混匀。 3.温育：用封板膜封板后置37℃温育30分钟。 4.配液：将30倍浓缩洗涤液用蒸馏水30倍稀释后备用 5.洗涤：小心揭掉封板膜，弃去液体，甩干，每孔加满洗涤液，静置30秒后弃去，如此 重复5次，拍干。
(2008)pcDNA3_hNGFb真核表达载体的构建及其表达
?学术交流?pcDNA3-hN GFb真核表达载体的构建及其表达 邓兴力 杨智勇 董坚 王廷华 徐丹 冯忠堂 【摘要】 目的 构建人神经生长因子β(N GF-β)基因真核表达载体并观察其在L929细胞内的表 达。方法 以R T-PCR从人脑组织总RNA中扩增N GF-βcDNA,将其克隆到真核表达载体pcD2 NA3中,经酶切鉴定和序列分析后,以Lipofectamine2000介导转染L929细胞,应用免疫细胞化学和 western blot鉴定N GF-β在细胞内的表达。结果 R T-PCR产物为750bp的特异片段,重组质粒 pcDNA3-hN GFb酶切后产生750bp和5.2kb的片段,DNA测序证实750bp片段的碱基序列与人 N GF-βcDNA完全一致。将其转染L929细胞后,免疫细胞化学、western blot结果表明N GF-β及其 前体proN GF-β能在真核细胞中正确表达。结论 成功构建了重组真核表达质粒pcDNA3-hN GFb, 为后续的研究奠定基础。 【关键词】 神经生长因子; 真核表达; 重组质粒; 转染 C onstru ction o f recombinant plasmid pcDN A32hNG Fb and its exp ression DEN G Xing2li,Yang Zhi2yong, DO N G J ian,et al. De partment of N eurosurgery,1st A f f iliated Hos pital of Kunming Medical College,Kunming650032,China 【Abstract】 Objective To construct the eukaryotic expression recombinant plasmid pcDNA3- hN GFb and investigate its expression in L929cells.Methods The cDNA of human nerve growth factor beta subunit(N GF-β)was amplified by R T-PCR from human brain tissue.By gene recombination technique,human N GF-βcDNA was inserted into eukaryotic expression vector pcDNA3.The recombi2 nant plasmid was verified with restriction enzyme digestion and DNA sequencing.Transfected the recom2 binant vector into L929cells with Lipofectamine2000transfection reagent.The expression of N GF-β was analyzed by immunocytochemistery as well as western blot.R esults The R T-PCR product is 750bp specific segment.By restriction enzyme digestion,the recombinant plasmid was digested into 750bp and5.2kb f ragments.DNA sequence result showed the750bp f ragment was identical with human N GF-βcDNA in GenBank.Immunocytochemistery and western blot showed the N GF-βwas expressed successfully in L929cells.Conclusions The recombinant plasmid pcDNA3-hN GFb was constructed successfully,which will provide the foundation for f urther research. 【K ey w ords】 Nerve growth factor(N GF);Eukaryotic expression;Recombinant plasmid;T ransfection 中图分类号:R741 文献标识码:A 文章编号:100926574(2008)022******* 神经生长因子(nerve growt h factor,N GF)是神经营养因子家族中最早被发现也是迄今为止研究得最为深入的细胞生长因子。N GF在体内首先以其前体的形式合成,随后在各种蛋白酶的作用下裂解为成熟体[1]。最近研究表明,N GF前体(p roN GF)具有与成熟N GF相反的生物学活性,其通过作用于p75N TR受体及sortilin受体介导包括神经细胞内的多种细胞凋亡[2-5]。本研究将构建人N GF-β基因真核表达质粒pcDNA3-hN GFb,并研究其在L929细胞中的表达情况,为进一步研究p roN GF的 作者单位:650032昆明医学院第一附属医院神经外科(邓兴力,杨志勇),生物治疗中心(董坚);昆明医学院神经科学研究所(王廷华、徐丹、冯忠堂) 作者简介:邓兴力(1979-),男,博士研究生。研究方向:细胞移植治疗帕金森病的研究。 通讯作者:冯忠堂 fzt@https://www.sodocs.net/doc/6d22517.html, 生物学功能奠定实验基础。 1 材料与方法 1.1 材料 1.1.1 质粒、细菌与脑组织 真核表达质粒载体pcDNA3由昆明医学院第一附属医院生物治疗研究中心保存;小鼠成纤维细胞系L929、E.coli D H5α由昆明医学院神经科学研究所保存;人脑肿瘤旁组织由昆明医学院第一附属医院神经外科提供。 1.1.2 主要试剂与工具酶 TRIzol,Lipofectamine 2000Reagent,G418,RPM I1640培养基,小牛血清(Invitrogen);Hind,xho,T4DNA Ligase,Calf In2 testine Alkaline Phosp hotase,First Strand cDNA Synt hesis K it,High Fidelity PCR Enzyme Mix(MB I Fermentas);凝胶回收试剂盒,DNA纯化试剂盒,质粒DNA提取试剂盒(OM EGA);DNA MA KER DL2000,DNA MA KERλ-Eco T14(Takara);6×
小鼠γ干扰素(IFN-γ)说明书
小鼠γ干扰素(IFN-γ)酶联免疫分析（ELISA） 试剂盒使用说明书 本试剂仅供研究使用目的：本试剂盒用于测定小鼠血清,血浆及相关液体样本中γ干扰素(IFN-γ)含量。 实验原理： 本试剂盒应用双抗体夹心法测定标本中小鼠γ干扰素(IFN-γ)水平。用纯化的小鼠γ干扰素(IFN-γ)抗体包被微孔板，制成固相抗体，往包被单抗的微孔中依次加入γ干扰素(IFN-γ)，再与HRP标记的γ干扰素(IFN-γ)抗体结合，形成抗体-抗原-酶标抗体复合物，经过彻底洗涤后加底物TMB显色。TMB在HRP酶的催化下转化成蓝色，并在酸的作用下转化成最终的黄色。颜色的深浅和样品中的γ干扰素(IFN-γ)呈正相关。用酶标仪在450nm波长下测定吸光度（OD值），通过标准曲线计算样品中小鼠γ干扰素(IFN-γ)含量。 试剂盒组成： 样本处理及要求： 1. 血清：室温血液自然凝固10-20分钟，离心20分钟左右（2000-3000转/分）。仔细收集上 清，保存过程中如出现沉淀，应再次离心。 2. 血浆：应根据标本的要求选择EDTA、柠檬酸钠或肝素作为抗凝剂，混合10-20分钟后， 离心20分钟左右（2000-3000转/分）。仔细收集上清，保存过程中如有沉淀形成，应该再次离心。 3. 尿液：用无菌管收集，离心20分钟左右（2000-3000转/分）。仔细收集上清，保存过程 中如有沉淀形成，应再次离心。胸腹水、脑脊液参照实行。 4. 细胞培养上清：检测分泌性的成份时，用无菌管收集。离心20分钟左右（2000-3000转/ 分）。仔细收集上清。检测细胞内的成份时，用PBS（PH7.2-7.4）稀释细胞悬液，细胞
干扰素(IFN)
干扰素（IFN）是一种广谱抗病毒剂，并不直接杀伤或抑制病毒，而主要是通过细胞表面受体作用使细胞产生抗病毒蛋白，从而抑制乙肝病毒的复制；同时还可增强自然杀伤细胞（NK细胞）、巨噬细胞和T淋巴细胞的活力，从而起到免疫调节作用，并增强抗病毒能力。干扰素是一组具有多种功能的活性蛋白质（主要是糖蛋白），是一种由单核细胞和淋巴细胞产生的细胞因子。它们在同种细胞上具有广谱的抗病毒、影响细胞生长，以及分化、调节免疫功能等多种生物活性。 目录 1干扰素 2干扰素简介 3治疗有效率 4干扰素多少钱 5发现 6什么叫干扰素（IFN） 7品种及价位 8作用机制 1.8.1 ①间接性 2.8.2 ②广谱性 3.8.3 ③种属特异性 4.8.4 ④发挥作用迅速 1干扰素 药物类别：抗肿瘤药，抗病毒药；所属类别：生物反应调节剂 药物名称：干扰素英文名称：Interferon 药物别名：序号中文别名英文别名 一．α干扰素 制剂/规格：序号制剂规格 1．注射剂5×10。单位（1 ml）；1×106。单位（1 ml）； 2．冻干剂l×10。单位 成份/化学结构：序号成份化学结构 药理作用：1.抗病毒作用：其抗病毒活性不是杀灭而是抑制病毒，它一般为广谱病毒抑制剂，对RNA和DNA病毒都有抑制作用。当病毒感染的恢复期可见干扰素的存在，另一方面用外源性干扰素亦可缓解感染。
2．抑制细胞增殖干扰素抑制细胞分裂的活性有明显的选择性，对肿瘤细胞的活性比正常细胞大500～1000倍。干扰素抗肿瘤效果可以是直接抑制肿瘤细胞增殖，或通过宿主机体的免疫防御机制限制肿瘤的生长。 3．诱导细胞凋亡：干扰素可以诱导肿瘤细胞凋亡，从而杀灭肿瘤细胞。 4.干扰素对体液免疫、细胞免疫均有免疫调节作用，对巨噬细胞及NK细胞也有一定的免疫增强作用。 药动学：干扰素在肌内注射或皮下注射后入血的速度较慢，需较长时间才能在血中测到。肌内注射后Tmax为5～8小时。一次肌注：106单位，血清浓度为100单位/ml，这比在病毒感染时自然产生的干扰素量为高。循环中的干扰素半衰期为2～4小时。只有少量干扰素能进入血脑屏障，脑脊液内的浓度约为血内浓度的l/30，只有在兔身上研究过排泄，排出量只有0.2%～2.0%。 适应症：1.用于多种恶性肿瘤，包括毛细胞白血病、慢性白血病、非何淋巴瘤、骨髓瘤、膀胱癌、卵巢癌、晚期转移性肾癌及胰腺恶性内分泌肿瘤、黑色素瘤和Kaposi肉瘤等。 2．与其他抗肿瘤药物并用。 3．作为放疗、化疗及手术的辅助治疗剂。 4．病毒性疾病的防治。 用法用量： 多采用皮下注射、肌注、脑脊髓腔内或腹腔内、局部灌注给药。一般剂量多用一次1×106～3×106单位，皮下注射或肌注，每周3次，可连用数月或更长。可根据病情逐渐增减剂量。该药有时间依赖性，长时间保持有效浓度，抗癌效果较好（即连续治疗为佳）。 不良反应： 1.全身反应主要表现为流感样症状，即寒战、发热和不适。剂量超过44×104单位/m2时，注射2～6小时后即可出现发热。随着疗程延长，发热可逐渐减轻，一般7天后可停止发热。为避免发热，事先可使用醋氨酚。若仍发热，与IF-α含杂质有关，不宜再用。 2．骨髓抑制在用药中可出现白细胞、血小板和网状红细胞减少。减少剂量在8.5×104单位/m2以下，可减轻骨髓抑制发生。 3．局部反应部分患者在注射部位可出现红斑，并有压痛，24小时后即可消退。 4．其他脱发、皮疹、血沉加快、嗜睡、一过性肝损伤。偶见过敏性休克，用药前作过敏试验。 相互作用：泼尼松或其他皮质激素有降低干扰素生物活性的作用，应予注意。 注意事项：1.心肌梗死、重症高血压、脑血管疾病慎用。 2．如发现冻干制剂萎缩、变色，液体制剂混浊、有异物或不溶性沉淀等均不宜使用。
IFN-试剂盒使用说明
1.原理: 1）单克隆抗体IFN-γ已经包被在PVDF-微孔板中 2）刺激的细胞被移入孔中,37℃ 5%CO2 孵育特定时间.在孵育过程中,抗体与分泌的IFN-γ结合 3）孵育后,所有的细胞以及未结合的物质被洗掉,加入生物素标记的IFN-γ抗体与细胞分泌的IFN-γ结合 4）孵育后,未结合的抗体被洗掉,加入链霉亲和素-HRP与生物素标记的IFN-γ抗体结合5）孵育后,未结合的链霉亲和素-HRP被洗掉,加入AEC显色底物,颜色建立一部分取决于最初IFN-γ的结合的数量 6）细胞因子存在的地方会形成红色斑点 2.材料与方法 提供的试剂 IFN-γ抗体包被板,96孔 生物素标记的IFN-γ浓缩物(1小瓶) 分析溶液 (25ml/瓶) HRP浓缩物（200倍）（60μL/小瓶） 清洗浓缩液（200倍）（50ml/瓶） AEC发色体（250μL/小瓶） AEC底物(25ml/瓶) 3.补充 1）显微镜或ELISpot读取器 2）移液枪和枪头 3）蒸馏水或去离子水 4.注意事项 1）样品和试剂在使用前需要在室温（20-25℃）准备。使用后冷藏。 2）IFN-γ抗体包被板应该在打开锡箔纸前达到室温 3）对照和重复实验的设置 4）每个孔要保证孵育时间一致 5）注意换枪头，避免交叉 6）不要将不同孔中的试剂混合 7）残余的清洗液必须用滤纸吸干，不要将纸直接深入孔中 5.试剂准备 A．用一个干净的塑料tube将生物素标记的IFN-γ浓缩物(1小瓶)1:250稀释（为最佳效果，稀释后直接使用） B.将清洗浓缩液（200倍）（50ml/瓶）温度达到室温，轻轻混合。一个干净的塑料tube 将其用蒸馏水稀释。用后冷藏，2周内用完。 C．将HRP浓缩物（200倍）（60μL/小瓶）稀释，稀释后30min用完。 D．20μL AEC发色体加入到1.0 mL AEC底物混合到干净的塑料tube。 6.步骤
IFN-γ
IFN-γ?IL-4?IL-10在川崎病患者血清中的表达和意义 【摘要】目的探讨IFN-γ、IL-4、IL-10在川崎病（KD）发病机制中的作用。方法采用病例-对照分析，使用双抗体夹心酶联免疫吸附试验（ELISA）方法，检测45例急性期KD患儿静脉注射丙种球蛋白（IVIG）前后血浆中的IFN-γ、IL-4、IL-10蛋白浓度与25例正常同年龄对照组的差异。结果急性期KD患儿血浆IFN-γ、IL-4、IL-10水平觉对照组明显上升，差异有统计学意义（P＜0．01）；给予IVIG治疗后，上述指标水平明显下降，差异有统计学意义（P＜0．05）。结论IFN-γ、IL-4、IL-10可能通过调节T细胞活化的细胞因子机制参与KD发病的自身免疫耐受过程。 【Abstract】Objective To investigate the effects of IFN-γ，IL-4，IL-10 in pathogenesis of Kawasaki disease.Methods Detect the concentration of IFN-γ，IL-4，IL-10 in 45 patients with Kawasaki disease in acute stage before and after IG intravenous injection.At meantime，detect same indexes in 25 health children.Then compared the indexes between patients and health children.Results The concentration of IFN-γ，IL-4，IL-10 of 45 patients with Kawasaki disease in acute stage were increased significantly in comparison of health children.The concentration was decreased after IG intravenous injection.Conclusion IFN-γ，IL-4，IL-10 participate immune tolerance process of Kawasaki disease morbidity with adjusting activation of T cell. 【Key words】Kawasaki disease；interferon-γ；interleukin-4；interleukin-10 川崎病（Kawasaki disease，KD）是儿童常见的免疫相关性血管炎性综合征。其对心血管系统的损害最为严重，可形成冠状动脉扩张和冠状动脉瘤，目前成为儿童后天性心脏病中最常见的病因[1]，但发病机制仍未揭示。超抗原学说认为T 细胞异常活化是川崎病血管免疫损伤的始动环节和关键步骤，本研究初步观察KD患儿血浆中IFN-γ、IL-4、IL-10的细胞因子蛋白浓度改变，探讨细胞因子在KD 免疫发病机制的作用。 1 资料和方法 1．1 研究对象观察对象来自广东医学院附属医院儿科病房2005-2006年KD住院患儿45例，均符合第3届国际川崎病会议修订的诊断标准[2]；病程在10 d以内，且未经任何治疗；既往健康，排除各种免疫缺陷病史。其中男26例，女19例；年龄1~10岁，平均4．5岁。对照（NC）组为25例健康体检儿童，男15例，女10例；年龄1~9岁，平均5岁。两组儿童在年龄性别方面差异无统计学意义（P＞0．05）。受试者家属对试验均知情，并经医院学术伦理委员会讨论通过。 1．2 方法
ifndefine的用法
ifndefine的用法 这两天看代码的时候突然之间发现在代码中间出现了#ifndefine 这样类似的语句 显然这是一个预处理语句虽然看到的时候就已经在那里想着这应该是与#define类似的一个语句，但是具体的还是需要在查看一下具体的用法的….. #ifndef x //if not define的简写 #define x … #endif 这是宏定义的一种，它可以根据是否已经定义了一个变量来进行分支选择，一般用于调试等等.实际上确切的说这应该是预处理功能中三种（宏定义，文件包含和条件编译）中的一种—-条件编译。C语言在对程序进行编译时，会先根据预处理命令进行“预处理”。C语言编译系统包括预处理，编译和链接等部分。 #ifndef x //先测试x是否被宏定义过 #define x 程序段1 //如果x没有被宏定义过，定义x，并编译程序段1 #endif 程序段2 //如果x已经定义过了则编译程序段2的语句，“忽视”程序段1。 条件指示符#ifndef 的最主要目的是防止头文件的重复包含和编译。了解：条件编译当然也可以用条件语句来实现。但是用条件语句将会对整个源程序进行编译，生成的目标代码程序很长，而采用条件编译，则根据条件只编译其中的程序段1或程序段2，生成的目标程序较短。如果条件选择的程序段很长，采用条件编译的方法是十分必要的。 编辑本段 内容 #ifndef 标识1 //判断”标识1″是否定义，如果被定义则返回假，如果没有被定义则返回真。/**********************************/ 语句1 #ifndef 标识1 语句2 #define 标识1 语句3 #endif 语句4 …… 语句5 …… 该段代码意思是：如果标识1没有被定义，则重定义标示1，即执行语句2、语句3;如果标识1已经被定义，则直接跳过语句2、语句3，直接执行语句4、语句5、…… /***********************************/ 备注：#ifndef 和#endif 要一起使用，如果丢失#endif，可能会报错。 千万不要忽略了头文件中的#ifndef，这是一个很关键的东西。比如你有两个C文件，这两个C文件都include了同一个头文件。而编译时，这两个C文件要一同编译成一个可运行文件，于是问题来了，大量的声明冲突。 还是把头文件的内容都放在#ifndef和#endif中吧。不管你的头文件会不会被多个文件引用，你都要加上这个。一般格式是这样的： #ifndef < 标识> #define < 标识> …… …… #endif
ELISPOT(IFN-γ检测)试验详细介绍
ELISPOT（IFN-γ检测）试验详细介绍 一、实验背景 IFN-γ对于抵抗细胞内病原体和控制肿瘤的免疫力至关重要。它主要由天然杀伤（NK）和天然杀伤T细胞（NKT）以及抗原特异性Th1 CD4 +和CD8 +效应T细胞产生。在研究针对病原体和癌细胞的免疫应答时，与治疗组相比，对病原体或癌细胞的抗原特异性IFN-γ反应的测量结果可定量评估治疗效果。 与没有肽的对照培养相比，测量抗原特异性IFN-γ反应涉及CD4 +或CD8 + T 细胞与抗原呈递细胞（APC）和来自靶病原体或癌细胞的特定肽一起培养。在合适的时间范围后，通过ELISPOT测定法测量释放的细胞因子。有和没有肽的分泌IFN-γ的细胞数量的差异是抗原特异性IFN-γ反应的量度。该测定法可以应用于其他细胞因子，例如IL10。 干扰素γ（IFN-γ）是二聚化的可溶性细胞因子，是II型干扰素的唯一成员。IFN-γ具有抗病原，免疫调节和抗肿瘤的特性，可增强NK细胞的活性，增加抗原呈递，激活诱导型一氧化氮合酶，诱导活化浆细胞中产生IgG2a，通过上调转录因子T-bet促进Th1细胞分化。鉴于这种细胞因子在免疫反应中的重要作用，有几种方案可以量化IFN-γ。也许最简单的方法是ELISA分析，该方法用于通过用抗体捕获细胞因子来测量血清样品和组织培养上清液中的细胞因子水平。还有一种基于流式细胞术的测定法，其中细胞通透性过后，通过流式细胞术检测细胞内IFN-γ。含有细胞因子的细胞百分比通常很低，并且不能表明该蛋白是否具有功能，是否可以分泌并且不能测量它是否对特定的靶抗原或多种抗原作出反应。酶联免疫斑点法（ELISPOT）是一种高度灵敏的免疫分析法，可在单细胞水平上测量分泌细胞因子的细胞的频率。抗原特异性ELISPOT 分析可量化特定细胞类型（CD4 +或CD8 + T细胞）的数量，该细胞可响应特定抗原而分泌IFN-γ。ELISPOT板上的IFN-γ特异性抗体从细胞分泌后立即捕获IFN-γ，其检测极限通常为100,000个细胞中的1个。该测定法的高灵敏度使其对于研究在特定免疫反应中发现的少量细胞特别有用。 二、实验原理图
人γ干扰素(IFN-γ)说明书
人γ干扰素(IFN-γ)酶联免疫分析（ELISA） 试剂盒使用说明书 本试剂仅供研究使用目的：本试剂盒用于测定人血清，血浆及相关液体样本中γ干扰素(IFN-γ)的含量。 实验原理： 本试剂盒应用双抗体夹心法测定标本中人γ干扰素(IFN-γ)水平。用纯化的人γ干扰素(IFN-γ)抗体包被微孔板，制成固相抗体，往包被单抗的微孔中依次加入γ干扰素(IFN-γ)，再与HRP标记的羊抗人受体结合，形成抗体-抗原-酶标抗体复合物，经过彻底洗涤后加底物TMB显色。TMB在HRP酶的催化下转化成蓝色，并在酸的作用下转化成最终的黄色。颜色的深浅和样品中的γ干扰素(IFN-γ)呈正相关。用酶标仪在450nm波长下测定吸光度（OD 值），通过标准曲线计算样品中人γ干扰素(IFN-γ)浓度。 试剂盒组成： 试剂盒组成48孔配置96孔配置保存说明书1份1份 封板膜2片（48）2片（96） 密封袋1个1个 酶标包被板1×48 1×96 2-8℃保存标准品：450ng/L 0.5ml×1瓶0.5ml×1瓶2-8℃保存标准品稀释液 1.5ml×1瓶 1.5ml×1瓶2-8℃保存酶标试剂 3 ml×1瓶 6 ml×1瓶2-8℃保存样品稀释液 3 ml×1瓶 6 ml×1瓶2-8℃保存显色剂A液 3 ml×1瓶 6 ml×1瓶2-8℃保存显色剂B液 3 ml×1瓶 6 ml×1瓶2-8℃保存终止液3ml×1瓶6ml×1瓶2-8℃保存浓缩洗涤液（20ml×20倍）×1瓶（20ml×30倍）×1瓶2-8℃保存 样本处理及要求： 1. 血清：室温血液自然凝固10-20分钟，离心20分钟左右（2000-3000转/分）。仔细收集上 清，保存过程中如出现沉淀，应再次离心。 2. 血浆：应根据标本的要求选择EDTA或柠檬酸钠作为抗凝剂，混合10-20分钟后，离心 20分钟左右（2000-3000转/分）。仔细收集上清，保存过程中如有沉淀形成，应该再次离心。 3. 尿液：用无菌管收集，离心20分钟左右（2000-3000转/分）。仔细收集上清，保存过程 中如有沉淀形成，应再次离心。胸腹水、脑脊液参照实行。 4. 细胞培养上清：检测分泌性的成份时，用无菌管收集。离心20分钟左右（2000-3000转/ 分）。仔细收集上清。检测细胞内的成份时，用PBS（PH7.2-7.4）稀释细胞悬液，细胞
人β干扰素IFN-β试剂盒使用方法
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