家猪I型干扰素多基因家族的结构和特性及干扰素-alpha对口蹄疫基因工程疫苗的免疫增强作用

【作者】 程功；

【导师】 郑兆鑫；

【作者基本信息】 复旦大学 ， 微生物学， 2008， 博士

【摘要】 第一章:猪干扰素-alpha多基因家族的结构和性质研究家猪基因组测序计划(Swine Genome Sequencing Project)提供的序列数据使得我们能够对猪IFN-α多基因家族的性质进行系统的分析。通过BLAST的方法,我们从已提交的序列工作草图中获得了16条IFN-α亚型的基因序列(Porcine interferon-α,PoIFN-α),其中包括14条功能基因和2条假基因。多序列比对发现,有6条功能基因编码的蛋白在C端出现了8个氨基酸的缺失。构建的PoIFN-α多基因家族系统发生树揭示各亚型之间的进化关系。除此以外,对PoIFN-α蛋白分子C端的进化速率分析和阳性选择分析说明C末端的缺失不是某些亚型向假基因进化的标志,而是在基因组中保持进化优势的一种策略。为了进一步分析PoIFN-α家族的性质,从猪的肝脏基因组中分离出了8种不同的PoIFN-α亚型基因,然后将这些基因克隆入真核表达载体中,筛选阳性克隆后重组质粒转染BHK-21细胞并表达48小时收集上清液。转染效率通过荧光定量RT-PCR标定,使其保持一致。分别利用WISH细胞/VSV病毒和PK-15细胞/PRV病毒两种干扰素检测系统测定细胞转染上清液中的抗病毒活性。实验结果表明完整的PoIFN-α亚型的抗病毒活性在WISH细胞中比C端缺失的亚型高2-50倍,在PK-15细胞中比C端缺失的亚型高15-55倍。但是没有发现尾部的缺失对PoIFN-α的酸稳定性产生影响。为了进一步分析PoIFN-α各亚型在不同病毒诱导条件下的表达情况,我们采用了两种PCR检测策略:对每种亚型设计特异性引物检测和设计PoIFN-α通用引物检测。实验结果表明,在Poly IC-DEAE-dextran诱导的PK15细胞中,PoIFN-α2、-α3、-α4、-α8、-α9五种亚型在诱导6hr/24hr后得到了表达;在PRV感染的PK15细胞中,PoIFN-α2、-α3、-α8、-α9、-α10、-α13六种亚型的表达出现上调;在SFV弱毒株感染的PK15细胞中PoIFN-α2、-α3、-α4、-α8、-α9、-α10六种亚型在诱导后得到表达。荧光定量PCR分析的结果表明,Poly IC-DEAE-dextran和PRV诱导的PK15细胞中PoIFN-α的表达上调存在时间依赖性,而SFV弱毒株感染的PK15细胞中PoIFN-α的表达在6hr和24hr没有明显的不同。第二章:干扰素-θ,一种哺乳动物新型干扰素基因家族性质的研究通过对家猪基因组测序计划提交的工作草图序列进行生物信息学分析,我们发现了一组干扰素基因近似序列。该组基因编码的蛋白质与其他I型干扰素存在23%-59%的氨基酸序列同源性,SMART软件预测发现其具有I型干扰素的保守结构域,同时进化分析表明该基因家族与小鼠的IFN-ζ基因及家猪的IFN-δ具有较近的系统发生关系,综合以上几点我们认为该组基因是一个新型的I型干扰素基因家族,命名为IFN-θ。IFN-θ基因家族由8个亚型组成,其中有5个亚型编码具有生物功能的蛋白质,而其他3个亚型为假基因。在5个功能亚型中,IFN-θ2与IFN-θ3、IFN-θ4与IFN-θ5分别具有很高的序列同源性,而IFN-θ1与其他4个亚型均有80%左右同源性。我们根据基因组分析得到的序列设计引物并以家猪肝脏基因组DNA为模板,利用PCR的方法分离到了5条IFN-θ功能基因。将IFN-θ的三种亚型(IFN-θ1、IFN-θ2、IFN-θ5)克隆入真核表达载体并转染细胞,经过48小时表达后收集上清液,生物活性分析表明IFN-θ的三种亚型在PK-15细胞和IBRS-2细胞中均有抗病毒功能,但是三个亚型的抗病毒活性明显不同。在病毒诱导表达谱分析中,PRV、SFV减毒株及Poly IC均能够在不同时间点在IBRS-2细胞中诱导IFN-θmRNA的表达。受体结合分析及IFN-θ诱导ISGs基因分析表明,IFN-θ分子能与IFNαR胞外区发生特异性结合反应,激活下游JAK/STAT信号通路,产生ISGF3聚合物并与ISRE结合,最终引起ISGs基因的上调表达。最后我们分析了IFN-θ对其他细胞因子的影响,实验结果表明IFN-θ能够使IL-6、IL-12及IFN-γ三种细胞因子的表达上调,说明IFN-θ对免疫系统存在潜在的调节作用。综上所述,IFN-θ不仅在序列上与其他I型干扰素有较高的同源性,在抗病毒、受体结合、诱导表达等多个方面符合I型干扰素的共同特征,因此我们认为IFN-θ是I型干扰素基因家族中一个新的亚型。第三章:猪干扰素-alpha用作抗口蹄疫病毒重组多肽疫苗佐剂的研究在本项研究中,我们将猪干扰素-alpha(PoIFN-α)制备的核酸佐剂与抗口蹄疫基因工程多肽疫苗联合免疫动物,检测猪干扰素-alpha在动物体内的佐剂效果。首先,将PoIFN-α基因克隆入pcDNA3真核表达载体中,筛选阳性克隆后大量抽提质粒,通过脱水化-再水化步骤将重组质粒包裹入阳离子脂质体内制备成核酸佐剂(IFN-adjuvant)。该佐剂与低剂量的多肽疫苗联合注射家猪。检测结果表明,佐剂与疫苗联用可以强烈地诱导动物体产生FMDV中和抗体和T细胞介导的免疫反应;然而在只注射低剂量的多肽疫苗的动物体内,我们只检测到温和的细胞免疫和体液免疫反应。作为蛋白疫苗的佐剂,PoIFN-α能够诱导炎症因子(inflammatory cytokine)在动物体内的强烈表达,这个结果表明干扰素佐剂和多肽疫苗能够使原T辅助细胞向I型辅助细胞(Th1)分化。对动物体内PoIFN-α含量测定结果表明,重组蛋白疫苗能够与干扰素佐剂协同反应产生内源性IFN-α的表达。在病毒攻击实验中,所有的对照组动物均出现口蹄疫临床症状;然而佐剂和疫苗联合注射组的动物均得到保护,没有发现病毒血症和口蹄部水疱,对该组的病毒非结构蛋白抗体检测表明,攻毒14天后没有发现病毒在动物体内复制。根据以上结果,我们认为猪干扰素-alpha是抗口蹄疫病毒多肽重组疫苗的高效佐剂,能够有效的辅助多肽疫苗提高动物体内的免疫反应并保护动物免受口蹄疫病毒的感染。更多还原

【Abstract】 The availability of data on the pig genome sequence prompted us to characterize the porcine IFN-α(PoIFN-α) multigene family.Fourteen functional PoIFN-αgenes and two PoIFN-αpseudogenes were detected in the porcine genome. Multiple sequence alignment revealed a C-terminal deletion of eight residues in six subtypes.A phylogenetic tree of the porcine IFN-αgene family defined the evolutionary relationship of the various subtypes.In addition,analysis of the evolutionary rate and the effect of positive selection suggested that the C-terminal deletion is a strategy for preservation in the genome.Eight PoIFN-αsubtypes were isolated from the porcine liver genome and expressed in BHK-21 cells line.We detected the level of transcription by real-time quantitative RT-PCR analysis.The antiviral activities of the products were determined by WISH cells/Vesicular Stomatitis Virus(VSV) and PK 15 cells/Pseudorabies Virus(PRV) respectively.We found the antiviral activities of intact PoIFN-αgenes are approximate 2-50 times higher than those of the subtypes with C-terminal deletions in WISH cells and 15-55 times higher in PK 15 cells.There was no obvious difference between the subtypes with and without C-terminal deletion on acid susceptibility.For investigating the virus-inducing expression profile of PoIFN-αsubtypes,the expression of PoIFN-αwas detected using the two PCR strategies in three systems,namely,the Poly(I). Poly(C)-DEAE-dextran-induced PK 15 cells,the Pseudorabies Virus-infected PK 15 cells and the infected PK 15 cells with attenuated strain of Swine Fever Virus respectively.In Poly(I).Poly(C)-DEAE-dextran induced PK 15 cells,the expression of IFN-α2,-α3,-α4,-α8,-α9 after 6h/24h inducement in PK 15 cells were observed. In Pseudorabies Virus-infected PK 15 cells,the expression of PoIFN-α2,-α3,-α8,-α9, -α10,and -α13 was observed after 6h/24h infection and in attenuated strain of Swine Fever Virus-infected PK 15 cells,the upregulation of PoIFN-α2,-α3,-α4,-α8,-α9, and -α10 was detected.The results of Real-time quantitative PCR analysis suggested that the expression was time-dependent in Pseudorabies Virus/Poly(I). Poly(C)-DEAE-dextran induced PK 15 cells.But in attenuated strain of Swine Fever Virus-infected PK 15 system,the expression level of IFN-αsubtypes was not obviously time dependent. From the data of the pig genome sequence,we obtained a group of new interferon-like sequences,which have intact ORF to code 167AA-184AA peptides. The peptides have 23%-59%amino acid identities to the typeⅠinterferon as known. The prediction of conserved domain suggested those interferon-like peptides possess the typeⅠinterferon conserved domain,and then the phylogenetic analysis showed that those sequences have close relationship with mouse IFN-ζand porcine IFN-δ. Therefore,we classified those peptides to typeⅠinterferon family and nominated them as interferon-θ.IFN-θmultigene family consists of eight subtypes,including five functional subtypes and three pseudogenes.In functional genes,IFN-θ2/-θ3 and IFN-θ4/-θ5 have high sequence homology respectively.However,IFN-θ1 has about 80%sequence similarity to the other four subtypes.In this work,we designed the PCR primers and obtained the five IFN-θfunctional genes from porcine liver genomic DNA.Three subtypes(IFN-θ1、IFN-θ2、IFN-θ5) were chosen to be inserted into the pcDNA4 vactor,and then the reconstructed plasmids were used to transfect BHK-21 cell lines.After 48hr inoculation,we collected the supernatants to determine the antiviral activity of IFN-θ.The results showed that all IFN-θs possess antiviral activities in PK-15 and IBRS-2 porcine kidney cell lines,but the three IFN-θsubtypes had obvious difference in antiviral activity.For investigating the virus-inducing expression profile of IFN-θsubtypes,we induced IBRS-2 cell using PRV,attenuated strain of SFV and Poly IC.After 6hr and 18hr inducing,we detected the up-regulation of all IFN-θmRNA using quantitative RT-PCR analysis.The receptor analysis and ISGs gene inducing analysis suggested that IFN-θs especially bind to extra-cellular domain of IFNαR and activate the JAK/STAT signaling pathway to up-regulate ISGs expression.At last,we analyzed the cytokines expression after IFN-θinducement.The results showed that IFN-θstimulate the expression of IL-6、IL-12 and IFN-γ,which suggested that IFN-θcould have the regulative function to immune system.On the basis of our results,IFN-θnot only has the sequence homology to other typeⅠinterferons,but also matches the primary properties of typeⅠinterferons in antiviral, IFNαR receptor binding and virus inducing,etc.So we figured that IFN-θis a new subtype of typeⅠinterferon family. The adjuvant effect of porcine interferon alpha(PoIFN-α) was examined in swine vaccinated with a recombinant FMD protein vaccine named IgG-FMDV, which contains the swine IgG single heavy chain constant region and an immunogenic peptide of serotype O FMDV.The PoIFN-αgene was cloned into pcDNA3 vector and the recombinant plasmid was incorporated into cationic liposomes by a dehydration and rehydration procedure to use as an adjuvant,injected together with low-dose IgG-FMDV.This procedure resulted in strong induction of FMDV-specific neutralizing antibody and significant T cell-mediated immune responses,whereas only a modest humoral and cellular response was observed with low dose vaccine alone.As an adjuvant for the protein vaccine,PoIFN-αinduced strong inflammatory cytokine production in vivo and the results denoted that IFN-adjuvant and our vaccines could drive the immune response toward Th1 type responses.The data of ELISA suggests that the recombinant protein vaccine synergizes with the IFN-adjuvant to produce endogenous IFN in vivo.In response to viral challenge,all control animals developed viremia and lesions,whereas all animals received IFN-adjuvant+IgG-FMDV were protected and nonstructural protein antibody in this group could not be detected by 14 days post-challenge(dpc).Our studies indicate that porcine IFN-αis a powerful adjuvant for recombinant FMD protein vaccine and could aid in vaccination against FMDV in swine.更多还原