在昆虫细胞中高效表达蓝舌病毒VP7蛋白作为组特异性诊断抗原

对蓝舌病毒结构蛋白ＶＰ７作为组特异性诊断抗原进行了研究,将编码ＢＴＶ１３主要组特异性抗原ＶＰ７的Ｓ７ｃＤＮＡ插入杆状病毒表达载体ｐＦａｓｔＢａｃ１,通过同源重组获得了重组杆状病毒ｅｖＢａｃＢＴＶＰ７。用此重组病毒感染昆虫细胞获得ＶＰ７蛋白的高效表达,表达量可占细胞蛋白总量的１２．４％。     

同时表达蓝舌病毒四个主要结构蛋白可装配成病毒样颗粒

为研制蓝舌病毒（ｂｌｕｅｔｏｎｇｕｅ ｖｉｒｕｓ,ＢＴＶ）基因工程疫苗和进一步研究ＢＴＶ结构与功能的关系,对ＢＴＶ病毒样颗粒（ＶＬＰ）的装配进行了研究。同时在昆虫细胞中表达ＢＴＶ主要结构蛋白ＶＰ７、ＶＰ３、ＶＰ２与ＶＰ５,将细胞裂解液超速离心纯化后,发现主要存在两 形态的颗粒：一种与前文报道的病毒核心颗粒（ＣＬＰ）相同,直径约为６０ｎｍ￣７０ｎｍ,蛋白壳厚１０ｎｍ￣１５ｎｍ;另一种大小为７０ｎｍ￣     

重组家蚕病毒表达传染性法氏囊病病毒VP2蛋白

将传染性囊病病毒ＨＺ９６株主要宿主保护性抗原ＶＰ２的ｃＤＮＡ基因克隆到杆状病毒转移载体ｐＢａｃ－ＰＡＫ８中,获得重组转移载体ｐＢａｃＰＡＫ－ＶＰ２,载体ｐＢａｃＰＡＫ－ＶＰ２与修饰病毒Ｂｍ－ＢａｃＰＡＫ６线性化基因组ＤＮＡ共转染单层家蚕Ｂｏｍｂｙｘ ｍｏｒｉ（Ｂｍ）Ｎ细胞,经细胞内同源重组,筛选到重组病毒。ＥＬＩＳＡ和Ｗｅｓｔｅｒｎ免疫鲩迹结果表明,ＶＰ２在家蚕培养细胞和家蚕幼虫中均得到了表达。     

柯萨基B_3病毒对外周血单个核白细胞介素－2受体表达的影响

本文研究了柯萨基Ｂ３病毒（ＣｏｘｓａｃｋｉｅｖｉｒｕｓＢ３）对正常人ＰＢＭＣ白细胞介素一２受体（ｍＩＬ一２Ｒ）表达的影响,结果实验组为８９．８３±７．０３％,对照组为５２．５±６．１３％,表明ＣｏｘｓａｃｋｉｅｖｉｒｕｓＢ３能作用于ＰＢＭＣ,使其ｍＩＬ一２Ｒ表达明显减少（Ｐ＜０．０１）,由此影响ＩＬ－２发挥正常的生物学功能,如促使Ｔ细胞增殖,ＮＫ细胞活化等,本文认为ｍＩＬ－２表达减少可能是ＣｏｘｓａｃｋｉｅｖｉｒｕｓＢ组病毒所致心肌炎患者细胞免疫功能异常的原因之一。     

柯萨奇B3病毒基因在重组痘苗病毒中的表达

将编码柯萨奇Ｂ３病毒（ＣＶＢ３）衣壳蛋白ＶＰ１和ＶＰ２的基因,分别克隆到具有７．５ｋ启动子的痘苗病毒表达载体ｐＧＪＰ５上;将ＣＶＢ３衣壳蛋白全基因克隆到具有Ｔ７启动子的痘苗表达载体ｐＴＭ１上,并筛先到相应的重组痘苗病毒ＶＶＰ１、ＶＶＰ２和ＶＶＰ／４／２／３／１。ＶＶＰ１和ＶＶＰ２稳定表达产物为ＣＶＢ３衣壳蛋白ＶＰ１和ＶＰ２,而ＶＶＰ４／２／３／１为一无分泌性的多聚蛋白,且这三种表达产物均属无分泌性     

噬菌体T7溶菌酶及其融合蛋白在大肠杆菌中的表达

以噬菌体Ｔ７ＤＮＡ为模板,ＰＣＲ扩增Ｔ７溶菌酶基因,插入ｐＢｌｕｅｓｃｒｉｐｔＳＫ载体中,ＤＮＡ序列分析表明,克隆的Ｔ７溶菌酶基因和已报道的序列无氨基酸水平上的差异。将Ｔ７溶菌酶基因分别拼接在烟草病原相关蛋白（ＰＲ１ｂ）信号肽编码序列的３’末端和马铃薯卷叶病毒外壳蛋白（ＰＬＲＶＣＰ）基因靠近３’末端处,构建成两个融合蛋白基因。将Ｔ７溶菌酶及其融合蛋白基因插入大肠杆菌表达载体ｐＢＶ２２１,蛋白电泳及溶菌实验表明,Ｔ７溶菌酶基因在大肠杆菌中高效表达,其产物的表达量占菌体可溶性蛋白的２０％以上,ＰＬＲＶＣＰ的表达量并没有因Ｃ端融合Ｔ７溶菌酶而提高,高等植物的信号肽在大肠杆菌中也能起分泌信号作用。     

汉滩病毒核壳蛋白（NP）在杆状病毒系统的表达及其免疫原性研究

应用杆状病毒表达载体成功地表达了汉滩病毒７６－１１８株（ＨＴＮＶ）核壳蛋白,将ＨＴＮＶＳ基因插入杆状病毒转染质粒ｐＡｃＹＭＩＢ的多角体基因启动子下游附近,与经Ｂｓｕ３６１酶切线性化的杆状病毒（ＡｃＶＥＰＡ）ＤＮＡ共同转染Ｓ１９细胞,经空斑筛选获得了高效表达ＮＰ的重组杆状病毒（ＡｃＶＨａｎＳ）。经ＳＤＳ－ＰＡＧＥ和Ｗｅｓｔｅｒｎ ｂｌｏｔ证实,表达产物与ＨＴＮＶ毒粒ＮＰ分子量均为５０ＫＤ左右,紫外扫     

丙型肝炎病毒RNA多聚酶在昆虫细胞中的表达

ＨＣＶ ＮＳ５Ｂ基因片段克隆入ＢＡＣ－ＴＯ－ＢＡＣ＾ＴＭ重组杆状病毒表达系统的ｐＦＡＳＴＨＴｃ载体质粒,转化ＤＨ１０ＢＡＣ＾ＴＭ感受态细菌获得重组的Ｂａｃｍｉｄ质粒,将重组Ｂａｃｍｉｄ质粒转染Ｓｆ细胞,获得的重组杆状病毒可表达目的蛋白。免疫印迹和体外活性检测表明,所表达蛋白为ＨＣＶ ＮＳ５Ｂ蛋白,具有多聚酶活性。     

用细菌/杆状病毒系统在昆虫细胞中表达GFP与HCV抗原融合蛋白

用细菌／杆状病毒（Ｂａｃ－ｔｏ－Ｂａｃ）系统在昆虫细胞中高效表达了绿色荧光蛋白（ＧＦＰ）与ＨＣＶ抗原的双功能融合蛋白,经ＥＬＩＳＡ测定和荧光显微镜观查证实,表达产物既能发射易于检测的绿色荧光,又具有ＨＣＶ的抗原活性,实现了用绿色荧光蛋白等分子标记抗原,为免疫诊断新方法的建立打下了理论基础．     

柯萨奇B3病毒VP1基因在大肠杆菌中的表达

本文通过融合柯萨奇Ｂ３病毒（ＣＶＢ３）ＶＰ１基因与人凝血酶基因,使ＣＶＢ３的ＶＰ１在大肠杆菌中得到稳定的表达,经蛋白扫描仪等测定,表达的融合蛋白占体可溶性蛋白的１１％左右,表牵ＶＰ１产物与抗ＣＶＢ３ＶＰ１单克隆抗体和小鼠抗ＣＶＢ３血清产生较强的免疫反应。与天然的ＣＶＢ３蛋白抗原性相同,应用无关单抗和载体质粒对照均呈现阴性反应,应用表达的ＶＰ１作为抗原,我们对临床部分急性病毒性心肌炎患者血清进行了特     

Baculovirus multigene expression vectors and their use for understanding the assembly process of architecturally complex virus particles

The baculovirus expression vector is a eukaryotic DNA viral vector for the cloning and expression of foreign genes in cultured lepidopteran insect cells and insects. It has become an important tool for the large-scale production of recombinant proteins for a variety of applications including the structure-function analysis of genes and their gene products. We have developed a number of baculovirus multigene expression vectors and utilized these to understand the assembly process of multicomponent capsid structures of large viruses such as bluetongue virus (BTV), a member of the Orbivirus genus within the family Reoviridae. BTV is some 810 Å in diameter and comprised of two protein shells containing four major proteins, VP2, VP5, VP7 and VP3, surrounding a genome of ten double-stranded RNA segments and three minor proteins (VP2, VP4 and VP6). BTV is the etiological agent of a sheep disease that is sometimes fatal in certain parts of the world (e.g., Africa, Asia, and the Americas). Using baculovirus multigene vectors, we have co-expressed various combinations of BTV genes in insect cells and produced structures that mimic the various stages of BTV assembly. For example, co-expressed VP3 and VP7 form BTV core-like particles, while co-expressed VP2, VP5, VP7 and VP3 form BTV virus-like particles. Using deletion, point and domain switching analyses of each protein, we have been able to identify certain sequences in the VP7 and VP3 proteins that are essential for the assembly of core-like particles. These expression and biochemical studies have been complemented by collaboration studies using cryoelectron microscopy and image processing analyses to provide the three-dimensional structure of the expressed particles. In addition and with other associates, we have used X-ray crystallography of VP7 to deduce its atomic structure. Extensive studies on the immune responses elicited by these self-assembled particles, and chimeric derivatives involving various foreign antigens, have been carried out. Finally, using as little as 10 μg of the self-assembled virus-like particles, we have shown that they can confer long-lasting protection in sheep against BTV.     

Assembly and intracellular localization of the bluetongue virus core protein VP3

The bluetongue virus (BTV) core protein VP3 plays a crucial role in the virion assembly and replication process. Although the structure of the protein is well characterized, much less is known about the intracellular processing and localization of the protein in the infected host cell. In BTV-infected cells, newly synthesized viral core particles accumulate in specific locations within the host cell in structures known as virus inclusion bodies (VIBs), which are composed predominantly of the nonstructural protein NS2. However, core protein location in the absence of VIBs remains unclear. In this study, we examined VP3 location and degradation both in the absence of any other viral protein and in the presence of NS2 or the VP3 natural associate protein, VP7. To enable real-time tracking and processing of VP3 within the host cell, a fully functional enhanced green fluorescent protein (EGFP)-VP3 chimera was synthesized, and distribution of the fusion protein was monitored in different cell types using specific markers and inhibitors. In the absence of other BTV proteins, EGFP-VP3 exhibited distinct cytoplasmic focus formation. Further evidence suggested that EGFP-VP3 was targeted to the proteasome of the host cells but was dispersed throughout the cytoplasm when MG132, a specific proteasome inhibitor, was added. However, the distribution of the chimeric EGFP-VP3 protein was altered dramatically when the protein was expressed in the presence of the BTV core protein VP7, a normal partner of VP3 during BTV assembly. Interaction of EGFP-VP3 and VP7 and subsequent assembly of core-like particles was further examined by visualizing fluorescent particles and was confirmed by biochemical analysis and by electron microscopy. These data indicated the correct assembly of EGFP-VP3 subcores, suggesting that core formation could be monitored in real time. When EGFP-VP3 was expressed in BTV-infected BSR cells, the protein was not associated with proteasomes but instead was distributed within the BTV inclusion bodies, where it colocalized with NS2. These findings expand our knowledge about VP3 localization and its fate within the host cell and illustrate the assembly capability of a VP3 molecule with a large amino-terminal extension. This also opens up the possibility of application as a delivery system.     

Synthesis of bluetongue virus (BTV) corelike particles by a recombinant baculovirus expressing the two major structural core proteins of BTV.

The L3 and M7 genes of bluetongue virus (BTV), which encode the two major core proteins of the virus (VP3 and VP7, respectively), were inserted into a baculovirus dual-expression transfer vector and a recombinant baculovirus expressing both foreign genes isolated following in vivo recombination with wild-type Autographa californica nuclear polyhedrosis virus DNA. Spodoptera frugiperda insect cells infected with the recombinant synthesized large amounts of BTV corelike particles. These particles have been shown to be similar to authentic BTV cores in terms of size, appearance, stoichiometric arrangement of VP3 to VP7 (ratio, 2:15), and the predominance of VP7 on the surface of the particles. In infected insect cells, the corelike particles were observed in paracrystalline arrays. The formation of these structures indicates that neither the BTV double-stranded viral RNA species nor the associated minor core proteins are necessary for assembly of cores in insect cells. Furthermore, the three BTV nonstructural proteins NS1, NS2, and NS3, are not required to assist or direct the formation of empty corelike particles from VP3 and VP7.     

A single point mutation in the VP7 major core protein of bluetongue virus prevents the formation of core-like particles.

To understand the assembly process of bluetongue virus (BTV), we have established a functional assay which allows us to produce and manipulate BTV core-like particles (CLPs) composed of the viral VP7 and VP3 proteins. A cDNA clone encoding the 349-amino-acid VP7 protein has been manipulated to generate deletion, extension, and site-specific mutants. Each mutant was coexpressed with the BTV VP3 protein to generate CLPs. Deletion and extension mutants involving the VP7 carboxy terminus prevented CLP formation, while an extension mutant involving an 11-amino-acid rabies virus sequence added to the amino terminus of VP7 allowed CLP formation. Substitution of either of two cysteine residues of VP7 (Cys-15 or Cys-65) by serine also did not prevent CLP formation; however, substitution of the single lysine residue of VP7 (Lys-255) by leucine abrogated CLP formation, indicating a critical role for this lysine.     

Synthesis and characterization of chimeric particles between epizootic hemorrhagic disease virus and bluetongue virus: functional domains are conserved on the VP3 protein.

A functional assay has been developed to determine the conservative nature of the interacting sites of various structural proteins of orbiviruses by using baculovirus expression vectors. For this investigation, proteins of two serologically related orbiviruses, bluetongue virus (BTV) and the less studied epizootic hemorrhagic disease virus (EHDV), were used to synthesize chimeric particles. The results demonstrate that the inner capsid protein VP3 of EHDV-1 can replace VP3 protein of BTV in formation of the single-shelled corelike particles and the double-shelled viruslike particles. Moreover, we have demonstrated that all three minor core proteins (VP1, VP4, and VP6) can be incorporated into the homologous and chimeric corelike and viruslike particles, indicating that the functional epitopes of the VP3 protein are conserved for the morphological events of the virus. This is the first evidence of assembly of seven structural proteins of the virus by a baculovirus expression system. Confirmation at the molecular level was obtained by determining the EHDV-1 L3 gene nucleic sequence and by comparing it with sequences available for BTV. The analysis revealed a high degree homology between the two proteins: 20% difference, 50% of which is conservative. The consequences for Orbivirus phylogeny and the possibility of gene reassortments are discussed.     

Development of recombinant vaccines against bluetongue

Bluetongue virus is the aetiological agent of bluetongue, a disease of domestic and wild ruminants. Twenty-four serotypes are recognized. Novel subunit vaccines, that complement existing modified live polyvalent vaccines, are being developed. Serotype-specific viral neutralizing antibodies that are able to protect sheep against virulent homologous virus challenge can be induced by immunizing with the BTV outer capsid protein VP2 purified from virions or with VP2 expressed by baculovirus recombinants. Presentation of VP2 on virus-like particles, which assemble upon co-expression of the four major structural viral proteins (VP2, VP5, VP3 and VP7), improves the protective effect of VP2. Sheep immunized with core-like particles, comprised of VP3 and VP7, developed only limited clinical signs after virulent virus challenge, demonstrating that not only the outer capsid proteins, but also the core proteins are involved in protection against bluetongue.     

Functional dissection of the major structural protein of bluetongue virus: identification of key residues within VP7 essential for capsid assembly

A lattice of VP7 trimers forms the surface of the icosahedral bluetongue virus (BTV) core. To investigate the role of VP7 oligomerization in core assembly, a series of residues for substitution were predicted based on crystal structures of BTV type 10 VP7 molecule targeting the monomer-monomer contacts within the trimer. Seven site-specific substitution mutations of VP7 have been created using cDNA clones and were employed to produce seven recombinant baculoviruses. The effects of these mutations on VP7 solubility, ability to trimerize and formation of core-like particles (CLPs) in the presence of the scaffolding VP3 protein, were investigated. Of the seven VP7 mutants examined, three severely affected the stability of CLP, while two other mutants had lesser effect on CLP stability. Only one mutant had no apparent effect on the formation of the stable capsid. One mutant in which the conserved tyrosine at residue 271 (lower domain helix 6) was replaced by arginine formed insoluble aggregates, implying an effect in the folding of the molecule despite the prediction that such a change would be accommodated. All six soluble VP7 mutants were purified, and their ability to trimerize was examined. All mutants, including those that did not form stable CLPs, assembled into stable trimers, implying that single substitution may not be sufficient to perturb the complex monomer-monomer contacts, although subtle changes within the VP7 trimer could destabilize the core. The study highlights some of the key residues that are crucial for BTV core assembly and illustrates how the structure of VP7 in isolation underrepresents the dynamic nature of the assembly process at the biological level.     

Identification of domains in bluetongue virus VP3 molecules essential for the assembly of virus cores.

Bluetongue virus (BTV) cores consist of the viral genome and five proteins, including two major components (VP3 and VP7) and three minor components (VP1, VP4, and VP6). VP3 proteins form an inner scaffold for the deposition on the core of the surface layer of VP7. VP3 also encapsidates and interacts with the three minor proteins. The BTV VP3 protein consists of 901 amino acids and has a sequence that is a highly conserved among BTV serotypes and other orbiviruses (e.g., epizootic hemorrhagic disease virus and African horse sickness virus). To locate sites of interaction between VP3 and the other structural proteins, we have analyzed the effects of a number of VP3 deletion mutants representing conserved regions of the protein, using as an assay the formation of core-like particles (CLPs) expressed by recombinant baculoviruses. Five of the VP3 deletion mutants interacted with the coexpressed VP7 and made CLPs. These CLPs also incorporated the three minor proteins. One mutant, lacking VP3 amino acid residues 499 to 508, failed to make CLPs. Further mutational analyses have demonstrated that a methionine at residue 500 of VP3 and an arginine at residue 502 were both required for CLP formation.     

Assembly of double-shelled, viruslike particles of bluetongue virus by the simultaneous expression of four structural proteins.

Bluetongue is a disease of ruminants. The etiologic agent is bluetongue virus (BTV), a gnat-transmitted member of the Orbivirus genus of the Reoviridae. The virus has a genome of 10 double-stranded RNA species L1 to L3, M4 to M6, S7 to S10). The L2 and M5 genes of BTV which encode the outer capsid proteins VP2 and VP5, respectively, were inserted into a recombinant baculovirus downstream of duplicated copies of the baculovirus polyhedrin promoter. Insect cells coinfected with this virus plus a recombinant baculovirus expressing the two major core proteins VP3 and VP7 of BTV (T.J. French and P. Roy, J. Virol. 64:1530-1536, 1990) synthesized noninfectious, double-shelled, viruslike particles. When purified, these particles were found to have the same size and appearance as authentic BTV virions and exhibited high levels of hemagglutination activity. Antibodies raised to the expressed particles contained high titers of neutralizing activity against the homologous BTV serotype. The assembly of these bluetongue viruslike particles after the simultaneous expression of four separate proteins is indicative of the potential of this technology for the production of a new generation of viral vaccines and for the study of complex, multiprotein structures.