猪瘟病毒结构蛋白E~(rns)在E.coli中的高效表达及纯化

猪瘟(Classical swine fever,CSF)是由猪瘟病毒(Classical swine fever virus,CSFV)引起的猪的高度接触性传染病,是严重危害养猪业的传染病之一。CSFV基因组为单股正链RNA分子,长约12.3kb,仅编码一个开放性阅读框。位于5′端的囊膜糖蛋白Erns、E1和E2构成了CSFV的外壳,其中Erns     

猪瘟病毒结构蛋白E^ms在E．coli中的高效表达及纯化

猪瘟（Classical swine fever,CSF）是由猪瘟病毒（Classical swine fever virus,CSFV）引起的猪的高度接触性传染病,是严重危害养猪业的传染病之一。CSFV基因组为单股正链RNA分子,长约12．3kb,仅编码一个开放性阅读框。位于5’端的囊膜糖蛋白E^ms、E1和E2构成了CSFV的外壳,     

逆转录病毒载体介导的猪瘟病毒E2基因的真核表达

利用DNA重组技术将猪瘟病毒(Classical swine fever virus , CSFV)石门株囊膜蛋白E2基因插入逆转录病毒载体pBABE-puro 中构建成重组逆转录病毒载体pBABE-puro-E2,该重组逆转录病毒载体与pVSVg质粒经磷酸钙共转染法将其转入293GP细胞中包装逆转录病毒假病毒.用包装的假病毒感染PK-15细胞,经嘌呤霉素筛选阳性细胞后进行流式细胞技术(FACS)分析,结果表明CSFV E2基因在PK-15细胞膜上成功表达.将表达E2蛋白的PK-15细胞腹腔免疫Balb/c小鼠,成功诱导小鼠产生了抗E2蛋白的抗体.     

猪瘟病毒结构蛋白Erns在E.Coli中的高效表达及纯化

猪瘟(Classical swine fever,CSF)是由猪瘟病毒(Classical swinefever virus,CSFV)引起的猪的高度接触性传染病,是严重危害养猪业的传染病之一.CSFV基因组为单股正链RNA分子,长约12.3kb,仅编码一个开放性阅读框.位于5'端的囊膜糖蛋白Erns、E1和E2构成了CSFV的外壳,其中Erns和E2参与病毒感染细胞的过程,并能诱导宿主产生保护性免疫应答[1].目前研究的CSFV基因工程疫苗主要以E2蛋白作为抗原,并通过检测Erns的抗体来区分E2标记疫苗免疫猪和野毒感染猪,有利于剔除猪群中潜在的传染源,达到最终消灭猪瘟的目的.氨基酸序列比较发现,CSFV的Erns氨基酸序列中有地衣类与植物核苷酸酶家族的特征序列,属于胞外RNase家族,具有RNase活性,Erns可降解病毒和细胞的RNA,在研究CSFV的致病机制方面具有重要意义[2].本研究利用RT-nPCR技术,克隆到了Erns基因,并利用大肠杆菌表达系统高效表达了Erns蛋白,纯化后的蛋白具有良好的生物学活性,为进一步建立Erns抗体的检测方法和探讨Erns蛋白在CSFV致病过程中的作用奠定了基础.     

猪瘟病毒E2蛋白表位分析及其单克隆抗体可变区cDNA测序

猪瘟病毒(CSFV)囊膜表面结构糖蛋白E2(gp55)是诱导机体产生中和抗体及激发保护性免疫应答的主要抗原蛋白.E2和Erns与细胞表面受体的相互作用介导病毒对细胞的感染过程.本文采用抗CSFV(AlfortTubingen毒株)E2结构蛋白的单克隆抗体c2410和a18,淘选噬菌体展示的12肽随机肽库,对CSFV E2蛋白表位进行分析.研究发现单克隆抗体c2410和a18识别同一线性表位,定位于E2蛋白的832-837位氨基酸(SPTTLR),但二者在ELISA和免疫印迹分析中对不同表(拟)位的反应性存在差异.自杂交瘤细胞中提取总RNA,对单克隆抗体轻链和重链可变区cDNA进行序列分析.结果表明c2410和a18虽然来源于同一批次融合的杂交瘤细胞系,识别同一表位,但仍属于不同的单克隆抗体.     

猪瘟病毒E2蛋白表位分析及其单克隆抗体可变区cDNA测序

猪瘟病毒(CSFV)囊膜表面结构糖蛋白E2(gp55)是诱导机体产生中和抗体及激发保护性免疫应答的主要抗原蛋白。E2和E^ms与细胞表面受体的相互作用介导病毒对细胞的感染过程。本文采用抗CSFV(Alfort Tiibingen毒株)E2结构蛋白的单克隆抗体c2410和a18,淘选噬菌体展示的12肽随机肽库,对CSFV E2蛋白表位进行分析。研究发现：单克隆抗体c2410和a18识别同一线性表位,定位于E2蛋白的832-837位氨基酸(SPTTLR),但二者在ELISA和免疫印迹分析中对不同表(拟)位的反应性存在差异。自杂交瘤细胞中提取总RNA,对单克隆抗体轻链和重链可变区cDNA进行序列分析。结果表明：c2410和a18虽然来源于同一批次融合的杂交瘤细胞系,识别同一表位,但仍属于不同的单克隆抗体。     

猪瘟病毒与宿主天然免疫系统的相互作用

猪瘟(Classical swine fever,CSF)是由猪瘟病毒(Classical swine fever virus,CSFV)感染引起的一种高度接触性传染病,临床上以出血综合征与免疫抑制为主要特征。它在多个国家流行,给中国乃至世界养猪业造成巨大的经济损失。研究表明,猪瘟病毒感染能够诱导宿主的天然免疫应答,也能通过影响天然免疫效应分子的表达来抑制宿主的天然免疫功能。本文将对猪瘟病毒感染与天然免疫应答及其免疫抑制的现象与机理进行综述。     

猪瘟病毒C株兔脾毒在SK6细胞中的增殖培养及其鉴定

猪瘟病毒(Classical swine fever virus,CSFV)是猪的最重要传染病之一,给养猪业造成巨大经济损失.传统疫苗C株在猪瘟防制中曾发挥了巨大作用,但由于猪瘟病毒逐渐产生变异,同时使用传统疫苗无法区分自然感染动物和免疫动物,从而使传统疫苗的应用受阻.因此十分必要研制新型猪瘟疫苗.用适宜的宿主细胞培养猪瘟传统弱毒疫苗C株,通过灵敏可靠的方法检测病毒在宿主细胞中的感染,是研究猪瘟病毒C株的一个重要基础环节.     

黄病毒NS2-3/NS3蛋白的结构与功能

猪瘟病毒(Classical swine fever virus,CSFV)、牛病毒性腹泻病病毒(Bovine viral diarrhea virus,BVDV)和羊边界病病毒(Border disease virus,BDV)共同组成黄病毒科(Flaviviridae)的瘟病毒属(Pestivirus)。近年来,对该属病毒的核酸序列、蛋白结构、基因组片段及其表达产物功能     

猪瘟病毒E2基因真核表达质粒的构建及基因疫苗的研究

构建了猪瘟病毒(classical swine fever virus, CSFV)主要保护性抗原E2基因4种不同的真核表达质粒.小鼠免疫试验表明,E2基因上不同的功能区对基因疫苗的免疫应答有很大影响,有信号肽序列的E2基因可诱导产生特异性免疫反应,且无跨膜区序列的E2基因所诱导的免疫应答反应比有跨膜区序列的强,而无信号肽序列的E2基因则不能诱导产生CSFV特异性的免疫反应.攻毒保护试验表明,免疫家兔最少可抵抗10个最小感染剂量(MID)的猪瘟兔化弱毒苗(Hog cholera lap-inized virus, HCLV)的攻击;免疫猪可抵抗致死剂量的CSFV石门株强毒的攻击.     

表达猪瘟病毒E2蛋白的重组腺病毒的构建及其在兔体内的免疫原性分析

为了构建猪瘟重组腺病毒载体疫苗,通过细菌内同源重组法构建了含有猪瘟病毒E2基因的重组腺病毒rAdV-E2.测定其一步生长曲线,同时用间接免疫荧光试验和Western blotting检测外源基因表达,然后用rAdV-E2免疫家兔,免疫后6周用猪瘟兔化弱毒疫苗株(c株)进行攻击,攻毒后3 d取其脾脏,用实时荧光定量RT-PCR检测C株病毒RNA.结果表明,该重组腺病毒传至第10代时,毒价可达1.0×1010TCID<,50/mL;外源基因可在其中得到稳定表达;rAdV-E2接种兔免疫后2周产生猪瘟特异性抗体,免疫后5 W抗体达到峰值,攻毒后rAdV-E2接种兔和C株接种兔均未出现定型热反应,从其脾脏也未检测到C株病毒RNA,而野生型腺病毒接种兔均出现了定型热反应,并且从其脾脏检测大量C株病毒RNA,其含量达到了103拷贝/μL以上.由此表明,rAdV-E2可望开发为猪瘟候选疫苗.     

Generation and Efficacy Evaluation of Recombinant Classical Swine Fever Virus E2 Glycoprotein Expressed in Stable Transgenic Mammalian Cell Line

Classical swine fever virus (CSFV) is the causative agent of classical swine fever (CSF), which is a highly contagious swine disease that causes significant economic loses to the pig industry worldwide. The envelope E2 glycoprotein of CSFV is the most important viral antigen in inducing protective immune response against CSF. In this study, we generated a mammalian cell clone (BCSFV-E2) that could stably produce a secreted form of CSFV E2 protein (mE2). The mE2 protein was shown to be N-linked glycosylated and formed a homodimer. The vaccine efficacy of mE2 was evaluated by immunizing pigs. Twenty-five 6-week-old Landrace piglets were randomly divided into five groups. Four groups were intramuscularly immunized with mE2 emulsified in different adjuvants twice at four-week intervals. One group was used as the control group. All mE2-vaccinated pigs developed CSFV-neutralizing antibodies two weeks after the first vaccination with neutralizing antibody titers ranging from 1∶40 to 1∶320. Two weeks after the booster vaccination, the neutralizing antibody titers increased greatly and ranged from 1∶10,240 to 1∶81,920. At 28 weeks after the booster vaccine was administered, the neutralizing antibody titers ranged from 1∶80 to 1∶10240. At 32 weeks after the first vaccination, pigs in all the groups were challenged with a virulent CSFV strain at a dose of 1×10⁵ TCID₅₀. At two weeks after the challenge, all the mE2-immunized pigs survived and exhibited no obvious symptoms of CSF. The neutralizing antibody titer at this time was 20,480. Unvaccinated pigs in the control group exhibited symptoms of CSF 3–4 days after challenge and were euthanized from 7–9 days after challenge when the pigs became moribund. These results indicate that the mE2 is a good candidate for the development of a safe and effective CSFV subunit vaccine.     

Expression of major antigen domains of E2 gene of CSFV and analysis of its immunological activity

E2 is an envelope glycoprotein of Classical swine fever virus (CSFV) and contains sequential neutralizing epitopes to induce virus-neutralizing antibodies and mount protective immunity in the natural host. In this study, four antigen domains (ABCD) of the E2 gene was cloned from CSFV Shimen strain into the retroviral vector pBABE puro and expressed in eukaryotic cell (PK15) by an retroviral gene expression system, and the activity of recombinant E2 protein to induce immune responses was evaluated in rabbits. The results indicated that recombinant E2 protein can be recognized by fluorescence antibodies of CSFV and CSFV positive serum (Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, China) using Western blot, indirect immunofluorescence antibody test (IFAT) and ELISA, Furthermore, anti-CSFV specific antibodies and lymphocyte proliferation were elicited and increased by recombinant protein after vaccination. In the challenge test, all of rabbits vaccinated with recombinant protein and Chinese vaccine strain (C-strain) were fully protected from a rabbit spleen virus challenge. These results indicated that a retroviral-based epitope-vaccine carrying the major antigen domains of E2 is able to induce high level of epitope-specific antibodies and exhibits similar protective capability with that induced by the C-strain, and encourages further work towards the development of a vaccine against CSFV infection. Foundation items: The National “973” (2005CB523201); Key Technology R&D Programme (2006BAD06A03).     

表达猪圆环病毒2型Cap蛋白的重组猪瘟兔化弱毒疫苗株的构建与鉴定

猪瘟(CSF)是由猪瘟病毒(CSFV)引起的一种毁灭性传染病,给养猪业造成重大经济损失。猪瘟兔化弱毒疫苗(C株)是一株非常安全、有效的优秀弱毒疫苗,对各年龄和品种的猪都极其安全,同时对不同基因亚型的CSFV均能提供有效的免疫保护。在现地,CSFV和猪圆环病毒2型(PCV2)混合感染的现象时常发生,有必要研制针对这两种病毒混合感染的二价疫苗。本研究首次构建了表达PCV2 Cap蛋白的重组C株,并评价了其在体内外的特性。结果表明,该重组病毒与C株具有相近的体外增殖特征,能够稳定表达Cap蛋白,在家兔体内具有与C株相似的生物学表型,在免疫家兔后10 d,抗CSFV E2抗体全部转阳,然而抗Cap抗体未能转阳。本研究为进一步优化表达PCV2Cap蛋白的重组C株奠定了基础。     

Expression of Major Antigen Domains of Gene of E2 CSFV and Analysis of its Immunological Activity

E2 is an envelope glycoprotein of Classical swine fever virus (CSFV) and contains sequential neutralizing epitopes to induce virus-neutralizing antibodies and mount protective immunity in the natural host. In this study, four antigen domains (ABCD) of the E2 gene was cloned from CSFV Shimen strain into the retroviral vector pBABE puro and expressed in eukaryotic cell (PK15) by an retroviral gene expression system, and the activity of recombinant E2 protein to induce immune responses was evaluated in rabbits. The results indicated that recombinant E2 protein can be recognized by fluorescence antibodies of CSFV and CSFV positive serum (Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, China) using Western blot, indirect immunofluorescence antibody test (IFAT) and ELISA, Furthermore, anti-CSFV specific antibodies and lymphocyte proliferation were elicited and increased by recombinant protein after vaccination. In the challenge test, all of rabbits vaccinated with recombinant protein and Chinese vaccine strain (C-strain) were fully protected from a rabbit spleen virus challenge. These results indicated that a retroviral-based epitope-vaccine carrying the major antigen domains of E2 is able to induce high level of epitope-specific antibodies and exhibits similar protective capability with that induced by the C-strain, and encourages further work towards the development of a vaccine against CSFV infection.     

Expression and characterization of a recombinant porcinized antibody against the E2 protein of classical swine fever virus

Applied Microbiology and Biotechnology - Classical swine fever virus (CSFV) is the causative agent of classical swine fever (CSF), a highly contagious and economically important disease of pigs....     

猪瘟病毒囊膜糖蛋白E2中和表位的定位及其免疫反应性分析

猪瘟病毒(CSFV)囊膜结构糖蛋白E2(gp55)是激发保护性免疫应答的主要抗原蛋白。E^ms和E2与细胞表面受体的相互作用介导病毒对细胞的感染过程。采用抗CSFV中和性单克隆抗体c24／10,淘选噬菌体展示的12肽随机肽库,结合噬菌体拟位免疫反应性分析结果,对CSFV E2蛋白中和表位进行定位。结果表明：F2蛋白的SPTTLR基序(832～837位氨基酸)构成CSFV特异性线性中和表位,基序的第一、二、三位氨基酸是表位与单克隆抗体c24／10结合所必需的氨基酸,也是表位的关键性氨基酸。     

基于Erns和E2基因的猪瘟标记疫苗研究概述

猪瘟(Classical swine fever,CSF)是猪的一种急性、热性和致死性传染病。该病流行范围很广,而且致死率极高,给世界养猪业造成严重危害。目前,猪瘟流行地区或国家仍然采用接种弱毒疫苗的方法作为预防猪瘟的主要策略,但接种弱毒疫苗的传统预防控制方法无法区别猪瘟疫苗免疫抗体和野毒感染抗体。为了净化、消灭猪瘟,新型标记疫苗的研究已迫在眉睫。近些年,陆续有国内外研究者应用分子生物学和基因工程方法,对猪瘟野毒株或弱毒株进行基因修饰构建出新毒株,其中以Erns和E2为基础构建新毒株的方法占据着重要地位。部分候选疫苗具有较好的免疫效果,可用于区分免疫和自然感染动物,而且有望作为新一代疫苗来替代传统弱毒疫苗。     

猪瘟病毒糖蛋白E^rns中和表位的鉴定和比较

猪瘟病毒 (CSFV)囊膜结构糖蛋白Erns(gp4 8)是诱导机体产生中和抗体及激发保护性免疫应答的第二抗原蛋白。E2和Erns与细胞表面受体的相互作用介导CSFV感染细胞的过程。Erns具有RNA酶活性 ,影响病毒自身复制并涉及对病毒的中和效应。采用抗CSFValfortT櫣ｂｉｎｇｅｎ毒株Ｅｒｎｓ糖蛋白的 1B5 ,b4_2 2和 2 4 16单克隆中和抗体 ,筛选噬菌体展示的 12肽随机肽库 ,进行Erns中和表位的鉴定和比较 ,获得分别针对 1B5、b4_2 2和 2 4 16单克隆抗体的 3个主要中和表 (拟 )位基序WxNxxP、DKNR (Q)G和A(T)CxYxKN ,分别定位于Erns的 35 1位～ 35 6位或 348位～ 35 0位、384位～ 386及 32 2位～ 32 3位、380位～ 386位氨基酸区域。分析表 (拟 )位基序与单克隆抗体的免疫反应性差异。b4_2 2和 2 4 16单克隆抗体识别基序存在共有序列KN ,识别Erns中的相似抗原区 ,但其侧翼序列及免疫印迹、免疫荧光抗体抑制试验结果均存在显著差异