摘要
乙型肝炎病毒(hepatitis B virus, HBV)的感染会导致急慢性乙型肝炎的发生,是引起肝硬化、肝细胞癌的重要因素之一。尽管目前有核苷类药物和干扰素等抗乙型肝炎病毒制剂,但缺乏切实有效的长期应答效应以及受个体耐药等因素的制约,故慢性HBV感染的治疗仍然是目前面临的一道难题。
IFNα因其能抑制HBV-DNA的复制,常被用作治疗病毒感染的抗病毒药物。有研究报道,在慢性肝炎携带者中IFNα治疗有效性仅为30%,其中约有50%的患者在停止治疗后可出现复发。为了达到抗病毒效果,药物剂量常要加大几倍、几十倍,药物剂量的加大也加重了其不良反应,又容易产生干扰素抗体,从而影响疗效,限制了其在临床的广泛应用。我们通过将抗乙肝表面抗原抗体Fab片段与IFNα在分子水平上进行重组,并制备了重组的融合蛋白( anti-HBs Fab-IFNα),以期通过抗体与干扰素的双重作用,实现感染细胞水平上的抗病毒性肝炎的生物导向治疗。
第一部分人源性抗HBs Fab和IFNα融合蛋白的原核表达
基于前序制备的人源化抗HBs-Fab抗体具有免疫学活性,这部分实验的目的是构建和表达人源性抗-HBs Fab和IFNα融合蛋白。以pBAD IFNα质粒为模板,根据IFNα基因序列结构设计引物,在引物5’和3’端加入XbaI的酶切位点和含有5个氨基酸的连接肽序列。依据PCR产物两端所引入的限制性内切酶,以相同的酶分别酶切PCR产物和pBAD Fab载体,以T4连接酶连接。连接产物转化Top10大肠杆菌后,挑取单菌落增菌,经测序和PCR鉴定,筛选阳性克隆。
挑取正确插入的转化菌落,于100ml含Amp的LB培养基中生长,以阿拉伯糖诱导表达。表达上清经Ni-NTA柱纯化、浓缩后,12%的SDS-PAGE电泳并转印至硝酸纤维素膜上,经封闭后与辣根过氧化物酶标记的羊抗人IgG Fab反应,于25Kd和47.5Kd处可见明显蛋白区带,推测47.5Kd的区带为λ和IFNα的融合蛋白,25Kd处为重链的Fd段。Western blot和HBsAg特异性Dot blot结果表明融合蛋白中的轻链具有较好的抗原结合活性,IFNα活性达4.2×103~ 4.85×104IU/ml。
第二部分人源性抗-HBsλ和IFNα融合蛋白的真核表达
巴斯德毕赤酵母具有真核表达系统的诸多优点,可对表达的蛋白进行翻译后的加工、折叠与修饰,从而使表达出的蛋白具有生物活性;与其他真核表达系统相比,既具有原核表达系统操作简易、易于培养、生长速度快、表达量高、成本低等优点;且能利用甲醇作为唯一的碳源和能量来源。在P. pastoris中表达的蛋白既可存在于细胞内,又可分泌到胞外,自身分泌的蛋白非常少,十分有利于纯化。
将目的片段克隆到pPICZαC载体的AOX1启动子基因后,分离纯化质粒DNA,PCR、测序鉴定。将重组质粒以BstX I内切酶线性化,经电穿孔方法将质粒转化入X33菌株,在Zeocin浓度为100μg/ml的转化平板上挑取转化子,进行小规模培养。
培养上清中的λ-IFNα用BrCN-sepherose 4B柱纯化,纯化后的重组蛋白经SDS- PAGE电泳,表达蛋白的分子量约为50Kd,略大于其在原核中表达的分子量,用ELISA法检测融合蛋白IFNα的抗原性及其抗体亲和力,用WISH细胞检测IFNα生物学活性,证实融合蛋白既具有与抗HBs Fab相近的HBsAg的亲和力,又具有干扰素活性,干扰素活性达到7.8×104 ~5.1×105 IU/ml,高于原核表达系统(p<0.01);Western blot和HBsAg特异性Dot blot结果也表明表达产物具有较好的抗原结合活性。
第三部分抗HBsλ-IFNα对HBV感染作用的实验研究
目前体外研究HBV感染是以HBV基因组转染肝癌细胞作为细胞模型,HepG 2.2.15就是以HBV基因组转染HepG2而建立的。HepG2.2.15呈现持续表达HBV,并被证明在其培养上清中可检测到HBsAg, HBeAg和HBV DNA颗粒的释放。HBV DNA含量为1.2×105~3.7×105拷贝/ml,HBsAg浓度为16.2~ 20.5 COI(正常参考值为<1.0 COI),HBeAg浓度为230~320 Ncu/L(正常参考值为<30 Ncu/L)。HepG 2.2.15可以作为探索HBV病原体与宿主的相互作用的工具,是评估抗HBV新药的良好模型。
用于研究的λ-IFNα是含有人抗HBsλ抗体片段与IFNα的融合蛋白。本实验旨在比较IFNα和λ-IFNα两种不同的制剂对HepG2.2.15细胞的作用,检测细胞培养上清中病毒释放的HBV-DNA和抗原含量。分别收集培养9天的HepG 2.2.15细胞及上清,以ELISA法检测HBsAg和HBeAg的含量,以荧光定量PCR法检测病毒载量,融合蛋白的细胞毒性用MTT法进行检测。
Fas是细胞表面重要的介导凋亡的受体,是细胞凋亡的信号分子。Fas与配体FasL结合,活化并传导凋亡信号,是诱导细胞凋亡的重要途径,在机体的免疫监视过程中起重要作用。凋亡抑制是肿瘤细胞的基本特征之一,因此肿瘤细胞抵抗Fas介导的凋亡,可能是肿瘤细胞免疫逃逸的机制之一。本实验通过对HepG2.2.15细胞系Fas的表达及功能进行检测,了解重组蛋白λ-INFα对肝癌细胞凋亡的影响。
实验结果显示,经过9天的连续培养,λ-IFNα各个浓度组均能降低细胞培养上清中HBsAg、HBeAg及HBV-DNA的含量,且随时间的延长和药物浓度的增高,其作用也随之增强(P<0.05),而λ-IFNα和IFNα组间的抑制作用差异无统计学意义(P>0.05)。同时在细胞毒性实验中,λ-IFNα最高浓度组对细胞的毒性与阳性对照组差异无统计学意义(P> 0.05)。2 IU/ml、10 IU/ml浓度的λ-IFNα对HBV-DNA、HBsAg和HBeAg的抑制率差异无显著性(P>0.05),20 IU/ml、50 IU/ml、100 IU/ml浓度的λ-IFNα对HBV-DNA、HBsAg和HBeAg的抑制率有显著差异(P<0.05),λ-IFNα对HBsAg、HBeAg和HBV-DNA的抑制率高于同浓度的IFNα(P<0.05)。提示通过抗体片段的运载,使IFNα更易或更多的与靶细胞表面的IFNα受体结合,从而更有效发挥IFNα抗HBV效果。
HepG2.2.15细胞低表达Fas蛋白,经λ-IFNα处理后Fas表达增加。HepG2.2.15能抵抗Fas介导的凋亡,Fas激活抗体CH11不能诱导HepG2.2.15的凋亡。HepG 2.2.15经λ-IFNα处理后可上调Fas的表达,CH11能诱导其凋亡的发生(p<0.05)。
越来越多的证据证明体液免疫是保护机体免受HBV感染的重要途径。总之,本研究证实在体外抗HBsλ-IFNα能中和HBV病毒分泌的HBsAg,降低HBV- DNA的载量,其作用优于IFNα。同时λ-IFNα能上调HepG2.2.15细胞的Fas表达,促进CH11介导的凋亡。抗HBsλ-IFNα抗体在体外实验中显示了有效的中和病毒的作用,为今后的动物实验奠定了基础。
Infection with hepatitis B virus (HBV) can cause transient and chronic liver in?am- mation. A long-term chronic HBV infection can lead to liver cirrhosis and development of hepatocellular carcinoma. Although HBV infection can be prevented through vaccination, interferon-γ(IFNγ) and nucleosidic inhibitors such as lamivudine have been approved for the treatment of HBV. Low response rates to therapy, severe side-effects, high cost and indefinite outcomes of long-term therapy are the disadvantages of their therapy. HBV has still remained as one of the most significant viral pathogens.
Interferon-α(IFNα), an immunomodulatory antiviral drug, that inhibits HBV-DNA replication, is frequently used drugs in CHB. IFNαis effective only in less than 30% of the chronic carriers, and about 50% of IFNα-treated patients experience recurrence after cessation of treatment.Because the recovery on IFNαis limited in CHB, Anti-HBs Fab, a fully human monoclonal antibody against a surface antigen of hepatitis B virus (HBsAg), was generated and its immunological and biochemical characteristics were studied. The recombination of anti HBs Fab and IFNαmaybe has a synergistic activity for HBV-DNA and HBV antigen clearance.
Part 1 Construction and expression of the fusion protein consisting of IFNαand anti-HBs Fab in prokaryotic
Anti-HBs Fab have been shown its immunological and biochemical characteristics for against a surface antigen of hepatitis B virus. The purpose of this section is to construct and express the fusion protein consisting of IFNαand anti-HBs Fab. Using pBAD-IFNαplasmid as template, the IFNαwere amplified with corresponding endonuclease sites and artificial linker at 5’、3’termini by polymerase chain reaction (PCR) techniques. IFNαPCR products were digested with XbaI for gel purification. The vector pBAD-HBs Fab was digested with XbaI. Then two productions were ligation with T4 ligase at 16℃30min. Following bacterial transformation, the recombinant plasmid was identified with restriction enzyme digestion and PCR.
Single clones were picked from the resulting transformation, grown in 100ml of LB broth with ampicillin (100μg/L), and induced with arabinose. Fusion protein was purified by Ni2+ charged His tag affinity chromatography. After purification of the fusion protein, Human anti-HBs Fab-IFNαwere concentrated and separated in a 12% SDS-PAGE. The proteins were transferred to nitrocellulose, block, incubated with HRP-conjugated goat anti-human IgG Fab.
The molecular weight of the purified protein was about 47.5Kd and 25Kd. We supposed that the band about 47.5Kd fusion protein consistingλfragment and IFNα, another band about 25Kd containing heave chain Fd fragment. The fusion protein showed well bioactivity with western blot and Dot-blot, and HBsAg affinity which is similar to anti-HBs Fab fragment. Theλ-IFNαactivity was 4.2×103~4.85×104IU/ml.
The new constructed expression of the fusion protein with both HBsAg affinity andλ-IFNαactivity make it possible for immunobiotherapy drug targeted to Hepatitis B virus.
Part 2 Construction and expression of the fusion protein consisting of anti-HBsλfragment and IFNαin Pichia
As a eukaryote, Pichia pastoris has many of the advantages of higher eukaryotic expression systems such as protein processing, protein folding, and posttranslational modification, while being as easy to manipulate as E. coli. It is faster, easier, and less expensive to use than other eukaryotic expression systems and generally gives higher expression levels. Pichia pastoris is methylotrophic yeast, capable of metabolizing methanol as its sole carbon source. Heterologous expression in Pichia pastoris can be secreted. Secretion requires the presence of a signal sequence on the expressed protein to target it to the secretory pathway. Pichia may have an advantage in the glycosylation of secreted proteins because it may not hyperglycosylate.
After cloned and sequenced gene of interest into the pPICZαC behind the AOX1 promoter, isolated plasmid DNA by miniprep for restriction analysis and sequencing. The plasmid DNA was linearized with BstX I, then was transformed into X33 using electro- poration methods. Transformants are plated on YPDS plates containing 100μg/ml Zeocin to isolate Zeocin-resistant clones for small-scale expression.
λ-IFNαwas purified using BrCN-sepherose 4B from the culture media, and purifica- tion of recombination protein was analyzed by Western blot. The molecular weight of the purified protein was about 50Kd, which was higher than its expression in E.coli. Because of Pichia had many of the advantages of higher eukaryotic expression such as protein processing, protein folding, and posttranslational modification. The antigenicity and binding activity to HBsAg of fusion protein were dectected with ELISA and the bioactivity of IFNαwas detected with WISH cells. This anti HBsλ-IFNαantibody had the HBsAg affinity similar to that of anti-HBs Fab fragment, and exhibited high affinity which estimated by dot blot where the HBsAg concentration was 3μg/ml. Theλ-IFNαactivity was 7.8×104~5.1×105IU/ml, which was higher than prokaryotic expression.
Part 3 The study on anti-HBsλ-IFNαeffect in HBV infection
Current in vitro models for hepatitis B virus (HBV) are based on human hepato- blastoma cell lines transfected with HBV genome. HepG2.2.15 was established from the hepatoblastoma cell line HepG2. HepG2.2.15 exhibited prolonged expression of HBV, as was demonstrated by secreted levels of HBsAg, HBeAg, and HBV DNA in the culture medium of the growing cells. The quantity of HBV-DNA ranged between 1.2×105~3.7×105 copies/ml. The concentration of HBsAg was between 16.2~20.5 COI (normal<1.0 COI), HBeAg was between 230~320Ncu/L (normal<30 Ncu/L), which were evaluated with chemiluminescence immunoassay method. HepG2.2.15 can serve as an important tool for further exploration of HBV host–pathogen interaction, and for assessing new antiviral agents.
λ-IFNαused in this study is a recombination protein contained humanλantibody fragment and IFNα, which was raised against the surface antigen of hepatitis B virus. To assess our hypothesis that the HBV DNA detected in the cell culture medium originated from viral particles. Cytotoxicity of fusion protein was determined by mitochondrial toxicity testing (MTT) of cells. The inhibitory activities ofλ-IFNαwas further examined at different conditions. Different concentrations ofλ-IFNαand IFNαin DMEM were added to the wells with the monolayer growth of HepG2.2.15 cells and the cells and supernatant were collected at day 3, 6 and 9. The concentrations of HBsAg and HBeAg in the culture supernatant were determined by ELISA respectively, and the extracellular HBV DNA was measured by quantitative PCR with HBV specific primers.
Flow cytometry was used to determine the Fas expression of HepG 2.2.15 and which was treated withλ-IFNα. Resistance of HepG 2.2.15 to Fas-mediated apoptosis and the effect ofλ-IFNαon the apoptosis were studied using anti-Fas agonistic monoclonal antibody CHl1. Over a 9-day period in culture, groupλ-IFNαwith different concentration produced and secreted HBsAg and HBeAg and HBV- DNA at increasing concentrations at any time. The cytotoxicity ofλ-IFNαhad no statistical differences which treated under the same conditions (p>0.05).The inhibitory rate ofλ-IFNαon HBV- DNA and HBsAg and HBeAg showed no statistical differences between the concentration of 2 and 10 IU/ml (P>0.05). At the concentration of 20、50、100 IU/ml, the inhibitory rate ofλ-IFNαon HBV-DNA and HBsAg and HBeAg showed higher statistical differences(P<0.05). A significantly higher HBsAg and HBeAg and HBV- DNA inhibitory rate was byλ-IFNαcultures at every assayed conditions (p<0.05). The results proved that targetingλ-IFNαcan enhance the anti-HBV effect of IFNα.λ-IFNαmight take advantage of interaction both receptors of IFNαand HBsAg byλcarried. IFNαcould be easier to get to the targeting cells. The result makes it porsible to carry out further studies on targeted therapy.
Fas expression of HepG2.2.15 was low and increased after the cells were treated withλ-IFNα. The HepG2.2.15 could resist Fas-mediated apoptosis. CH11 could not induce HepG2.2.15 cells to apoptosis. But the apoptosis induced by CH11 increased when the cells up-regulated the Fas expression after treatment withλ-IFNα.
Increasing evidence demonstrates that humoral immunity is important for protection from HBV infection. In a word, this part of experiment confirmed thatλ-IFNαwas able to neutralize HBsAg in vitro.λ-IFNαis able to up-regulate the Fas expression and subsequently promotes the CHll-mediated apoptosis. Anti HBsλ-IFNαin vitro models of HBV infection have shown some promise, the efficacy of neutralizing antibodies in vivo requires evaluation in animal experiment.
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