猫ω-like干扰素在家蚕中的表达和生物活性检测

干扰素在畜牧养殖业和宠物治疗中可以用于治疗病毒性传染病和提高疫苗免疫效力。用杆状病毒表达系统在家蚕中表达猫ω-like干扰素,将猫ω-like干扰素基因进行优化后合成,克隆到杆状病毒转移载体p VL1393上,与病毒复制基因失活拯救型Bm Bacmid病毒DNA共转染Bm N细胞系,获得重组Bm NPV病毒,猫ω-like干扰素基因位于多角体基因启动子下游,用重组病毒感染家蚕收获表达产物。采用细胞病变法利用干扰素抑制VSV-GFP感染猫肾细胞的方法来测定干扰素活性,结果显示干扰素效价可以达到4.53×106IU/m L以上。研究结果有望为研制新型动物干扰素疫苗提供参考。     

表达猪瘟病毒E2蛋白的BacMam病毒的构建及其免疫原性分析

含具有哺乳动物细胞活性的启动子的重组杆状病毒(BacMam病毒)可有效转导多种哺乳动物细胞,并被广泛用于开发新型非复制型载体疫苗．将水泡性口炎病毒G蛋白(VSV-G)基因插入多角体启动子下游,得到经修饰的杆状病毒转移载体,将对虾白斑综合症病毒(WSSV)ie1启动子控制下的猪瘟病毒E2基因表达盒插入此载体中,构建了BacMam病毒BacMam/G-ie1-E2,以其感染Sf9细胞和转导HeLa细胞,通过间接免疫荧光试验和Western blot分析检测E蛋白的表达,同时用BacMam病毒直接免疫小鼠,用检测猪瘟病毒抗体的间接ELISA方法检测免疫小鼠血清抗体,用基于CFSE和WST-8的淋巴细胞增殖试验评价其细胞免疫应答．结果显示,BacMam/G-ie1-E2能同时在昆虫细胞和哺乳动物细胞中高效表达E2蛋白,免疫小鼠能诱导产生针对猪瘟病毒的特异性抗体,免疫小鼠脾细胞经猪瘟病毒刺激后能诱导特异性的淋巴细胞增殖．这表明,由BacMam病毒介导的基因转移有望用于开发针对猪瘟病毒的非复制型载体疫苗．     

蚕杆状病毒载体表达系统及其

就杆状病毒的分子生物学特性及其作为表达载体以蚕体为表达外源基因的特点和优点进行综述,并对该表达系统在动物疫苗生产中的应用作一简要概述.     

人乳头瘤病毒16型和11型L1基因双价重组杆状病毒转移质粒的构建及鉴定

目的：构建人乳头瘤病毒16型与11型L1基因双价重组杆状病毒转移质粒并对其进行鉴定。方法：采用：PCR法从尖锐湿疣组织标本中扩增人乳头瘤病毒11型晚期基因L1,并对其进行克隆测序;利用基因重组技术将人乳头瘤病毒16型和11型L1晚期基因共同装入杆状病毒转移载体中,分别位于强启动子Ppolh和弱启动子P10之下;利用酶切和PCR技术对双价重组杆状病毒转移质粒进行鉴定。结果：PCR扩增法获得尖税湿疣组织中感染的人乳头瘤病毒11型的L1基因,得到人乳头瘤病毒16型L1和11型L1基因双价重组杆状病毒转移质粒,经酶切电泳鉴定验证重组成功。结论：本研究成功构建了双价重组杆状病毒转移质粒,为进一步构建双价L1蛋白表达系统进而建立双价基因工程亚单位疫苗打下基础。     

蚕杆状病毒载体表达系统及其在动物疫苗生产中的应用

就杆状病毒的分子生物学特性及其作为表达载体以蚕体表达外源基因的特点和优点进行综述,并对该表达系统在动物疫苗生产中的应用作一简要概述,。     

昆虫杆状病毒表达载体系统在疫苗研究中的应用进展

昆虫杆状病毒表达载体系统(Baculovirus expression vector system,BEVS)已成功应用于多种蛋白的表达,并为疫苗开发提供了充足的原材料。相比其他表达系统,BEVS具有许多优势:杆状病毒专一寄生于无脊椎动物,安全性高;重组蛋白表达水平高;可对重组蛋白进行正确折叠和翻译后修饰,获得具有生物活性的蛋白;适应于多基因表达如病毒样颗粒(Virus-like particle)的复杂设计;适用于大规模无血清培养等。为了更好地理解BEVS在疫苗研究中的应用前景,文中将从BEVS的发展及其在疫苗研究中的应用等方面进行综述。     

杆状病毒疫苗载体的应用研究进展

人源病毒基因载体存在的记忆免疫和毒性阻碍了其在临床上的应用.自从发现杆状病毒能够有效进入多种哺乳动物细胞系和原代细胞后,其在基因治疗中的应用引人注目,候选杆状病毒载体迅速兴起.在哺乳动物细胞中,杆状病毒既不能复制也不能引起明显的细胞毒性作用,不存在哺乳动物病毒载体的相关记忆免疫.另外,含有新的穿梭启动子且包膜修饰的重组...     

杆状病毒表达系统——有效的VLP构建工具

杆状病毒表达系统是以杆状病毒为外源基因载体,昆虫细胞或活体昆虫为受体的真核表达系统。相对于其他表达系统,杆状病毒表达系统具有特殊的优势:杆状病毒基因组作为表达载体可以容纳更多外源基因;杆状病毒极晚期启动子能有效调控外源蛋白的表达;昆虫细胞作为受体能够对外源蛋白进行加工修饰;杆状病毒通常只感染节肢动物,不会对人畜构成危害。因此,该系统越来越受到人们的重视,并已应用于亚单位疫苗的研发与生产,特别其对于构建病毒样颗粒,即由一种或多种病毒结构蛋白自行装配而成且不含病毒基因组的蛋白颗粒,具有不可比拟的优势。对此做详细评述并展望病毒样颗粒疫苗的发展趋势。     

RNA病毒作为疫苗载体的研究与应用

在目前的疫苗研究中,对新疫苗免疫效果的要求越来越高。减毒或无毒的病毒疫苗载体能够激发高效持久的系统和黏膜免疫,并且具有较高的安全性,为研究新疫苗提供了一条途径。RNA病毒作为疫苗载体,在可操作性和免疫效果方面有着显著的优势,近年来已成为疫苗研究领域的热点。综述了几种RNA病毒载体目前的研究状况及其在疫苗载体方面的应用。     

杆状病毒介导外源基因在哺乳动物细胞中表达的研究进展

杆状病毒(Baculovirus)是一种以昆虫为唯一宿主的病毒, 可用做生物杀虫剂或作为表达载体在昆虫细胞中大量表达外源蛋白, 制备疫苗。研究发现, 在哺乳动物细胞中携带哺乳动物启动子的重组杆状病毒能启动下游外源基因的表达但病毒不能在哺乳动物细胞中增值, 对细胞毒性小, 转导成功的细胞可以稳定传代并有效表达外源基因, 哺乳动物细胞比昆虫细胞对蛋白质具有更好的翻译后修饰, 表达出的蛋白结构更接近天然蛋白。因此, 杆状病毒可作为一种新型的哺乳动物细胞基因转移载体, 用于表达外源基因及作为一种基因治疗载体, 具有巨大潜力, 日益受到人们的关注。本文对杆状病毒作为一种表达载体在哺乳动物细胞中表达的研究进展进行了综述。     

Baculovirus capsid display in vaccination schemes: effect of a previous immunity against the vector on the cytotoxic response to delivered antigens

The baculovirus Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) infects lepidopteran invertebrates as natural hosts, although it also has been used as display vector for vaccine development. In this work, we evaluated the effectiveness of repetitive doses of AcMNPV-based vectors on the cytotoxic immune response specific to the capsid-displayed heterologous antigen ovalbumin (OVA). Our results demonstrate that baculovirus vectors induce a boosting effect in the cytotoxic immune response to OVA, making possible to recover the levels obtained in the primary response. Moreover, mice preimmunized with wild-type baculovirus showed a complete lack of antigen-specific CD8 cytotoxic T lymphocytes (CTLs) that may be related to the presence of antibodies directed to baculoviral surface proteins, particularly to GP64. However, baculovirus was able to induce the innate immune response in spite of a previous response against this vector, although some quantitative differences reflect a distinct activation of the immune cells in prime and boost. This is the first report in which the novel capsid display strategy is evaluated in prime-boost schemes to improve efficient CTL responses.

     

Baculovirus display strategies: Emerging tools for eukaryotic libraries and gene delivery.

Recombinant baculoviruses have been extensively used as vectors for abundant expression of a large variety of foreign proteins in insect cell cultures. The appeal of the system lies essentially in easy cloning techniques and virus propagation combined with the eukaryotic post-translational modification machinery of the insect cell. Recently, a novel molecular biology tool was established by the development of baculovirus surface display, using different strategies for presentation of foreign peptides and proteins on the surface of budded virions. This eukaryotic display system enables presentation of large complex proteins on the surface of baculovirus particles and has thereby become a versatile system in molecular biology. Surface display strategies play an important role, as they may be used to enhance the efficiency and specificity of viral binding and entry to mammalian cells. In addition, baculovirus surface display vectors have been engineered to contain mammalian promoter elements designed for gene delivery both in vitro and in vivo. Moreover, baculovirus capsid display has recently been developed; this holds promise for intracellular targeting of the viral capsid and subsequent cytosolic delivery of desired protein moieties. Finally, the viruses can accommodate large insertions of foreign DNA and replicate only in insect cells. Together, these are attributes that are very likely to make them important tools in functional genomics and proteomics.     

Complement inhibition rescued mice allowing observation of transgene expression following intraportal delivery of baculovirus in mice

BACKGROUND: The baculovirus Autographa californica nucleo-polyhedrosis virus (AcNPV) is an alternative to other viral vectors for hepatic gene delivery. A barrier to AcNPV being used in vivo is its susceptibility to inactivation by serum complement 1. In vivo utility has only been demonstrated using methods that avoid contact with serum 2-5. We have studied the complement pathways involved in baculovirus inactivation in vitro and the systemic administration of baculovirus vectors in vivo, with the co-administration of the complement inhibitor, soluble complement inhibitor 1 (sCR1). RESULTS: EDTA increased baculovirus survival in human serum more than EGTA showing that both the alternative and classical pathways of complement are activated. Depleting serum of IgM increased survival, whereas reconstitution with pooled IgM restored activity against baculovirus, suggesting naturally occurring IgM antibodies with affinity for baculovirus may be partially responsible for complement activation. Intraportal administration of baculovirus led to hepatic expression when the complement inhibitor sCR1 (soluble complement receptor type 1) was co-administered by tail vein injection; however, liver histology showed hepatic necrosis. Without co-administration of sCR1, intraportal infusion of baculovirus was fatal within 24 h. Histology demonstrated massive hepatic necrosis. Yolk sac vein injection of baculovirus was associated with fetal death. CONCLUSIONS: Transgene expression was demonstrated following intraportal infusion of recombinant baculovirus vectors in combination with sCR1; however, our experiments suggest a significant associated toxicity.     

Baculovirus-mediated gene silencing in insect cells using intracellularly produced long double-stranded RNA

Double-stranded RNA-mediated interference (RNAi) has recently emerged as a powerful reverse genetics tool to silence gene expression in multiple organisms, including plants, nematodes and insects. In this study, DNA vectors capable of promoting the synthesis of long hairpin dsRNAs in vivo from a DNA template to suppress gene expression in insect cells have been successfully constructed. The inhibition of the expression of a gene encoding enhanced green fluorescent protein (eGFP) in insect cells was demonstrated by using plasmid or baculovirus vectors. Both plasmid and baculovirus vectors were able to inhibit eGFP expression in a dose dependent manner. Complete inhibition was obtained when co-transfection ratios of target plasmid to inhibition plasmid were 1:1 and 1:0.1. Eighty percent suppression was still maintained even when the ratio of eGFP plasmid to 'hairpin' plasmid was as high as 1:0.01. When the hairpin dsRNAs were encoded in a baculovirus, the suppression was about 50% when the ratio of 'target' baculovirus to 'inhibition' baculovirus reached 1:10. Therefore, the designed plasmid and baculovirus vectors are useful to induce RNAi in insect cell systems.     

Use of baculovirus expression vectors: development of diagnostic reagents, vaccines and morphological counterparts of bluetongue virus

The productivity and flexibility of insect baculovirus expression vectors and the ability of the baculovirus genome to incorporate (and express) large amounts of foreign DNA allows this system to be used for both single and multiple gene expression. Using the system, bluetongue virus (BTV) genes have been expressed to develop diagnostic reagents and vaccines as well as to understand the basic structures of the virions. BTV which causes disease in ruminants in many parts of the world, consists of 10 double-stranded RNA segments enclosed by double capsids that are composed by 7 structural proteins. Since each protein is encoded by a single RNA species, DNA clones of all 10 RNA species were synthesized and individually expressed in baculovirus vectors at high levels. This has yielded proteins that have been shown to be excellent diagnostic and vaccine reagents. In addition, multiple expression vectors have been used to synthesize morphological structures (viral and subviral) representing BTV.     

Use of baculovirus expression vectors: development of diagnostic reagents, vaccines and morphological counterparts of bluetongue virus

Abstract The productivity and flexibility of insect baculovirus expression vectors and the ability of the baculovirus genome to incorporate (and express) large amounts of foreign DNA allows this system to be used for both single and multiple gene expression. Using the system, bluetongue virus (BTV) genes have been expressed to develop diagnostic reagents and vaccines as well as to understand the basic structures of virions. BTV which causes disease in ruminants in many parts of the world, consists of 10 double-stranded RNA segments enclosed by double capsids that are composed by 7 structural proteins. Since each protein is encoded by a single RNA species, DNA clones of all 10 RNA species were synthesized and individually expressed in baculovirus vectors at high levels. This has yielded proteins that have been shown to be excellent diagnostic and vaccine reagents. In addition, multiple expression vectors have been used to synthesize morphological structures (viral and subviral) representing BTV.     

In vitro and in vivo gene delivery by recombinant baculoviruses

Although recombinant baculovirus vectors can be an efficient tool for gene transfer into mammalian cells in vitro, gene transduction in vivo has been hampered by the inactivation of baculoviruses by serum complement. Recombinant baculoviruses possessing excess envelope protein gp64 or other viral envelope proteins on the virion surface deliver foreign genes into a variety of mammalian cell lines more efficiently than the unmodified baculovirus. In this study, we examined the efficiency of gene transfer both in vitro and in vivo by recombinant baculoviruses possessing envelope proteins derived from either vesicular stomatitis virus (VSVG) or rabies virus. These recombinant viruses efficiently transferred reporter genes into neural cell lines, primary rat neural cells, and primary mouse osteal cells in vitro. The VSVG-modified baculovirus exhibited greater resistance to inactivation by animal sera than the unmodified baculovirus. A synthetic inhibitor of the complement activation pathway circumvented the serum inactivation of the unmodified baculovirus. Furthermore, the VSVG-modified baculovirus could transduce a reporter gene into the cerebral cortex and testis of mice by direct inoculation in vivo. These results suggest the possible use of the recombinant baculovirus vectors in combination with the administration of complement inhibitors for in vivo gene therapy.     

Baculovirus surface display of SARS coronavirus (SARS-CoV) spike protein and immunogenicity of the displayed protein in mice models

The baculovirus surface display technique has provided an ideal tool to display foreign proteins with natural conformation, functions, and immunogenicity. In this work, we explored the application of this technique on SARS-associated coronavirus (SARS-CoV) spike (S) protein, and further analyzed the immunogenicity of displayed S protein. The entire ectodomain of S protein was fused between the gp64 signal peptide and the VSV-G membrane anchor and successfully displayed on the baculovirus surface. Subcutaneous injection with purified S-displayed baculoviruses without adjuvant elicited highly effective production of specific and neutralizing antibodies against S protein in mice. These results confirmed a successful surface display of S protein on baculoviruse, and suggested a potential role of S-displayed baculoviruses as a novel live virus-based vaccine candidate for SARS-CoV.     

FluBlok,a next generation influenza vaccine manufactured in insect cells

FluBlok, a recombinant trivalent hemagglutinin (rHA) vaccine produced in insect cell culture using the baculovirus expression system, provides an attractive alternative to the current egg-based trivalent inactivated influenza vaccine (TIV). Its manufacturing process presents the possibility for safe and expeditious vaccine production. FluBlok contains three times more HA than TIV and does not contain egg-protein or preservatives. The high purity of the antigen enables administration at higher doses without a significant increase in side-effects in human subjects.The insect cell–baculovirus production technology is particularly suitable for influenza where annual adjustment of the vaccine is required. The baculovirus–insect expression system is generally considered a safe production system, with limited growth potential for adventitious agents. Still regulators question and challenge the safety of this novel cell substrate as FluBlok continues to advance toward product approval. This review provides an overview of cell substrate characterization for expresSF cell line used for the manufacturing of FluBlok.In addition, this review includes an update on the clinical development of FluBlok. The highly purified protein vaccine, administered at three times higher antigen content than TIV, is well tolerated and results in stronger immunogenicity, a long lasting immune response and provides cross-protection against drift influenza viruses.     

Development of viral vectors and the application for viral entry mechanisms

Virus is identified as one of the obligate intracellular parasites, which only amplify in cells of specific living things. Viral vectors, which are developed by utilizing these properties, are available in the various fields such as basic research of medical biology or application of gene therapy. Our research group has studied development of viral vectors using properties of baculovirus or vesicular stomatitis virus (VSV). Due to the development of new baculoviral vectors for mammalian cells, it is possible to be more efficient transduction of foreign gene in mammalian cells and animals. Furthermore, pseudotype or recombinant VSV possessing the envelope proteins of hepatitis C virus, Japanese encephalitis virus or baculovirus were constructed, and characteristics of the envelope proteins or entry mechanisms of these viruses were analyzed.