【作者】 赵骞；

【导师】 方六荣；

【作者基本信息】 华中农业大学 ， 预防兽医学， 2008， 硕士

【摘要】 杆状病毒载体是目前应用十分广泛的病毒载体之一,它被广泛的应用于基因治疗、基因功能研究、疫苗开发等领域。长期以来,由于其宿主特异性,这种高表达的载体仅限用于介导外源基因在其受纳昆虫细胞内表达,近年来的研究发现,杆状病毒可进入某些哺乳动物细胞,但进入细胞后并不发生复制和基因转录。因此在杆状病毒载体中引入可在哺乳动物细胞中作用的启动子如RSV,CMV等,可通过杆状病毒将外源基因转入哺乳动物细胞中进行表达。SFV复制子载体是基于Semliki森林病毒的真核表达载体,主要包括基于RNA的SFV复制子系统和基于DNA的SFV复制子系统,后者由于其更高的安全性和稳定性而被更广泛的采用。基于DNA的SFV复制子载体系统构建策略为将SFV的cDNA置于真核表达载体启动子下游(如人巨细胞病毒(CMV)的早期启动子/增强子),用外源基因代替结构蛋白基因,一旦由强启动子驱动的甲病毒非结构蛋白编码的复制酶复合物合成,便可介导细胞浆内重组RNA的大量复制,从而导致外源基因编码的mRNAs的高水平表达(Hsu et al,2004),同时,甲病毒复制子不仅具有自主复制功能,而且具有与完整甲病毒相同的诱导宿主细胞凋亡的特性,使被转染细胞在短时间内发生凋亡。有鉴于此,本研究对杆状病毒载体的设计进行了新的探索,构建了插入SFV复制子的重组杆状病毒载体,并利用模式基因对载体的特性进行了研究,主要研究内容如下:1重组杆状病毒的构建及体外表达酶切质粒pSCA1,回收包含CMV-IE、SFV复制子和26s亚启动子的大片段,插入pFastBac Dual上,用以替换杆状病毒的p10及ployhedrin(pPolh)启动子,获得载体质粒pFastBac-CMV/SFV。以EGFP为报告基因,插入到pFastBac-CMV/SFV载体上位于26S启动子下游的BamHI位点,得到转移质粒pFB-CMV/SFV-EGFP,转化DH10Bac大肠杆菌,通过蓝白斑菌落及抗性筛选,获得重组穿梭载体,提取其基因组并转染sf9昆虫细胞,获得重组杆状病毒Bac-CMV/SFV-EGFP。同时酶切质粒pc-EGFP,回收CMV-EGFP表达盒,插入到载体pFastBac1上,获得转移质粒pFastBac-CMV-EGFP,并通过相同途径获得用于对照的杆状病毒Bac-CMV-EGFP。通过接种不同哺乳动物细胞,比较了重组杆状病毒Bac-CMV/SFV-EGFP与Bac-CMV-EGFP的表达差异。发现插入SFV复制子的杆状病毒能够高效介导外源基因的转导和表达,并具有明显的细胞选择性。我们进一步对杆状病毒Bac-CMV/SFV-EGFP在哺乳动物细胞上的最适表达条件进行了探索,分析了接种温度、接种剂量、接种时间等条件对表达的影响。并分别通过DNA-ladder和Caspase-3活性检测两种手段验证了受重组杆状病毒Bac-CMV/SFV-EGFP转导的哺乳动物细胞能产生明显的凋亡现象。2修饰的重组杆状病毒的构建及其免疫原性的研究杆状病毒由于不能在哺乳动物细胞内复制,具有很高的生物安全性,因而还可以用来作为体内基因投送的载体。但研究证实血清补体系统是杆状病毒体内基因转导过程中的主要抑制因素。因此,为了消除杆状病毒的补体敏感性,在本研究中利用水疱性口膜炎病毒G蛋白修饰杆状病毒。我们在pFastBac-G载体VSV-G基因的尾端引入SV40的“尾巴”,并引入酶切位点供SFV复制子的插入,获得中间质粒pFastBac-G-SV40,酶切质粒pSCA-EGFP,插入到pFastBac-G-SV40,从而获得转移质粒pFB-G-CMV/SFV-EGFP,并通过相同途径获得用于修饰后的重组杆状病毒Bac-G-CMV/SFV-EGFP。通过接种哺乳动物细胞发现修饰后的杆状病毒对转导条件的依赖性显著降低,对哺乳动物吸附及进入的能力有显著提高。将重组病毒肌肉注射免疫小鼠,试验结果表明,在首免后第三周,重组病毒Bac-G-CMV/SFV-EGFP免疫组ELISA抗体水平明显升高,加强免疫之后,Bac-G-CMV/SFV-EGFP免疫组ELISA平均抗体滴度达到1:1440,显著的高于其它免疫组。细胞免疫水平表现出与体液免疫水平相似的趋势,不管是体液免疫水平还是细胞免疫水平,Bac-G-CMV/SFV-EGFP免疫组表现出最佳的免疫效果,表明所构建的重组杆状病毒载体是一种具有良好发展前景的疫苗载体。3杆状病毒介导的表达HA基因的重组杆状病毒的构建以及免疫原性的研究为进一步验证所构建的重组杆状病毒载体在体内基因递送的效率,我们以禽流感病毒主要保护性抗原基因HA为目标基因,构建了重组杆状病毒Bac-G-CMV/SFV-HA,通过间接免疫荧光法证实它在哺乳动物细胞上的表达。将重组病毒肌肉注射免疫小鼠,并以本室所构建的自杀性DNA疫苗pSCA-HA为对照。实验结果表明,在体液免疫方面,重组杆状病毒并没有表现出显著优势,两者差异并不显著,但在细胞免疫方面,杆状病毒组极大的促进了IFN-γ的转录与表达,在转录水平上,与核酸疫苗的差异达到10倍之多。这一结果进一步表明本研究所构建的杆状病毒表达载体可以在疫苗的开发和生产上进行深入的研究。更多还原

【Abstract】 The Baculovirus Autographa californica multiple Nucleopolyhedrovirus(AcMNPV) has long been used as a biopesticide and as a tool for high-level expression of recombinant protein in insect cells.Its host specificity has been considered to be restricted to cells derived from arthropods,However,Hofmann et al.,first in 1995 reported that recombinant baculovirus containing cytomegalovirus immediate-early(CMVoIE) promoter can drive the expression of a luciferase reporter gene in human hepatocytes. Since this initial report,the baculovirus/mammalian expression system has been employed with different mammalian cell-active promoters,such as Rous sarcoma virus (RSV) promoter or a hybrid CAG promoter that constituted a CMV-IE enhancer, chickenβ-actin promoter,and rabbitβ-globin polyadenylation signal,to deliver genes into numerous mammalian cells.Alphavirus,including Sindbis virus,Semliki Forest virus(SFV),and Venezuelan Equine encephalitis(VEE) virus,has also catched considerable attention for use as expression vectors.The significant characterizations of alphavirus vector are self-amplification,high-level expression ofheterologous protein,and the induction of apoptosis in the infected cells.The key component of alphavirus for these propertes lies in its RNA "replicon",the RNA replicase complex,which is encoded by the viral nonstructural genes(nsPs) and is required for replication of the genomic RNA and transcription of subgenomic RNA.In this report,we constructed a baculovirus vector containing the SFV replicon under the transcriptional control of CMV-IE enhancer/promoter,enhanced green fluorescence protein(EGFP),which is immediately under the SFV 26S subgenomic promoter,was generated to evaluate the gene delivery efficiencies and heterogenous protein expression in mammalian cell lines.The most research works were as following:1.Construction and expression of recombinant baculovirusThe DNA fragment containing CMV-IE enhancer/promoter-SFV replicon-26S promoter was released from pSCA1(kindly provided by Dr.Bremner,University of Toronto,Canada) with SphI and SpeI digestion,and inserted into the pFastBac DUAL (Invitrogen) backbone to replace the pl0 and ployhedrin(pPolh) promoters of Baculovirus,resulting in pFastBac-CMV/SFV.EGFP reporter gene was obtained from pc-EGFP by BglⅡand BamHI digestion and then inserted into the unique BamHI site of pFastBac-CMV/SFV,immediately downstream of SFV subgenomic promoter 26S,to generate pFB-CMV/SFV-EGFP.The DNA fragment containing CMV-IE enhancer/promoter-EGFP was obtained from pc-EGFP by BamHI and NotI digestion and then inserted into pFastBacl to generate pFB-CMV-EGFE The recombinant baculoviruses were subsequently generated by using the Bat-to-Bat(?) system(Invitrogen) following the manufacturer’s instructions.The virus was further amplified by propagation in Sf-9 cells.By compared with Bac-CMV-EGFP,Bac-CMV/SFV-EGFP hereafter is a recombinant baculovirus,capable of expressing the reporter gene,an enhanced green fluorescence protein(EGFP) under control of this hybrid promoter,exhibiting high transduction efficiency and high-level expression of reporter protein in mammalian cells. Optimal transduction conditions,including transduction temperature,time and dose, were also investigated.Additionally,the recombinant baculovirus can induce apoptosis in mammalian cells in the course of transduction,demonstrated by observed DNA laddering patterns and increased caspase-3 activity.2.Construction and immune effect of modified recombinant baculovirusAlthough AcMNPV are failing to replicate in vertebrate cells,it does express aline genes that are dependent on the strength of the promoter used to drive transcription of the foreign gene.Follwing these findings,baculovirus have emerged as a vector with great potential for gene transfer in mammalian cells.However,in vivo gene delivery by systemic administration is hindered by the vector inactivation mediated by the complement system.Therefore,in this study we describes the generation of a recombinant baculovirus in which the vesicular stomatitis virus glycoprotein G(VSV-G) is present in the viral envel6pe.We inserted the SV40 fragment into the plasmid pFastBac-G,which in order to offer the restricted enzyme sites for the SFV replicon,to generate the plasmid pFastBac-G-SV40,and then the DNA fragment containing CMV-IE enhancer/SFV-EGFP was released from pSCA-EGFP with SphI and SpeI digestion,and inserted into the pFastBac-G-SV40,resulting in pFB-G-CMV/SFV-EGFP.The recombinant baculoviruses were subsequently generated by using the Bac-to-Bac(?) system(Invitrogen) following the manufacturer’s instructions. The virus was further amplified by propagation in Sf-9 cells.To characterize the induction of antigen-specific immune response mediated by baculovirus,mice were subjected to intramuscular with baculovirus,ELISA analysis showed that relativatly hifher EGFP-specific antiboby was detected in mice immunized with Bac-G-CMV/SFV-EGFP after the primary immunization.Following a boost at week 3,’ antibodies increased significantly and the mean ELISA antibody level was reach to 1:1440,In agreement with the humoral immune response,at 6 weeks after primary immunization,the highest celluar immune response was found in restimulated splenocytes from mice immunized with Bac-G-CMV/SFV-EGFP. 3.Construction and immune effect of recombinant baculovirus expressing the HA geneFurther more,HA gene was employed to construct the recombinant baculovirus Bac-G-CMV/SFV-HA.Expressed HA protein was confirmed with indirect immunofluorescent assay on Mammalian cells,mice were subjected to intramuscular with this baculovirus,compared with the plasmid pSCA-HA.The result showed that the recombinant baculovirus Bac-G-CMV/SFV-HA was as good as plasmid pSCA-HA on the humoral immune response,however,the recombinant baculovirus was much more better than pSCA-HA on the celluar immune response.In summary,the recombinant baculovirus/mammalian vector described in this study combines the safety and high infectivity of the baculovirus vector with the high level of foreign gene expression and the proapoptotic properties of SFV replicon,which will facilitate the development of baculovirus/mammalian gene delivery system for future gene therapy and vaccine researches.更多还原