【作者】 钟志成；

【导师】 赵卫； 万成松；

【作者基本信息】 南方医科大学 ， 病原生物学， 2010， 硕士

【摘要】 一、研究背景和目的马传染性贫血病毒(EIAV)与人免疫缺陷病毒(HIV)、猴免疫缺陷病毒(SIV)、猫免疫缺陷病毒(FIV)、牛免疫缺陷病毒(BIV)、绵羊梅迪-维斯纳病毒(MVV)和山羊关节炎-脑炎病毒(CAEV)同属于反转录病毒科慢病毒属,是严重危害人类和动物健康的病原体。它们在基因组结构、病毒复制的分子机制、抗原漂移、细胞噬性、病毒的生活周期、免疫机理及病毒与宿主相互作用等方面都极为相似。其共同特点是侵害宿主免疫系统,在单核—巨噬细胞或淋巴细胞内持续增殖。但EIAV作为慢病毒属的一员,除具有一些慢病毒共同特性外,还有其独具的特性。EIAV潜伏期只有几天至几周而不像其他慢病毒属高达几年,而且,可产生高滴度的病毒血症、剧烈的临床病理学表现,在EIAV感染过程中,新的抗原变异株的出现与疾病复发相关,因此其是研究慢病毒与宿主相互作用过程中抗原变异的极好模型；与其它慢病毒感染明显不同的是,慢性EIAV感染经历一年左右的时间变为隐性感染,病毒的表达长期被抑制,大部分马在感染EIAV后有正常的寿命,部分感染马最终能控制住病毒的复制。因此,鉴定这种免疫保护机制,对于慢病毒疫苗的研制具有重要的指导意义。我国科学家在20世纪70年代利用经典的异体传代和细胞工程方法,将一株来源于马体的EIAV超强毒力毒株LN经过驴体连续传代115余代使病毒毒力增强后获得了驴强毒株DV(对马和驴均100%致死),然后将DV株连续通过驴白细胞进行体外培养驯化,使得病毒毒力完全丧失,而保持了良好的免疫原性,最终成功培育出EIAV驴白细胞弱毒疫苗DLA[6](沈荣显等,1979),该疫苗免疫接种动物不仅能产生强大持久的免疫力,而且能抵抗同源和异源强毒株的攻击。即使强毒株攻击免疫动物,其也不受感染。经过严格的安全检验,此疫苗在中国广泛推广使用,使得马传染性贫血病在中国得以有效控制。到目前为止,该疫苗是世界上唯一成功应用的慢病毒弱毒疫苗。接种该疫苗后的马能耐受致死剂量的EIAV的攻击。这也使得我国EIAV弱毒疫苗免疫保护及致弱的机理研究成为当今慢病毒免疫基础理论研究的热点之一。其传代培养致弱的分子机制及弱毒、强毒与接种动物相互作用的致病、免疫机制都将丰富慢病毒的基本理论,并将为包括HIV在内的其它慢病毒疫苗的研制提供重要的理论依据。自上世纪八十年代在发现HIV-1拱后,全球更是对EIAV的分子生物学展开了广泛研究并取得了很多成果,尤其是反向遗传技术的发展为慢病毒的研究提供了新的思路。利用该技术在1998年成功构建的几株不同毒力的EIAV感染性分子克隆,为揭示病毒毒力及其与宿主相互作用的机制打下坚实基础。马慢病毒受体1(ELR1)是近年来发现的(2005),其属于肿瘤坏死因子受体超家族蛋白,目前认为它是EIAV的唯一受体。EIAV的env基因编码gp90和gp45糖蛋白。表面糖蛋白存在于病毒囊膜中,大小约为90kDa,是高度糖基化的蛋白,构成病毒纤突的柄,与宿主细胞膜上的受体相互作用。EIAV进入靶细胞的方式是：病毒囊膜中的gp90与受体ELR1结合,在低pH环境下(pH4.8-5.3)经由细胞内吞作用进入细胞。其能够单独支持EIAV血清强毒和细胞适应毒进入非EIAV靶细胞进行增殖,但研究表明其发挥效应时可能需要其它辅助受体的共同作用,同时是否像HIV一样有辅助受体的存在尚无定论。在感染性克隆上加入荧光基团标记物标记将有助于潜在受体的确定。本文对已有的疫苗株感染性克隆进行GFP分子标记,并将构建的感染性克隆进行拯救包装出活的病毒颗粒,藉此研究马传染性贫血病毒与宿主细胞之间潜在的受体相互作用的致病机制以及疫苗保护效应的原因。此外,论文内容还将实验中片段连接的SOE-PCR技术做了部分的拓展和延伸,通过我们的实验改进,可以实现在单管反应中的多片段连接。希望本文中的部分内容可以为相关研究的实验人员提供参考。二、研究方法利用SOE-PCR技术设计重叠的互补碱基的引物实现扩增过程中的两片段延伸反应,快速的实现基因的人工合成。该技术在多个领域得到广泛的应用,主要包括感染性克隆的构建、病毒基因组测序、病毒的遗传变异分析、限制性片段多态性分析以及基因分型等方面。我们在实验设计时也是基于此技术进行的。利用SOE-PCR技术在已有的EIAV感染性分子克隆中加入带有荧光标记物EGFP进行标记。为研究增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)基因在活细胞中作为报告基因和筛选标记的可行性,利用分子克隆技术,将质粒pIRES2 EGFP片段定点插入到EIAV感染性克隆低拷贝表达载体上,构建为重组质粒PLGgfg-3-8。GFP是一种27k Da的单体,由238个氨基酸构成,本身就是一个生物发光系统,附带有激发后能发射生物荧光的发光色基,其发光过程不同于其它生物发光组织,是不需荧光素酶参与的。GFP色基是由丝氨酸酪氨酸甘氨酸(SerTyrGly)形成的环化三肽所构成,并且只有色基包被在完整的GFP蛋白中才能发出荧光(Cody et al,1993),被切断的GFP(即使是C末端的少数几个氨基酸)亦能导致GFP失去发光能力。光激发GFP荧光是一个特异性的独立过程,并不需要任何的协同因子、底物或其它来自于水母的基因表达产物。当能量由Ca2+-激活光蛋白(Aequorin)传给GFP时引发荧光(Ward et al,1980)。当GFP在原核或真核细胞中表达并受到蓝光或紫外光辐照时就可发出亮绿色荧光。EGFP是GFP突变系。EGFP发生了双氨基酸取代,Leu(亮氨酸)取代Phe64(苯丙氨酸),Thr(苏氨酸)取代Ser65(丝氨酸)。EGFP在488nm处激发后灯荧光强度为wtGFP的35倍。一般真核mRNA的翻译都需要5’帽子来介导核糖体结合,微小RNA病毒家族则例外。这类病毒在其mRNA前没有帽子位点,但却有600-1200bp的5’非翻译区,其中含有多个非起始AUG,这段长的非翻译区叫做内部核糖体进入位点序列(Internal ribosome entry site,IRES)。IRES能招募核糖体对mRNA进行翻译。将IRES与外源cDNA融合,发现IRES能独立地起始翻译。在构建好带有荧光标记的感染性克隆株后,我们进一步的采用293FT细胞进行转染包装拯救使病毒恢复为具有感染力的病毒颗粒。293细胞是转染腺病毒E1A基因的人肾上皮细胞系,293T以及293FT细胞由293细胞派生,同时表达SV40大T抗原,含有SV40复制起始点与启动子区的质粒可以复制。用穿孔法进行转染操作可以方便的进行转染。蛋白表达水平高,转染后可以通过分析荧光表达强度可较容易地检测到表达的蛋白。瞬时转染293FT细胞是过表达蛋白并获得细胞内及细胞外(分泌的或膜)蛋白的便捷方式。慢病毒表达载体包含了包装、转染、稳定整合所需要的遗传信息。慢病毒包装质粒可提供所有的转录并包装RNA到重组的假病毒载体所需要的所有辅助蛋白。为产生高滴度的病毒颗粒,需要利用表达重组后的感染性克隆载体转染细胞,在细胞中进行病毒的包装,包装好的假病毒颗粒分泌到细胞外的培养基中,离心取得上清液后,可以直接用于宿主细胞的感染,目的基因进入到宿主细胞之后,经过反转录,整合到基因组,从而高水平的表达效应分子。为后续打下基础,藉此研究病毒抗原和受体之间相互作用的致病机制。三、结果与结论本研究对SOE-PCR技术进行了改进,在加入一定稀释度的中间引物后,能够实现3个片段的直接连接。利用该技术对已有的马传染性贫血病毒疫苗株感染性克隆进行EGFP分子标记,测序结果表明长度为4.2Kb的大片段其突变的碱基数为一个碱基。在后续的对该技术的进一步研究表明,利用该技术可完成5片段的总长度为8Kb的大片段连接,扩大了多片段连接范围,在国内外文献中尚无进行如此大片段多片段连接成功的报道。将构建的带EGFP分子标记的马传染性贫血病毒疫苗株感染性克隆进行拯救,以包装出能表达EGFP的具有感染性的病毒颗粒,藉此研究马传染性贫血病毒与宿主细胞之间潜在的受体相互作用的致病机制以及疫苗保护效应的原因。实验尚在进行中,初步实验发现,采用电穿孔的方法对293FT细胞进行质粒穿孔操作,穿孔细胞培养24小时,在荧光显微镜下观察,发现细胞可释放出了荧光,为下一步实验打下了基础。更多还原

【Abstract】 1.Background and purposeEquine infectious anemia virus (EIAV) and human immunodeficiency virus (HIV), simian immunodeficiency virus (SIV), cats immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), sheep Mehdi-Visna virus (MVV) and caprine arthritis-encephalitis virus (CAEV),belong to anti-retroviral lentivirus genus, is seriously endangering the health of human and animal pathogens.Their genome, the molecular mechanism of viral replication, antigen drift, cell macrophages, virus’s life cycle, immune mechanism and the interaction between virus and host are very similar. Their common feature is against the host immune system, monocytes-macrophages or lymphocytes continued proliferation. As a member of lentivirus genu, EIAV have some unique features.EIAV incubation period only a few days to weeks, not years, and can produce high-titer viremia, severe clinical and pathological manifestations,during EIAV infection process, the new emergence of antigenic variants associated with disease recurrence, therefore, it is an excellent model to study the slow process of interaction between virus and host of antigenic variation; and a more special is chronic EIAV infection experiencing years or so into a subclinical infection, the virus-suppressed long-term infected horses have normal life, most infected horses eventually control the virus replication. Therefore, the identification of the immune protection mechanisms is important for lentiviral vaccines designe.Taking the classic 70’s use of allograft passage and cell engineering method, Chinese scientists in the 20th century obtained donkey virulent DV (on horses and donkeys are 100% fatal) from a horse the super-virulent strain of EIAV LN ass body through the continuous passage of 115 on behalf of the enhanced virulence of the virus, and then acquired complete loss of virulence straight through the DV strains were cultured in vitro leukocyte domesticated donkey, while maintaining good immunogenicity and ultimately foster a successful EIAV Donkey Leukocyte attenuated vaccine DLA (Shen Rong-xian,1979), the vaccine immunized animals can not only strong and lasting immunity, and resistance to homologous and heterologous virulent attacks made virulent attacks from infected animals after the immunization. Undergo a rigorous safety inspection of widely used in China, it certificates EIAV can be effectively controlled in China. Chinese EIAV attenuated vaccine is the first time success of the slow virus diseases immunization. So far, the vaccine is the world’s only successful application of slow-attenuated vaccine virus.It also makes our EIAV attenuated vaccine protection and the mechanism of attenuated lentivirus immunization becomes a hot topic in basic theoretical research. The subculture of the molecular mechanisms induced by the weak and the attenuated and virulent pathogenic interactions with animal inoculation, the immune system will enrich the basic theory of slow virus, and to include other slow viruses, including HIV vaccines provide an important theory. After the discovery of HIV-1,EIAV molecular biology research have made a lot of results, especially reverse genetics in the development of slow virus research provides a new idea.Using this technique successfully in 1998,several strains of different virulence EIAV infectious molecular clone has been built, which lay a solid foundation to reveal the virulence mechanisms of interaction with the host.EIAV receptor 1 (ELR1)is a recently discovered (2006),it belongs to tumor necrosis factor receptor superfamily proteins, till now it is the only EIAV receptor. The env gene of EIAV encodes gp90 and gp45 glycoproteins. Surface glycoprotein present in the virus membrane, size of approximately 90kDa, is a highly glycosylated protein, it constitute the virus spike in the handle, with the host cell membrane receptor interaction. The way EIAV into the target cells is:the gp90 virus membrane binding and receptor ELR1,introducing low pH conditions (pH4.8-5.3)by endocytosis into the cell.Alone could support its virulent EIAV serum and cell-adapted EIAV drug into the target cells of non-proliferation, but research shows that the reaction may require auxiliary receptor interaction, whether as receptors for HIV do exist is unconclusive.Our aim is to construct fluorescent marker into the infectious clone hopes which will help to identify potential receptors.In this paper, the existing vaccine strains of infectious clones for GFP marker, and infectious clone constructed in live rescue packaged virus particles, to study equine infectious anemia virus and host cell receptor interaction potential between The pathogenic mechanism and the reasons for vaccine protective effect. In addition, the paper part of the experiment, the fragments will be connected to SOE-PCR technique made part of the expansion and extension, through the improvement of our experiment can be completed in a single reaction tube to connect multiple segments. Hope that part of this article can bring relevant research reference for personnel laboratory.2.Research methodsSOE-PCR technique esign of overlapping primers with complementary bases in the process of realization of the two fragments amplified extension reaction, and rapid realization of synthetic genes.The technology widely in many fields of applications, include the construction of infectious clone, virus gene group sequencing the virus’s genetic variation analysis, restriction fragment polymorphism analysis of genetic sub-types etc. The basis of our experimental design is also based on this.Using SOE-PCR technology, we tag a fluorescent marker EGFP to the existing infectious molecular clone of EIAV.To study the enhanced green fluorescent protein gene in living cells as a reporter gene and selection marker of the feasibility of using molecular cloning technique, the plasmid pIRES2 EGFP was cloned into EIAV infectious clone Low copy expression vector to construct the recombinant plasmid PLGgfp-3-8.GFP is a 27k Da monomer,encodes 238-amino acid,is itself a bioluminescence system, with a biological fluorescence emission after excitation to the luminescence base, the light emission process is different from other biological tissue, it needs not luciferase participation. GFP chromophore of tyrosine by serine glycine (SerTyrGly) form a ring consisting of three peptides, and only the color base coat protein in the integrity of GFP fluorescence can be issued, cut off the GFP (Even the few amino acids C terminal) can also lead to loss of light-emitting GFP capacity. GFP fluorescence excitation is independent of a specific process, does not require any cofactors, substrates or other from the jellyfish gene expression product. The Ca2+-activated light protein (Aequorin) which passed to GFP irradiates fluorescence. When GFP in prokaryotic or eukaryotic cells and are subject to Blu-ray or ultraviolet irradiation can be issued when the bright green fluorescence.EGFP is a GFP mutant with two amino acid replacement, Leu (leucine) to replace Phe64 (Phe), Thr (threonine) to replace Ser65 (Ser). The EGFP fluorescence intensity excitatied at 488nm wtGFP lamps 35 times.Eukaryotic mRNA translation generally requires 5’cap to mediate ribosome binding, except for small RNA virus family. This virus is not in its pre-mRNA cap site, but it is 600～1200bp 5’untranslated region, which contains a number of non-initiation AUG,during a long untranslated region called the internal ribosome entry site sequence (Internal ribosome entry site, IRES).IRES can recruit ribosomes on mRNA translation. The IRES and the integration of exogenous cDNA and found that IRES translation initiation independently.With a fluorescent marker in building a good infectious clone, we further carried out using 293T cells transfected rescue package to restore the virus infectious virus particles.293 cells were transfected with adenovirus E1A gene in human renal epithelial cell line,293T cells derived from 293 cells, while expression of SV40 large T antigen, SV40 replication origin and contains the promoter region of the plasmid can be replicated.Transfection kit with transfected operation can be easily transfected. High levels of protein expression after transfection by analysis of fluorescence expression can be detected more easily expressed protein. Transiently transfected 293T cells over-expression and access to intracellular proteins and extracellular (secreted or membrane) proteins and convenient way. Lentiviral expression vector contains the packaging, transfection, stable integration of genetic information needed. Lentiviral packaging plasmid can provide all of the transcription and RNA packaging into recombinant viral vectors need to leave all the auxiliary proteins. To a high titer of virus particles, need to use the same expression vector and packaging plasmids were transfected into cells, the cells for virus packaging, packing a good fake virus particles released into the extracellular medium,the centrifugation supernatant obtained After the host cells can be directly used for infection of the target gene into the host cell and following the reverse transcription, integration into the genome to a high level of expression of effector molecule. 3.ResultsSuccessful use of SOE-PCR technique was improved by adding diluted in the middle of the amount of primer, it can directly connect a 3 fragment of the existing vaccine strains of infectious molecular marker GFP cloning and sequencing results showed that a large segment of length 4.2Kb The mutation of a base number of bases. And follow-up study shows that use of the technology available into five segments of the large fragment length 8Kb connection, expanding the scope of multi-segment connection at home and abroad to conduct such a large fragment of the literature and more clips to connect not been reported, it can achieve more fragment of a large fragment of the mutation can connect the mutation rate control range.Construction of infectious clones to rescue packages in live virus particles, to study equine infectious anemia virus and host cell receptor interaction potential between the pathogenic mechanism and protective effect of the vaccine because the experiment requires further study.更多还原