转基因植物作为生物反应器生产口蹄疫疫苗的研究进展

利用转基因植物作为生物反应器表达重组蛋白,生产外源蛋白质作为动物疫苗是一个很有吸引力的廉价生产系统,它有可能代替生产成本较高的传统疫苗的发酵生产系统。通过口蹄疫病毒VPI结构蛋白基因在转基因植物中的表达,口蹄疫疫苗已在植物中产生。在植物中生产的抗原能够保持其自身的免疫原性。本文简要综述了近十年来用转基因植物作为生物反应器生产口蹄疫疫苗的研究进展、特点及其应用前景。     

转基因植物作为生物反应器生产口蹄疫疫苗的研究进展

利用转基因植物作为生物反应器表达重组蛋白,生产外源蛋白质作为动物疫苗是一个很有吸引力的廉价生产系统,它有可能代替生产成本较高的传统疫苗的发酵生产系统。通过口蹄疫病毒VP1结构蛋白基因在转基因植物中的表达,口蹄疫疫苗已在植物中产生。在植物中生产的抗原能够保持其自身的免疫原性。本文简要综述了近十年来用转基因植物作为生物反应器生产口蹄疫疫苗的研究进展、特点及其应用前景 。     

猪瘟表面抗原E2-Erns蛋白在亚麻芥种子中的表达及活性分析

猪瘟病毒能导致猪的高度接触性传染病,对养猪业危害极大。由于传统猪瘟疫苗在使用及生产中存在诸多不足,促使科研工作者研制开发新型猪瘟疫苗。猪瘟病毒主要保护性抗原是E2蛋白和Erns蛋白,能够引起机体的免疫反应。植物表达体系具有表达效果好和生产成本低等优点,随着近些年开始出现利用转基因植物生产蛋白产品的技术,植物生物反应器越来越引起广泛关注。植物生物反应器具有完整的真核细胞表达系统,能够进行准确的蛋白翻译后加工,能较好的保留蛋白生物活性。为利用转基因植物生物反应器制备猪瘟的亚单位可饲疫苗及其商品化提供实验依据,本研究拟构建种子特异性启动子驱动的目的基因的表达载体,并转导至亚麻芥生物反应器中,表达猪瘟病毒E2-Erns融合蛋白。利用表达的猪瘟病毒E2-Erns蛋白以口服接种的方式进行小鼠模型的免疫实验,验证目标蛋白的抗原性。     

植物生物反应器表达药用蛋白研究新进展

植物生物反应器被称为"分子农田",它具有无限生产重组蛋白的巨大潜力。利用转基因植物表达的重组蛋白具备原有的理化性质和生物活性,从而为人类提供了一种大量生产药用蛋白的安全可靠、经济、方便的新生产体系。目前已广泛运用于工业、农业尤其是生命科学以及医学制造领域。用植物生物反应器产重组疫苗、重组抗体和其他药用蛋白已成为国内外基因工程研究热点之一。然而,转基因植物产物的表达量、下游加工等问题却也成为利用植物生物反应器应用的限制因素。本文就其优势、近三年内国内外转基因植物生产药用蛋白的研究进展、存在问题及对策作一综述。     

以油体作为生物反应器的研究进展

获得安全、经济、稳定具有生物活性的重组蛋白,应用于基础研究及临床应用是一个重大的战略课题,现在可以利用酵母、细菌和动物细胞生产多种药物蛋白,但这些蛋白的生产过程还存在许多问题.利用植物作为生物反应器生产药用蛋白和疫苗是目前生物反应器研究的热点.油体蛋白在油料作物种子中高水平表达且易于分离,经过改造后是生产目的蛋白的一种理想栽体.介绍了油体、油体蛋白的结构以及利用植物油体蛋白表达体系这一新型植物生物反应器生产目的蛋白的研究进展和前景.     

利用植物生物反应器生产药用蛋白的研发现状

<正>植物生物反应器具有成本低、安全性高等优点,且植物具有真核细胞表达体系,能进行准确的蛋白修饰,使产品的免疫原性及生物活性较高,因此,植物生物反应器应用日益广泛。文章就现阶段植物生物反应器生产药用蛋白的研发及应用现状进行了综述,分析了目前本领域存在的主要技术瓶颈问题,并对利用植物生物反应器生产药用蛋白的发展前景进行了展望。     

口蹄疫病毒基因组RNA结构与功能研究进展

1 概述 口蹄疫病毒(foot-and-mouth disease virus,FMDV)属小RNA病毒科FMDV属,根据动物交叉保护和血清学试验分为O、A、C、SAT1、SAT2、SAT3和Asial 7个血清型,型间无交叉反应.每型又根据抗原亲缘关系分为不同亚型.小RNA病毒科包括鼻病毒、肠道病毒、甲肝病毒、心病毒和口蹄疫病毒5个属.     

植物生物反应器研究现状、瓶颈及策略

近10年,植物作为重组蛋白生产系统是生命科学中研究最活跃领域之一。植物系统具有低成本、安全和易规模化优势,其表达生物活性药用蛋白能力已被许多研究所证实;同时,植物药用蛋白产品还表现出潜在的市场和广阔应用前景。鉴于此,回顾了植物生物反应器兴起,介绍了植物表达系统和重组蛋白研究现状,综述了植物生物反应器面临瓶颈问题、解决对策和未来一段时间内研究热点;在展望植物生物反应器前景同时,对我国研究现状、与国外差距和未来发展应采取策略进行了讨论。     

植物生物反应器在分子医药农业中的应用

植物生物反应器作为分子医药农业的核心内容,与动物反应器和微生物反应器相比具有操作简便、成本低、规模化、周期短等优势,越来越多地被人们认识和应用。阐述了植物生物反应器转化载体构建和基因优化的方法、常用的植物受体种类,以及植物表达系统类型三个方面的研究进展。随着药用蛋白需求量的大幅度增加,植物生物反应器以其低成本的优势将显示出广阔的应用前景。     

抗FLAG标签抗体在植物中的高效表达

植物生物反应器是一种新兴的重组蛋白表达系统,是分子农业的核心内容之一。本研究在本氏烟草(Nicotiana benthamiana)中表达了抗八肽(DYKDDDDK, FLAG)标签抗体,并对其进行纯化与鉴定。通过多次免疫小鼠获得高效价抗FLAG抗体并测出其编码序列,然后亚克隆至植物DNA病毒表达载体,最后通过农杆菌介导转染烟草叶片。经Western blotting检测了转染后2−9 d抗体的表达情况：3 d后FLAG抗体开始在烟草叶片中表达,5 d后表达量达到峰值,每千克鲜叶估计可表达66 mg FLAG抗体。抗体经过分离纯化后浓缩为1 mg/mL,按1:10 000稀释仍可识别1 ng/mL的抗原,表明植物生产的FLAG抗体具有高亲和力。植物生物反应器可用于生产高亲和力抗体,并具有简易、成本低和生产周期短等特点,具有很高的应用价值。     

Advances in novel vaccines for foot and mouth disease: focus on recombinant empty capsids

Foot and mouth disease (FMD) is a highly contagious disease of cloven-hoofed animals, which causes severe economic losses in the livestock industry. Currently available vaccines are based on inactivated FMD virus (FMDV). Although inactivated virus vaccines have proved to be effective in FMD control, they have a number of disadvantages, including the need for high bio-containment production facilities and the lack of induction of immunological memory. Novel FMD vaccines based on the use of recombinant empty capsids have shown promising results. These recombinant empty capsids are attractive candidates because they avoid the use of virus in the production facilities but conserve its complete repertoire of conformational epitopes. However, many of these recombinant empty capsids require time-consuming procedures that are difficult to scale up. Achieving production of a novel and efficient FMD vaccine requires not only immunogenic antigens, but also industrially relevant processes. This review intends to summarize and compare the different strategies already published for the production of FMDV recombinant empty capsids, focusing on large-scale production.     

Transient Gene Expression in Serum-Free Suspension-Growing Mammalian Cells for the Production of Foot-and-Mouth Disease Virus Empty Capsids

Foot-and-mouth disease (FMD) is a highly contagious disease of cloven-hoofed animals. It produces severe economic losses in the livestock industry. Currently available vaccines are based on inactivated FMD virus (FMDV). The use of empty capsids as a subunit vaccine has been reported to be a promising candidate because it avoids the use of virus in the vaccine production and conserves the conformational epitopes of the virus. In this report, we explored transient gene expression (TGE) in serum-free suspension-growing mammalian cells for the production of FMDV recombinant empty capsids as a subunit vaccine. The recombinant proteins produced, assembled into empty capsids and induced protective immune response against viral challenge in mice. Furthermore, they were recognized by anti-FMDV bovine sera. By using this technology, we were able to achieve expression levels that are compatible with the development of a vaccine. Thus, TGE of mammalian cells is an easy to perform, scalable and cost-effective technology for the production of a recombinant subunit vaccine against FMDV.     

Evaluation of a genetically modified foot-and-mouth disease virus vaccine candidate generated by reverse genetics

ABSTRACT: BACKGROUND: Foot-and-mouth disease (FMD) is the most economically important and highly contagious disease of cloven-hoofed animals worldwide. Control of the disease has been mainly based on large-scale vaccinations with whole-virus inactivated vaccines. In recent years, a series of outbreaks of type O FMD occurred in China (including Chinese Taipei, Chinese Hong Kong) posed a tremendous threat to Chinese animal husbandry. Its causative agent, type O FMDV, has evolved into three topotypes (East-South Asia (ME-SA), Southeast Asia (SEA), Cathay (CHY)) in these regions, which represents an important obstacle to disease control. The available FMD vaccine in China shows generally good protection against ME-SA and SEA topotype viruses infection, but affords insufficient protection against some variants of the CHY topotype. Therefore, the choice of a new vaccine strain is of fundamental importance. RESULTS: The present study describes the generation of a full-length infectious cDNA clone of FMDV vaccine strain and a genetically modified virus with some amino acid substitutions in antigenic sites 1, 3, and 4, based on the established infectious clone. The recombinant viruses had similar growth properties to the wild O/HN/CHA/93 virus. All swine immunized with inactivated vaccine prepared from the O/HN/CHA/93 were fully protected from challenge with the viruses of ME-SA and SEA topotypes and partially protected against challenge with the virus of CHA topotype at 28 days post-immunization. In contrast, the swine inoculated with the genetically modified vaccine were completely protected from the infection of viruses of the three topotypes. CONCLUSIONS: Some amino acid substitutions in the FMDV vaccine strain genome did not have an effect on the ability of viral replication in vitro. The vaccine prepared from genetically modified FMDV by reverse genetics significantly improved the protective efficacy to the variant of the CHA topotype, compared with the wild O/HN/CHA/93 virus. Thus, the full-length cDNA clone of FMDV can be a useful tool to develop genetically engineered FMDV vaccine candidates to help control porcinophilic FMD epidemics in China.     

Development of novel strategies to control foot-and-mouth disease: Marker vaccines and antivirals

Foot-and-mouth disease (FMD) is economically the most important viral-induced livestock disease worldwide. The disease is highly contagious and FMD virus (FMDV) replicates and spreads extremely rapidly. Outbreaks in previously FMD-free countries, including Taiwan, the United Kingdom, and Uruguay, and the potential use of FMDV by terrorist groups have demonstrated the vulnerability of countries and the need to develop control strategies that can rapidly inhibit or limit disease spread. The current vaccine, an inactivated whole virus preparation, has a number of limitations for use in outbreaks in disease-free countries. We have developed an alternative approach using a genetically engineered FMD subunit vaccine that only contains the portions of the viral genome required for virus capsid assembly and lacks the coding region for most of the viral nonstructural (NS) proteins including the highly immunogenic 3D protein. Thus, animals inoculated with this marker vaccine can readily be differentiated from infected animals using diagnostic assays employing the NS proteins not present in the vaccine and production of this vaccine, which does not contain infectious FMDV, does not require expensive high-containment manufacturing facilities. One inoculation of this subunit vaccine delivered in a replication-defective human adenovirus vector can induce rapid, within 7 days, and relatively long-lasting protection in swine. Similarly cattle inoculated with one dose of this recombinant vector are rapidly protected from direct and contact exposure to virulent virus. Furthermore, cattle given two doses of this vaccine developed high levels of FMDV-specific neutralizing antibodies, but did not develop antibodies against viral NS proteins demonstrating the ability of FMD subunit vaccinated animals to be differentiated from infected animals. To stimulate early protection prior to the vaccine-induced adaptive immune response we inoculated swine with the antiviral agent, type I interferon, and induced complete protection within 1 day. Protection can last for 3-5 days. The combination of the FMD marker vaccine and type I interferon can induce immediate, within 1 day, and long-lasting protection against FMD. Thus, this combination approach successfully addresses a number of concerns of FMD-free countries with the current disease control plan. By rapidly limiting virus replication and spread this strategy may reduce the number of animals that need to be slaughtered during an outbreak.     

型内嵌合口蹄疫病毒全长感染性cDNA克隆的构建

【目的】近年来,O型口蹄疫的不断暴发严重危害了我国畜牧业的发展,其病原——O型口蹄疫病毒已演化出3种谱系:中国型猪毒系、泛亚系和缅甸98系。其中中国型猪毒系病毒高度嗜猪,对养猪业危害最大。目前应用的疫苗已不能有效保护中国型猪毒系变异株的流行,这给我国猪口蹄疫的防控带来了极大的困难。为了进一步发展免疫原性好、抗原谱广的猪O型口蹄疫疫苗候选株,本研究以O/HN/93现用疫苗毒株的感染性克隆为骨架,用流行的新猪毒系病毒的部分VP3和VP1基因(主要是替换VP1蛋白上的B-C环和G-H环)替换疫苗毒株的相应部分,构建了嵌合的FMDV全长cDNA克隆。【方法】线化的嵌合全长质粒和表达T7 RNA聚合酶的真核质粒pcDNAT7P共转染BHK-21细胞,体内转录拯救嵌合病毒。【结果】嵌合全长质粒转染BHK-21细胞36h后,出现明显的FMDV致细胞病变效应。对收获的病毒分别用RT-PCR、间接免疫荧光、电子显微镜观察结果证实成功拯救到嵌合的FMDV。拯救的病毒乳鼠致病性试验结果表明该拯救病毒对乳鼠的致病力减弱。该嵌合病毒的成功拯救为研制口蹄疫新型疫苗等奠定了基础。     

口蹄疫病毒非结构蛋白3AB双抗体夹心ELISA方法的建立

抗原纯净度是口蹄疫 (Foot-and-mouth disease,FMD) 灭活疫苗质量检验的一项重要内容,一般采用疫苗2–3次免疫动物后,检测非结构蛋白 (Non-structural protein,NSP) 抗体是否阳转,判断疫苗抗原的纯净度。文中旨在建立定量检测FMD灭活疫苗抗原中NSP 3AB含量的ELISA方法,为疫苗质量控制提供参考方法。利用口蹄疫病毒 (Foot-and-mouth disease virus,FMDV) NSP 3A单克隆抗体和辣根过氧化物酶 (Horseradish peroxidase,HRP) 标记的3B单克隆抗体,建立定量检测NSP 3AB含量的双抗体夹心ELISA检测方法。采用原核表达并纯化的3AB蛋白作为标准品,标准品系列稀释,绘制标准曲线,以标准品与未加抗原的阴性对照吸光值 (OD) 的比值大于2.0的标准品最低浓度为最低检测限。标准品浓度介于4.7–600.0 ng/mL之间时,测得的OD值与浓度呈线性相关,回归曲线呈直线,相关系数R2=0.99,确定最低检测限为4.7 ng/mL。检测12份未纯化灭活抗原中3AB蛋白含量介于9.3–200.0 ng/mL之间;而纯化后的病毒抗原中3AB蛋白残留量低于最低检测限;33份来自不同厂家的成品疫苗抗原中9份疫苗抗原3AB蛋白含量在9.0–74.0 ng/mL之间,其余24份疫苗抗原中3AB蛋白残留量低于最低检测限。检测3AB蛋白含量的双抗体夹心ELISA方法能够特异、敏感地检测疫苗抗原中的3AB蛋白含量,为疫苗质量控制与纯净度检验提供了一种可供选择的检测方法。     

Expression of foot-and-mouth disease virus capsid proteins in silkworm-baculovirus expression system and its utilization as a subunit vaccine

Background

Foot-and-mouth disease (FMD) is a highly contagious disease of livestock that causes severe economic loss in susceptible cloven-hoofed animals. Although the traditional inactivated vaccine has been proved effective, it may lead to a new outbreak of FMD because of either incomplete inactivation of FMDV or the escape of live virus from vaccine production workshop. Thus, it is urgent to develop a novel FMDV vaccine that is safer, more effective and more economical than traditional vaccines.

Methodology and Principal Findings

A recombinant silkworm baculovirus Bm-P12A3C which contained the intact P1-2A and 3C protease coding regions of FMDV Asia 1/HNK/CHA/05 was developed. Indirect immunofluorescence test and sandwich-ELISA were used to verify that Bm-P12A3C could express the target cassette. Expression products from silkworm were diluted to 30 folds and used as antigen to immunize cattle. Specific antibody was induced in all vaccinated animals. After challenge with virulent homologous virus, four of the five animals were completely protected, and clinical symptoms were alleviated and delayed in the remaining one. Furthermore, a PD₅₀ (50% bovine protective dose) test was performed to assess the bovine potency of the subunit vaccine. The result showed the subunit vaccine could achieve 6.34 PD₅₀ per dose.

Conclusion

The results suggest that this strategy might be used to develop the new subunit FMDV vaccine.     

口蹄疫病毒单克隆抗体的制备及应用研究进展

口蹄疫是严重影响全球政治经济的烈性动物传染病,快速诊断及有效防治对口蹄疫的防控具有重要意义。单克隆抗体具有高特异性、均质、活性单一等优点,在生物医学领域中有广泛用途。目前,国内外学者制备了多种抗口蹄疫病毒的单克隆抗体,并应用于口蹄疫病毒抗原定型、疫苗量化、抗体水平监测、自然感染与疫苗免疫动物的鉴别诊断,以及口蹄疫病毒抗原表位分析等方面。我们简要综述口蹄疫病毒单克隆抗体的制备及应用进展。     

The Expression of IL6 and 21 in Crossbred Calves Upregulated by Inactivated Trivalent FMD Vaccine

Foot and mouth disease (FMD) is an economically important disease and a whole-virus inactivated trivalent virus vaccine is the mainstay for controlling the disease in India. The protective humoral immune response to FMD vaccination is a complex, but, tightly regulated process mediated by the interplay of interleukins (IL). Based on the specific role of IL6 and 21 in adaptive immune response, we hypothesized that inactivated trivalent FMD vaccine would stimulate IL6 and 21 expression in the circulating lymphocytes. The expressions of IL6 and 21 were assayed on 0, 28, 60, 90, and 120 d post-vaccination (DPV) by quantitative PCR (qPCR) with simultaneous assessment of FMDV antibody titer by liquid phase blocking ELISA. The results revealed that the peak expression of IL6 and 21 was on DPV 28 which correlated well with the FMDV antibody titer and plummeted to the prevaccination titer level by 60 DPV. As IL21 is the final effector of antibody production as compared to IL6, we investigated the expression of IL21 in calves that had protective titer (>1.8) with the unprotected group (<1.8). Expression of IL21 on 28 DPV was numerically higher in the protected than that of the unprotected group of calves.