构建基于杆状病毒的BV-T7杂合表达体系及利用其在哺乳动物和禽类细胞中表达eGFP基因

【目的】研究重组杆状病毒BV-T7杂合表达体系能否有效转导禽类细胞并在禽类细胞中表达外源基因(eGFP),从而构建能在禽类细胞中高效稳定表达外源基因的重组杆状病毒表达系统。【方法】本研究利用Bac-to-Bac杆状病毒表达系统,结合T7表达系统,通过对eGFP表达水平的调控来把握噬菌体T7 RNA聚合酶(T7 RNAP)的功能。利用两支重组杆状病毒,pFastBac-CMV-T7 RNAP重组杆状病毒为哺乳动物细胞启动子CMV调控的噬菌体T7 RNA聚合酶的cDNA;pFB-T7pro-IRES-GFP-T7ter重组杆状病毒为T7启动子控制的eGFP报告基因。将两支重组杆状病毒共同侵染哺乳动物OL(oligodendrocyte)细胞、鸡胚成纤维细胞和鸡胚骨骼肌细胞。【结果】两支重组杆状病毒利用T7启动子和T7 RNAP,在OL细胞、鸡胚成纤维细胞和鸡胚骨骼肌细胞中成功表达eGFP报告基因,而且未引起细胞病变,但在鸡胚原代细胞中eGFP的表达相对弱于在OL细胞中的表达。在OL细胞中重组杆状病毒对细胞的转导效率为59.5%,在鸡胚成纤维细胞和鸡胚骨骼肌细胞中转导效率分别为23.2%和33.1%。【结论】本研究构建的基于杆状病毒、T7RNA聚合酶、T7启动子(BV-T7)杂合表达体系能够在哺乳类细胞及禽类细胞中表达T7 RNAP,并利用T7RNAP继续高效而稳定地表达外源基因。这为难于体外操作的RNA病毒提供了有效的研究方法,并对新型基因工程疫苗的研制提供了一个高效而稳定的表达载体系统。     

以杆状病毒为载体在鸡原代骨骼肌细胞中表达IBDV VP2基因

摘要：【目的】本研究旨在构建在鸡原代骨骼肌细胞中表达IBDV病毒VP2基因的重组杆状病毒。【方法】从IBDV适应细胞毒中提取RNA,用RT-PCR技术扩增VP2基因,将其克隆到自主构建的杆状病毒转移载体的CMV启动子之下,通过Bac-to-Bac系统获得VP2重组Bacmid,并将其转染Sf9昆虫 细胞,获得了VP2重组杆状病毒。重组病毒经扩增后以50个MOI感染鸡原代骨骼肌细胞,接种72h后裂解细胞收获蛋白。【结果】蛋白样品经SDS-PAGE和Western blot证实VP2蛋白获得表达,分子量约48kDa,与预测蛋白大小一致,且能被IBDV阳性血清所识别。【结论】重组杆状病毒可以有效地将VP2基因导入鸡原代细胞,并在CMV的启动下表达具有抗原性的VP2蛋白,本研究为研制IBDV及其他重要禽类传染病的杆状病毒载体疫苗奠定了基础。     

利用Gateway技术构建慢病毒载体及其在鸡囊胚细胞中的表达

目的:建立慢病毒载体介导的外源基因在动物中表达的模式。方法:通过PCR扩增出带EGFP基因和特异性结合位点的DNA序列,并应用Gateway技术构建了带EGFP基因的pLenti6/v5-DEST慢病毒载体。利用磷酸钙介导慢病毒4质粒系统在包装细胞中转染,收集并浓缩产生的病毒颗粒,通过感染293FT细胞测定慢病毒滴度。结果:通过PCR扩增后测序分析,表明慢病毒载体构建正确。病毒滴度测定结果为2×107TU/mL。并用慢病毒对鸡囊胚细胞进行感染,获得了较强的表达效果。结论:慢病毒载体系统所产生的病毒颗粒对动物细胞具有较强的感染能力,为慢病毒载体在转基因家禽研究中的应用奠定了基础。     

慢病毒GFP质粒的构建及脂质体介导鸡胚细胞中瞬时表达动力学初步研究

GFP(green fluorescent protein)是一种用于检测细胞的基因表达和蛋白定位的报告基因,pLenti6/V5-D-TOPO是高效的慢病毒类表达载体.实验利用PCR(polymerase chain reaction)从质粒pEGFP-N1中扩增了EGFP 基因片段,再利用高效、快速、定向的TOPO克隆法将扩增片段克隆到pLenti6/ V5-D-TOPO载体中,构建了既能高效转染细胞又能方便观察蛋白表达情况的pLEGFP重组载体.通过PCR法鉴定及瞬时转染,结果显示,GFP基因正确地插入载体中,并能够在Hela细胞及鸡胚X期细胞中瞬时表达,但重组和对照质粒瞬时转染效果存在明显差异.通过该研究证实分子量大小、质粒和脂质体比例是影响转染的关键因素,也为转基因的研究提供了一种方法.     

重组含有Epstein-Barr病毒潜伏膜蛋白1(LMP1)基因的杆状病毒在昆虫细胞中的表达

用杆状病毒表达系统重组病毒,在昆虫细胞中表达了完整的含有ＥＢＶ－ＬＭＰ１基因３个外显子开放读码框架的长２．３ｋｂ的ｃＤＮＡ片段。用重组病毒感染Ｓｆ９细胞,用免疫荧光染色,结果表明：４８小时表达重组蛋白,７２小时细胞较完整,免疫荧光染色强阳性,９６小时后细胞出现破碎。我们采集７２小时的组织培养上清和细胞破碎裂解液,分别采用ＳＤＳ－ＰＡＧＥ、ＨＰＬＣ分子筛法,用免疫蛋白印迹法实验证明,表达的蛋白能被抗ＬＭＰ１的单克隆抗体所识别,测定表达蛋白的分子量为６０ｋＤ。经蛋白含量扫描图分析,采用Ｓｅｐｈａｄｅｘ－７５柱初步纯化表达的ＬＭＰ１蛋白,将后者进行裸鼠体内致瘤实验,未见肿瘤生长。     

腺病毒载体介导密码子优化型HPV 16 L1基因在哺乳动物细胞中的高效表达及病毒样颗粒的装配

为研究重组腺病毒载体作为HPV16预防性疫苗的可行性,构建了含密码子优化型HPV 16 L1基因的重组腺病毒,并对优化基因在哺乳动物细胞中的表达进行研究。首先按照哺乳动物密码子偏好对野生型HPV16 L1基因进行改造并合成优化基因,命名为mod.HPV16L1。将mod.HPV16L1基因克隆到穿梭质粒PDC316上,与骨架质粒共转染293细胞,在细胞内包装重组腺病毒rAd-mod.HPV16L1。用免疫印迹法检测病毒感染的293T细胞中HPV16L1蛋白的表达。通过Optiprep密度梯度超速离心法纯化HPV16 L1病毒样颗粒(VLPs)。用磷钨酸负染,在电子显微镜下观察HPV16 L1蛋白自我装配形成的VLPs。结果显示,重组腺病毒载体可介导mod.HPV16 L1基因在哺乳动物细胞内的高效表达,L1蛋白可自我装配形成VLPs。     

抗病毒基因MxA真核表达载体的构建及在鸡细胞中的表达

将人抗病毒蛋白基因MxA与携带增强型绿色荧光蛋白(EGFP)基因的真核表达质粒重组后构建MxA基因真核表达载体pEGFP-C1-MxA.经PCR和酶切方法鉴定后,重组质粒在脂质体介导下转染鸡成纤维细胞和睾丸组织原代细胞,通过荧光观察,RT-PCR及细胞免疫组化检测目的基因的表达.结果表明,MxA基因片段已经被克隆到pEGFP-C1表达载体,成功构建了MxA基因真核表达载体pEGFP-C1-MxA.经该重组质粒转染后的鸡细胞的胞质中呈现颗粒状分布的绿色荧光,RT-PCR扩增出EGFP和MxA基因的特异性片断,免疫组化结果显示EGFP报告基因在细胞内的阳性表达,并表现出MxA的表达特征,间接证明了MxA可在鸡细胞中表达.MxA基因真核表达载体的成功构建以及在鸡细胞中的表达为进一步研究MxA基因在抗禽病毒性疾病中的应用打下了基础.     

利用人摄金蛋白(METALLOTHIONEIN)启动子和牛乳突瘤病毒在哺乳动物细胞中表达乙型肝炎表面抗原

用人Metallothioenin-Ⅱ启动子、乙型肝炎表面抗原基因,SV40早期基因的编接位点和多聚A位点构成了表达组件,然后插入到经改造过的BpV-1质粒中。所得质粒pdMTsAg-5转染小鼠C127细胞得到转化克隆。Ausria Ⅱ检测证明13株中有12株能产生HBsAg。对其中一株进行HBsAg收率观察,隔天收获为292.6～525.8μg/升,每天收获为200.9～369.0μg/升,可连续收获60天以上。经重金属离子诱导后,收率增至583～854.4μg/升。培养液经超滤浓缩和两次密梯离心后,可集中为一个狭窄的峰,顶峰的Ausria Ⅱ cpm为1.05×10~7,密度为1.20g/ml。     

基于对虾白斑综合症病毒极早期启动子的新型杆状病毒表达载体的构建与分析

摘要：【目的】构建携带有受杆状病毒多角体启动子控制的疱疹性口腔炎病毒糖蛋白(vesicular stomatitis virus glycoprotein, VSV G)和受白斑综合症病毒极早期基因(immediately-early gene 1,ie1)启动子控制的绿色荧光蛋白(enhanced green fluorescent protein, EGFP)两个表达阅读框的新型重组病毒vAc-G-EGFP,分析其在无脊椎动物和脊椎动物细胞系中表达报道基因的能力。【方法】 利用Bac-To-Bac 系统构建重组杆状病毒,利用病毒感染或转导实验介导报道基因在待测细胞系中的表达,用荧光显微镜和免疫印迹技术分析报道基因在待测细胞系中的实时表达情况。 【结果】成功构建了分别含VSV G 和 ie1启动子两个阅读框的重组杆状病毒vAc-G-EGFP,发现vAc-G-EGFP可以在无脊椎和脊椎动物细胞系中有效表达报道基因EGFP,免疫印迹实验显示,在不同时间点EGFP于这两类细胞中的表达存在差异。【结论】 基于白斑综合症病毒ie1启动子并携带有VSV G表达框的单一杆状病毒载体可以实现同时在不同种类细胞系中有效表达外源基因。本文构建的新型杆状病毒表达载体有希望普遍应用于基础和应用生物学研究。     

利用多基因缺失型杆状病毒在昆虫细胞中生产腺相关病毒

腺相关病毒(adeno-associated virus, AAV)是基因治疗领域最常使用的病毒载体之一,产量低、成本高是该产业面临的关键瓶颈问题。本研究旨在基于多基因缺失型杆状病毒,建立双病毒感染昆虫细胞以生产AAV的技术体系。首先,进行AAV生产用多基因缺失型重组杆状病毒的构建和扩增,并检测杆状病毒滴度及其感染细胞的效果;然后,使用双杆状病毒共感染昆虫细胞,并优化感染条件;最后,基于优化条件进行AAV生产,并检测评估产量、质量等相关指标。结果表明,AAV生产用多基因缺失型杆状病毒滴度较野生型无差异,感染后细胞存活率下降明显减缓。使用双病毒路线进行AAV优化生产,Bac4.0-1的基因组滴度为1.63×10¹¹ VG/mL,Bac5.0-2的基因组滴度为1.02×10¹¹ VG/mL,较野生型产量分别提升了240%和110%。电镜下,3组均具有正常的AAV病毒形态,且转导活性相近。本研究建立了基于多基因缺失型杆状病毒感染昆虫细胞的AAV生产体系,显著提高了AAV产量,具有一定的应用价值。     

Baculovirus as a highly efficient gene delivery vector for the expression of hepatitis delta virus antigens in mammalian cells

Baculovirus has been employed for a wide variety of applications. In this study, we further expanded the application to the high-level expression of hepatitis delta virus (HDV) antigens and the formation of virus-like particles (VLP) in transduced mammalian cells. To this end, two recombinant baculoviruses were constructed to express large hepatitis delta antigen (L-HDAg) and hepatitis B surface antigen (HBsAg) under mammalian promoters. With a simplified transduction protocol using unconcentrated virus, high transduction efficiencies were achieved in hepatoma cells, in which L-HDAg and HBsAg were expressed abundantly, allowing for easy colorimetric detection in Western blots. L-HDAg alone was nucleus-bound and HBsAg alone was secreted; formation and secretion of HDV-like particles were readily detected upon coexpression, indicating that the baculovirus-expressed proteins were processed correctly as the authentic proteins. Quantitative real-time PCR (Q-PCR) analyses quantitatively revealed that baculovirus transduction was more efficient than plasmid transfection with respect to DNA uptake and DNA transport to the nucleus. Furthermore, superinfection introduced more baculovirus DNA into cells in the long-term culture as revealed by Q-PCR, thereby enhancing and prolonging the expression. In summary, baculovirus transduction can be an attractive method as an alternative to the plasmid transfection commonly employed for HDV research thanks to the significantly higher gene delivery efficiencies as well as the abundant expression and proper processing. Baculovirus can also be envisaged as a useful tool for investigating protein-cell interactions and virus assembly.     

Isolation and analysis of a baculovirus vector that supports recombinant glycoprotein sialylation by SfSWT-1 cells cultured in serum-free medium

The inability to sialylate recombinant glycoproteins is a critical limitation of the baculovirus-insect cell expression system. This limitation is due, at least in part, to the absence of detectable sialyltransferase activities and CMP-sialic acids in the insect cell lines routinely used as hosts in this system. SfSWT-1 is a transgenic insect cell line encoding five mammalian glycosyltransferases, including sialyltransferases, which can contribute to sialylation of recombinant glycoproteins expressed by baculovirus vectors. However, sialylation of recombinant glycoproteins requires culturing SfSWT-1 cells in the presence of fetal bovine serum or another exogenous source of sialic acid. To eliminate this requirement and extend the utility of SfSWT-1 cells, we have isolated a new baculovirus vector, AcSWT-7B, designed to express two mammalian enzymes that can convert N-acetylmannosamine to CMP-sialic acid during the early phase of infection. AcSWT-7B was also designed to express a model recombinant glycoprotein during the very late phase of infection. Characterization of this new baculovirus vector showed that it induced high levels of intracellular CMP-sialic acid and sialylation of the recombinant N-glycoprotein upon infection of SfSWT-1 cells cultured in serum-free medium supplemented with N-acetylmannosamine. In addition, co-infection of SfSWT-1 cells with AcSWT-7B plus a conventional baculovirus vector encoding human tissue plasminogen activator resulted in sialylation of this recombinant N-glycoprotein under the same culture conditions. These results demonstrate that AcSWT-7B can be used in two different ways to support recombinant N-glycoprotein sialylation by SfSWT-1 cells in serum-free medium. Thus, AcSWT-7B can be used to extend the utility of this previously described transgenic insect cell line for recombinant sialoglycoprotein production.     

The kinetics of baculovirus adsorption to insect cells in suspension culture

The influence of various culture parameters on the attachment of a recombinant baculovirus to suspended insect cells was examined under normal culture conditions. These parameters included cell density, multiplicity of infection, and composition of the cell growth medium. It was found that the fractional rate of virus attachment was independent of the multiplicity of infection but dependent on the cell density. A first order mathematical model was used to simulate the adsorption kinetics and predict the efficiency of virus attachment under the various culture conditions. This calculated efficiency of virus attachment was observed to decrease at high cell densities, which was attributed to cell clumping. It was also observed that virus attachment was more efficient in Sf900II serum free medium than it was in IPL-41 serum-supplemented medium. This effect was attributed to the protein in serum which may coat the cells and so inhibit adsorption. A general discussion relating the observations made in-these experiments to the kinetics of recombinant baculovirus adsorption to suspended insect cells is presented.     

The establishment of new cell lines from Pseudaletia unipuncta with differential responses to baculovirus infection

Summary Six insect cell lines from Pseudaletia unipuncta embryos were established and characterized, and their susceptibility to Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) infection was investigated. These embryonic P. unipuncta cell lines had characteristics distinct from each other in morphology and growth, and showed differential responses to AcMNPV infection. Among the six cell lines, two were highly susceptible to virus infection. One of these two cell lines, BTI-Pu-A7S, produced over 100 AcMNPV occlusion bodies per cell, on average. Three cell lines showed an apoptotic response following AcMNPV infection. One cell line did not support complete virus replication through the late phase of virus growth and did not exhibit apoptosis. The P. unipuncta cell lines could be distinguished from SF21 and BTI-Tn-5B1-4 cells by their isozyme markers.