昆虫杆状病毒AcMNPV ORF38的功能研究及特异性转导B淋巴细胞的重组杆状病毒的构建

【作者】 葛晶；

【导师】 钟江；

【作者基本信息】 复旦大学 ， 微生物学， 2007， 博士

【摘要】 第一部分杆状病毒科（Baculoviridae）的成员是一类在自然界中仅感染节肢动物的DNA病毒。苜蓿银纹夜蛾核形多角体病毒（Autographa californica multiplenucleopolyhedrovirus,AcMNPV）是该科病毒的模式种,在科研与实践中都具有广泛的作用。近年,人们对杆状病毒开展了大量的研究,对病毒的复制机制、基因组结构及其表达和调控、蛋白的结构和功能、与宿主相互作用的分子生物学基础等有了较为深入的认识。自从1994年AcMNPV全基因组测序工作完成以来,很多必需基因的功能已经被阐释。除了必需基因之外,在杆状病毒的基因组中还有一些基因虽然对于病毒的复制来说并不是必需的,但它们却对病毒的复制起着种调节作用。因此,这些基因对病毒的正常复制同样是重要的。AcMNPV的AcORF38就是一个这样的基因。通过生物信息学分析发现,AcORF38蛋白带有焦磷酸水解酶（Nudix水解酶）的保守结构域。克隆表达制备了重组AcORF38蛋白,体外酶活分析证实其具Nudix水解酶的活性,可水解ADP-ribose。在pH=8.0,Mg²⁺浓度为5mM和AcORF38为28μM时,水解ADP-ribose的K_m值为204μM,K_cat=6.96 s^-1。RT-PCR证实,该基因确实在病毒感染的早期表达。进一步构建了orf38缺失的重组病毒vAcGFP-Δ38,证实该基因并不是病毒完成复制所必须的。但同时也发现vAcGFP-Δ38感染Sf9细胞后,出芽型病毒的效价仅为对照病毒的1/300,表明病毒的正常复制受到影响。因此,AcORF38对AcMNPV的正常复制有重要作用。为了研究AcORF38蛋白的作用机理,用电子显微镜比较了缺陷病毒和野生型病毒感染细胞的细胞病理学,发现缺陷病毒感染的细胞中病毒粒子数量远多于野生型病毒。进一步又用实时定量PCR法,分析了病毒早期基因表达,和病毒基因组复制的情况,发现缺陷病毒基因组合成水平及四个早期基因,lef-2,lef-3,dnapol和p143的转录水平也远高于野生型病毒。这提示AcORF38可以降低感染早期病毒基因的转录水平和DNA合成水平。进一步用AcORF38表达质粒和报告基因质粒共转染昆虫Sf9细胞,发现AcORF38表达不仅能降低报告基因egfp的表达和细胞荧光强度,同时还能降低细胞actin基因的mRNA水平。用CMV启动子在哺乳动物细胞HEK293等5株细胞中表达AcORF38,也观察到同样的结果。这些结果表明AcORF38可以非特异性地降低mRNA的水平。考虑到AcORF38作为Nudix水解酶的特性,初步推测AcORF38可能参与真核mRNA帽子结构的降解。为此,分别用ADP-ribose和mRNA帽子结构模拟物m7GpppG作为AcORF38的底物,进行水解反应,测得的米氏常数显示相对ADP-Ribose而言,m7GpppG更适合作为AcORF38的底物。总之,AcORF38对细胞和病毒的基因的表达有负调控作用,且这种调控作用没有专一性,推测可能是通过水解mRNA帽子结构来实现的。AcORF38的这种调控作用,有可能可以避免早期调控蛋白的过度表达,保证病毒正常复制。其确切的机理还有待深入研究。第二部分杆状病毒作为哺乳动物细胞基因转移的载体有很多独特的优点,包括:安全性好,在哺乳动物中不能复制;对外源基因的容量大,其核衣壳至少可以容纳100kb的外源基因;毒副作用小,即使在高MOI感染时也不会引起细胞毒性等等。但它也存在对一些细胞,包括B淋巴细胞,侵入效率低等问题。为了提高杆状病毒对B淋巴细胞的转导效率,本课题首次通过杆状病毒表面展示技术,对病毒的表面蛋白GP64分子进行修饰。采用"贪婪展示"的策略,即每一分子的GP64都是插入了外源短肽的),将人类EBV的表面蛋白GP350/220中能和成熟B淋巴细胞表面分子CD21相互结合的一段十肽,EDPGFFNVEI,成功地展示在重组病毒表面,得到重组病毒vAc-gp350EGFP。用重组病毒转导Raji细胞能使其EGFP的阳性率从0.05%上升至12.6%,同时用其它四株无论是EBV阳性或是EBV阴性的B淋巴细胞HR1,B95-8,BJAB,和DG75也都得到了类似的结果。进一步的实验发现用CD21的抗体处理B淋巴细胞,能竞争性地抑制该重组病毒对细胞的转导,提示重组病毒确实是通过与B淋巴细胞表面CD21蛋白以配-受体结合的方式相互作用,提高病毒转导效率的。本研究的结果大大提高了杆状病毒转导B淋巴细胞的效率和外源基因的表达水平,使杆状病毒有可能用于转导B淋巴细胞。此外,本研究的实验途径,可以广泛地应用于利用已知的受-配体关系改造杆状病毒,扩大其可以转导的哺乳动物细胞的范围,获得高效特异的病毒载体。更多还原

【Abstract】 PartⅠBaculoviridae is a group of viruses that only infects arthropod in nature.The type species of Baculoviridae,Autographa californica nucleopolyhedrovirus （AcMNPV）,has been extensively used in research and application.More and more knowledge about the Baculovirus,such as its life cycle,genome structure and replication,the regulation of gene expression,protein structure and function,and the interaction between virus and host,have been accumulated during last few decades.After the publication of the whole genome sequence of AcMNPV in 1994,many essential viral genes were identified.However,apart from these essential genes,there are still many other genes that play important roles in the life cycle of the virus, although not being essential for virus replication.Without them,virus replication would be impaired.AcORF38 of AcMNPV is one of such genes.Bioinformatics analysis revealed that it contains a conserved motif of Nudix superfamily.In the current study,the recombinant AcORF38 protein was prepared and shown to have the activity to hydrolyze ADP-Ribose in vitro,with a K_m of 204μM,and K_catof 6.96 s^-1at pH 8.0 and 5mM MgCl₂ when the concentration of AcORF38 was 28μM.This result implied that AcORF38 is truly a member of Nudix superfamily.The results of RT-PCR showed that orf38 gene is transcribed in the early phase of AcMNPV infection.An orf38 gene-deleted mutant virus,vAcGFP-△38,was constructed.The virus produced progeny virus,which meant that orf38 was non-essential for virus replication.However,the extracellular virus produced by the mutant virus was only 1/300 of that of the wild-type virus.This result indicated that orf38 played an important role in the infection cycle of virus.To further study the function of AcORF38,infected Sf9 cells were examined with electron microscopy.In the showed much more virus particles were observed in cells infected with vAcGFP-△38 than that infected with vAcGFP.In consistence with electronic microscopy,quantitation of viral DNA and mRNAs by real-time PCR found that the level of viral DNA,as well as four viral early mRNAs,including lef-2, lef-3,dnapol and p143,were significantly high in vAcGFP-△38-infected cells than vAcGFP-infected cells.The results suggested that AcORF38 might reduce the viral DNA synthesis and viral gene expression in the early phase of virus infection. Transient-expression assays further confirmed that AcORF38 reduced the expression of reporter gene egfp,as well as the mRNA level of cellular gene actin. Similar results were observed in transient assay in other 5 mammalian cells using AcORF38-expression vector driven by CMV promoter,indicating that AcORF38 might non-specifically reduce mRNA level in various cells.Taking the properties of Nudix hydrolase into account,it was postulated that AcORF38 might be an enzyme involved in degrading the cap structure at the 5’ end of eukaryotic mRNA.To test it,both the analog of the cap,m7GpppG,and ADP-ribose were used as substrate for AcORF38.The K_m value was lower when m7GpppG was used as substrate,than ADP-Ribose,indicating that m7GpppG mignt be a more appropriate substrate than ADP-Ribose.Taking together,the current study suggested that AcORF38 was a negative regulator of viral and cellular gene expression.It did that in a non-specific way, possibly through hydrolyzing the cap structure of mRNAs.Such a negative regulator could prevent the over expression of viral early proteins and ensure the normal replication of the virus.The detailed mechanism of AcORF38 is still to be understoodPartⅡAs a mammalian gene transfer vector,baculovirus has several unique advantages.For example,it is highly safe to use since it can not replicate in mammalian cells,it has a large capacity to hold foreign genes,since its rod-shaped nucleocapsid can accommodate an additional 100kb or more of DNA,and it has little cytotoxicity even at high multiplicity of infection.However,baculovirus also has its disadvantages such as the low transduction efficiency in some cells,including B-lymphocytes.In order to enhance baculovirus transduction of B cells,baculovirus surface display technology was applied to modify the virus surface protein of baculovirus. A short peptide motif from GP350/220 of Epstein-Barr virus,EDPGFFNVEI,which was known to bind to CD21,a surface protein on B-lymphocyte,was inserted into GP64,the main surface glycoprotein of AcMNPV.A recombinant virus, vAc-gp350EGFP,was constructed with the "avidity display" strategy,in which all copies of GP64 molecules on the virion had the motif.The recombinant virus, compared with a control virus with wild type GP64,showed increased transduction efficiency in B cell lines Raji,HR1,B95-8,BJAB,and DG75,regardless of their being EBV-positive or EBV-negative.The rate of EGFP positive Raji cell increased from 0.05%when transduced with the control virus,to 12.6%when transduced with vAc-gp350EGFP.The transduction of Raji cells by vAc-gp350EGFP was dose-dependently inhibited by pre-treatment of cells with anti-CD21 antibody, indicating that the improvement of the transduction was due to the interaction between the GP350/220 peptide and the CD21 molecules on the surface of B lymphocytes.The work significantly improved the transduction efficiency of baculovirus in B-lymphocytes.The approach used in the work might be applied to explore the known interactions of ligands and receptors,so as to expand the range of susceptible cells of baculovirus-mammalian cells vectors,or to increase the specificity of those vectors.更多还原