CN112831477A - 表达猪瘟病毒E2蛋白和猪圆环病毒Cap蛋白的重组伪狂犬病病毒株及其构建方法和用途 - Google Patents
表达猪瘟病毒E2蛋白和猪圆环病毒Cap蛋白的重组伪狂犬病病毒株及其构建方法和用途 Download PDFInfo
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Abstract
本发明公开了表达猪瘟病毒E2蛋白和猪圆环病毒Cap蛋白的重组伪狂犬病病毒株及其构建方法和用途。本发明通过fosmid基因操纵系统,将PRV TJ株基因组TK和gE/gI基因部分缺失,将Cap与gG(US4)融合,在US9插入E2表达框,拯救表达猪瘟病毒E2蛋白和猪圆环病毒Cap蛋白的重组伪狂犬病病毒株,其在体外的复制和生长动力学与亲本病毒相当,外源基因的插入不影响重组病毒株的遗传稳定性;用该重组伪狂犬病毒株免疫猪,能诱导出较强的PRV特异性体液免疫应答,未显示出任何PRV特异性的临床症状。本发明所构建的重组伪狂犬病病毒株能用于制备预防伪狂犬病、猪瘟病或/和猪圆环病毒所导致疾病疫苗。
Description
技术领域
本发明涉及重组伪狂犬病病毒株,本发明进一步涉及表达猪瘟病毒 E2蛋白和猪圆环病毒Cap蛋白的重组伪狂犬病病毒株及其构建方法和用 途,属于重组伪狂犬病病毒株的构建和应用领域。
背景技术
伪狂犬病(Pseudorabies,PR)是由伪狂犬病病毒(Pseudorabies virus, PRV)引起猪、羊等多种家畜和野生动物的以发热、奇痒(猪除外)、脑脊 髓炎、呼吸系统和神经系统障碍为主要特征的一种传染病(Mettenleiter,T.C., Aujeszky's disease(pseudorabies)virus:the virus and molecular pathogenesis-state of the art,June 1999.Veterinary Research 2000,31,(1), 99-115.)。PRV属于疱疹病毒科疱疹病毒甲亚科水痘病毒属。其基因组为双 链线性DNA分子,长约143kb(Pomeranz,L.E.;Reynolds,A.E.;Hengartner, C.J.,Molecular biology of pseudorabies virus:impact onneurovirology and veterinary medicine.Microbiology and Molecular BiologyReviews.2005,69,(3), 462-500.),基因组由70个开放阅读框(ORFS)组成,至少编码70-100个 病毒蛋白:包括结构蛋白、非结构蛋白、复制和毒力相关蛋白(An,T.Q.; Peng,J.M.;Tian,Z.J.;Zhao,H.Y.;Li,N.;Liu,Y.M.;Chen,J.Z.;Leng,C.L.;Sun, Y.;Chang,D.,Pseudorabies virus variant in bartha-k61–vaccinated pigs,china, 2012.EmergingInfectious Diseases 2013,19,(11),1749.)。伪狂犬病以新生仔 猪死亡率高,育肥猪呼吸困难以及母猪流产和死胎为主要特征。伪狂犬病基 因缺失疫苗是一种安全有效的疫苗,已经在许多国家广泛应用,对控制和根 除伪狂犬病起到了关键作用(Freuling,C.;Müller,T.;Mettenleiter,T.,Vaccines against pseudorabies virus(PrV).VeterinaryMicrobiology 2017,206,3-9.)。与 其他疱疹病毒一样,PRV可作为多种外源基因稳定表达的疫苗载体,用于 伪狂犬病和其他重大疾病多价疫苗的研究(Chen,Y.;Guo,W.;Xu,Z.;Yan,Q.; Luo,Y.;Shi,Q.;Chen,D.;Zhu,L.;Wang,X.,A novel recombinant pseudorabiesvirus expressing parvovirus VP2 gene:Immunogenicity and protective efficacyin swine.Virology Journal 2011,8,(1),307.)。研究表明,gE/gI和gE/gI/TK缺 失的PRV重组病毒,对易感动物安全,且能完全保护接种动物免受致死性 PRV TJ株的攻击(Wang,C.H.;Yuan,J.;Qin,H.Y.;Luo,Y.;Cong,X.;Li,Y.; Chen,J.;Li,S.;Sun,Y.;Qiu,H.J.,Anovel gE-deleted pseudorabies virus(PRV) provides rapid and completeprotection from lethal challenge with the PRV variant emerging in Bartha-K61-vaccinated swine population in China.Vaccine 2014,32,(27),3379-3385.Cong,X.;Lei,J.L.;Xia,S.L.;Wang,Y.M.;Li,Y.;Li, S.;Luo,Y.;Sun,Y.;Qiu,H.J.,Pathogenicityand immunogenicity of a gE/gI/TK gene-deleted pseudorabies virus variant insusceptible animals.Veterinary Microbiology 2016,182,170-177.)。
猪瘟(CSF)是由猪瘟病毒(CSFV)引起的一种猪的重要疫病,对 世界范围的养猪业有重大经济影响。该病以发热、出血、免疫抑制和共济 失调为主要特征。CSFV属于黄病毒科瘟病毒属成员。CSFV基因组由一 个开放阅读框(ORF)组成,编码一个大的多聚蛋白,此多聚蛋白被加工 成12种病毒蛋白:Npro、C、Erns、E1、E2、p7、NS2、NS3、NS4A、NS4B、 NS5A和NS5B(Weiland,F.;Weiland,E.;Unger,G.;Saalm,A.;Thiel,H., Localization of pestiviralenvelope proteins E(rns)and E2 at the cell surface and on isolatedparticles.Journal of General Virology 1999,80,(5),
1157-1165.)。CSFV E2糖蛋白具有诱导保护性免疫应答的功能,多数猪 瘟基因工程疫苗正是基于E2蛋白研制的。目前,疫苗接种是许多国家控 制和根除猪瘟的重要策略。C株疫苗在猪瘟的控制中起着重要作用,但该 疫苗并不能区分感染的动物和接种过疫苗的动物(DIVA)(Dong,X.-N.; Chen,Y.-H.,Marker vaccine strategies and candidateCSFV marker vaccines. Vaccine 2007,25,(2),205-230.)。因此,为了净化猪瘟,需要研制新型猪 瘟标记疫苗。
猪圆环病毒2型(PCV2)是导致断奶后仔猪多系统衰竭综合症(PMWS) 的主要病原。PMWS是一种多因素导致的疾病,主要影响仔猪和育肥猪 (Allan,G.M.;Ellis,J.A.,Porcine circoviruses:a review.Journal of Veterinary Diagnostic Investigation2000,12,(1),3-14.)。除PMWS外,PCV2可导致许 多疾病,这些通常被认为是猪圆环病毒相关疾病(PCVAD),对全世界的养 猪业造成巨大的经济损失。此外,PCV2容易与其他猪病相关病毒混合感 染,例如PRV,猪繁殖和呼吸综合征(PRRSV),CSFV和猪细小病毒病 (PPV)。PCV2是一种无包膜的小型单链DNA病毒,属于圆环病毒科 (Circoviridae)圆环病毒属。PCV2基因组为环状DNA,大小约为1.7kb。 PCV2基因组主要编码两个ORFs。ORF1编码Rep蛋白,其在病毒复制中起 关键作用。ORF2编码衣壳(Cap)蛋白,主要负责病毒衣壳的形成(Truong,C.;Mahé,D.;Blanchard,P.;Le Dimna,M.;Madec,F.;Jestin,A.;Albina,E.,Identification of an immunorelevant ORF2 epitope from porcine circovirus type2 as a serological marker for experimental and natural infection.Archives ofVirology 2001,146,(6),1197-1211.)。疫苗是防控PCV2感染的主要策略。 Cap作为PCV2唯一的结构蛋白是诊断和开发PCV2疫苗的主要抗原(Wu, P.C.;Lin,W.L.;Wu,C.M.;Chi,J.N.;Chien,M.S.;Huang,C.,Characterization of porcine circovirus type 2(PCV2)capsid particle assembly and its application to virus-like particle vaccinedevelopment.Applied Microbiology and Biotechnology 2012,95,(6),1501-1507.)。Cap蛋白可在大肠杆菌或杆状病毒中表达,并形 成病毒样颗粒(VLPs)(Liu,L.J.;Suzuki,T.;Tsunemitsu,H.;Kataoka,M.;Ngata, N.;Takeda,N.;Wakita,T.;Miyamura,T.;Li,T.C.,Efficient production of type 2 porcine circovirus-like particles by arecombinant baculovirus.Archives of Virology 2008,153,(12),2291.)。目前,针对PCV2的市售亚单位疫苗是基 于杆状病毒表达系统表达的Cap蛋白,该疫苗在控制PCV2感染,减少病毒 血症和消除PCVAD方面非常有效(Chae,C.,Commercial porcine circovirustype 2vaccines:Efficacy and clinical application.The Veterinary Journal 2012,194,(2),151-157.Martelli,P.;Ferrari,L.;Morganti,M.;De Angelis,E.;Bonilauri,P.;Guazzetti,S.;Caleffi,A.;Borghetti,P.,One dose of a porcine circovirus 2subunit vaccine induces humoral and cell-mediated immunity and protectsagainst porcine circovirus-associated disease under fieldconditions.Veterinary Microbiology 2011,149,(3-4),339-351.Alarcon,P.;Rushton,J.;Wieland,B., Cost of post-weaning multi-systemic wasting syndrome andporcine circovirus type-2subclinical infection in England–An economic diseasemodel.Preventive Veterinary Medicine 2013,110,(2),88-102.)。但是PCV2疫苗接种的成本仍 然是大多数生猪企业的主要关注点。
最近获得许可的商业疫苗已被证明是高度有效的,免疫猪后能够提供较 强的免疫保护性。但是,疫苗接种不能提供100%的免疫保护,免疫后的猪 仍可能感染新的PCV2毒株(Kekarainen,T.;McCullough,K.;Fort,M.;Fossum, C.;Segalés,J.;Allan,G.,Immuneresponses and vaccine-induced immunity against Porcine circovirus type2.Veterinary Immunology and Immunopathology 2010,136,(3-4),185-193.)。PCV2突变频率较高(约1.2×10-3/年),几乎与 RNA病毒的进化速度相似,这可能是造成疫苗接种猪再感染的原因。此 外,疫苗免疫可能导致PCV2毒株进化,新的毒株具有逃逸现有PCV2疫苗 免疫的趋势(Segalés,J.,Best practice and future challenges for vaccinationagainst porcine circovirus type 2.Expert Review of Vaccines 2015,14,(3), 473-487.Kekarainen,T.;Gonzalez,A.;Llorens,A.;Segales,J.,Genetic variability ofporcine circovirus 2in vaccinating and non-vaccinating commercialfarms.Journal of General Virology 2014,95,(8),1734-1742.)。研究证明,新 毒株的Cap基因突变是导致免疫后再感染的原因。因此,有必要在目前流行 的PCV2毒株基础上开发新型疫苗。此外,研制PCV2和其他猪病的多价疫 苗,可简化免疫程序并降低成本。
尽管已经对伪狂犬和猪瘟的控制与根除做出重大努力,但这些疾病在 猪群中仍有流行(Lei,J.L.;Xia,S.L.;Wang,Y.;Du,M.;Xiang,G.T.;Cong,X.; Luo,Y.;Li,L.F.;Zhang,L.;Yu,J.,Safety and immunogenicity of a gE/gI/TK gene-deletedpseudorabies virus variant expressing the E2 protein of classical swine fevervirus in pigs.Immunology Letters 2016,174,63-71)。已有报道表明 PRV、CSFV和PCV2常在猪群中出现混合感染(Beach,N.M.;Meng,X.J., Efficacy and future prospects ofcommercially available and experimental vaccines against porcine circovirustype 2(PCV2).Virus Research 2012,164, (1-2),33-42.Huang,Y.L.;Pang,V.F.;Lin,C.M.;Tsai,Y.C.;Chia,M.Y.;Deng, M.C.;Chang,C.Y.;Jeng,C.R.,Porcine circovirustype 2(PCV2)infection decreases the efficacy of an attenuated classical swinefever virus(CSFV)vaccine. Veterinary Research 2011,42,(1),115.Zhang,L.;Li,Y.;Xie,L.;Wang,X.;Gao, X.;Sun,Y.;Qiu,H.J.,Secreted expression of the Cap gene ofporcine circovirus type 2in classical swine fever virus C-strain:potential ofC-strain used as a vaccine vector.Viruses 2017,9,(10),298.),导致猪发生严重消耗性疾病。因 此,多联苗的发展在解决混合感染、简化免疫程序、单次接种的过程中具有重大意义,并且可以降低这三种疾病在猪群中带来的经济损失。
PRV基因组含有独特长区段(UL)、独特短区段(US)、长反向重复 序列、内部重复序列(IRs)和末端重复序列(TRs)。它包含多个非必需基 因,外源基因插入不影响病毒体内和/或体外的复制(Qiu,H.J.;Tian,Z.J.; Tong,G.Z.;Zhou,Y.J.;Ni,J.Q.;Luo,Y.Z.;Cai,X.H.,Protective immunity induced by a recombinant pseudorabies virusexpressing the GP5 of porcine reproductive and respiratory syndrome virus inpiglets.Veterinary Immunology and Immunopathology 2005,106,(3-4),309-319.Qian,P.;Li,X.M.;Jin,M.L.; Peng,G.Q.;Chen,H.C.,An approach to a FMDvaccine based on genetic engineered attenuated pseudorabies virus:oneexperiment using VP1 gene alone generates an antibody responds on FMD andpseudorabies in swine.Vaccine 2004, 22,(17-18),2129-2136.Jiang,Y.;Fang,L.;Xiao,S.;Zhang,H.;Pan,Y.;Luo,R.;Li,B.;Chen,H.,Immunogenicity and protectiveefficacy of recombinant pseudorabies virus expressing the two major membrane-associated proteins of porcine reproductive and respiratory syndromevirus.Vaccine 2007,25,(3), 547-560.),使它成为表达其他猪病抗原的合适载体。虽然已报道gI和gE 是毒力相关基因,但却是PRV免疫原性的非必需基因。缺失TK和gE/gI基 因减弱PRV的毒力但却未影响它的免疫原性(6.Wang,C.H.;Yuan,J.;Qin, H.Y.;Luo,Y.;Cong,X.;Li,Y.;Chen,J.;Li,S.;Sun,Y.;Qiu,H.J.,A novel gE-deleted pseudorabiesvirus(PRV)provides rapid and complete protection from lethal challenge withthe PRV variant emerging in Bartha-K61-vaccinated swine population inChina.Vaccine 2014,32,(27),3379-3385.Cong,X.;Lei,J.L.; Xia,S.L.;Wang,Y.M.;Li,Y.;Li,S.;Luo,Y.;Sun,Y.;Qiu,H.J.,Pathogenicity and immunogenicity of a gE/gI/TK gene-deleted pseudorabies virus variant in susceptible animals.VeterinaryMicrobiology 2016,182,170-177.Jiang,Y.;Fang, L.;Xiao,S.;Zhang,H.;Pan,Y.;Luo,R.;Li,B.;Chen,H.,Immunogenicity and protective efficacy of recombinantpseudorabies virus expressing the two major membrane-associated proteins ofporcine reproductive and respiratory syndrome virus.Vaccine 2007,25,(3),547-560.)。大量基因缺失的重组PRV已发展成 表达外源基因的疫苗载体(Hong,Q.;Qian,P.;Li,X.M.;Yu,X.L.;Chen,H.C., A recombinant pseudorabies virus co-expressingcapsid proteins precursor P1-2A of FMDV and VP2 protein of porcineparvovirus:a trivalent vaccine candidate. Biotechnology Letters 2007,29,(11),1677-1683.Song,Y.;Jin,M.;Zhang,S.;Xu, X.;Xiao,S.;Cao,S.;Chen,H.,Generationand immunogenicity of a recombinant pseudorabies virus expressing cap proteinof porcine circovirus type 2.Veterinary Microbiology 2007,119,(2-4),97-104.He,Y.;Qian,P.;Zhang,K.;Yao,Q.; Wang,D.;Xu,Z.;Wu,B.;Jin,M.;Xiao,S.;Chen,H.,Construction and immune response characterization of a recombinantpseudorabies virus co-expressing capsid precursor protein(P1)and amultiepitope peptide of foot-and-mouth disease virus in swine.Virus Genes2008,36,(2),393-400.)。因此,PRV常被 作为有发展前景的活病毒载体疫苗。
综上所述,PRV、PCV2和CSFV三种病毒的感染均可通过疫苗接种 来防控,因此,有必要开发针对这三种病的疫苗来降低疫苗成本,并简化 疫苗接种程序。
发明内容
本发明的目的之一是提供一株表达猪瘟病毒E2蛋白和猪圆环病毒 Cap蛋白的重组伪狂犬病病毒株;
本发明的目的之二是提供一种构建所述表达猪瘟病毒E2蛋白和猪圆 环病毒Cap蛋白的重组伪狂犬病病毒株的方法;
本发明的目的之三是将所构建的达猪瘟病毒E2蛋白和猪圆环病毒 Cap蛋白的重组伪狂犬病病毒株应用于制备预防猪伪狂犬病、猪瘟病或/ 和猪圆环病毒所导致疾病疫苗。
本发明的上述目的是通过以下技术方案来实现的:
本发明提供了一株表达猪瘟病毒E2蛋白和猪圆环病毒Cap蛋白的重 组伪狂犬病病毒株,该重组伪狂犬病病毒株以PRV TJ株基因组为骨架, 在PRV TJ株基因组中插入CSFVE2基因和PCV2 Cap基因;优选的,该 重组伪狂犬病病毒株以缺失了TK和gE/gI基因的PRVTJ株基因组为骨 架,将CSFV E2基因插入PRV TJ株基因组US9基因,另外,将猪圆环 病毒Cap基因与PRV TJ株gG(US4)基因的C末端融合。
本发明将所构建的表达猪瘟病毒E2蛋白和猪圆环病毒Cap蛋白的重 组伪狂犬病病毒株提交到中国普通微生物菌种保藏管理中心进行保藏,其 保藏信息如下:
保藏编号:CGMCC No.18220;
分类命名:表达猪圆环病毒CAP蛋白和猪瘟病毒E2蛋白的重组伪狂 犬病病毒;
保藏时间:2019年10月28日;
保藏单位:中国微生物菌种保藏管理委员会普通微生物中心;
保藏地址:北京市朝阳区北辰西路1号院,中国科学院微生物研究所。
本发明进一步提供了一种构建所述表达猪瘟病毒E2蛋白和猪圆环病 毒Cap蛋白的重组伪狂犬病病毒株的方法,包括:
(1)将PRV TJ株的fosmid系统中含TK和gE/gI基因的粘粒进行缺 失突变;
(2)将猪圆环病毒Cap蛋白基因与PRV TJ株的fosmid系统中 gG(US4)基因融合,在PRV TJ株的US9基因中插入猪瘟病毒E2蛋白基因 表达框;
(3)将fosmid质粒转染细胞,拯救病毒,即得。
其中,步骤(3)中将fosmid-f-ΔTK、fosmid-s-ΔgE/gI、 fosmid-s-ΔgE/gI-US9E2-US4Cap和两个包含其余PRVTJ基因组的fosmid 质粒共转染Vero细胞,拯救病毒。
本发明通过Red/ET重组介导的fosmid基因操纵系统,对TK和gE/gI 基因部分缺失,Cap与gG(US4)融合,在US9插入了E2表达框,构建了 一株达猪瘟病毒E2蛋白和猪圆环病毒Cap蛋白的重组伪狂犬病病毒株 rPRVTJ-delgE/gI/TK-E2-Cap。
该重组伪狂犬病病毒株rPRVTJ-delgE/gI/TK-E2-Cap在体外的复制和 生长动力学与亲本病毒相当,而且外源基因的插入不影响该重组伪狂犬病 病毒株的遗传稳定性;用该重组伪狂犬病病毒株免疫猪,可诱导出较强的 PRV特异性体液免疫应答,未显示出任何PRV特异性的临床症状;因此, 将本发明构建的重组伪狂犬病病毒株rPRVTJ-delgE/gI/TK-E2-Cap应用于 制备预防猪伪狂犬病、猪瘟病或/和猪圆环病毒所导致疾病疫苗。
由此,本发明提供了将所构建的重组伪狂犬病病毒株 rPRVTJ-delgE/gI/TK-E2-Cap应用于制备预防猪伪狂犬病、猪瘟病或猪圆 环病毒所导致疾病疫苗中的用途,包括:
(1)重组伪狂犬病病毒株rPRVTJ-delgE/gI/TK-E2-Cap在制备预防猪 伪狂犬病疫苗中的用途;
(2)重组伪狂犬病病毒株rPRVTJ-delgE/gI/TK-E2-Cap在制备预防猪 瘟病疫苗中的用途;
(3)重组伪狂犬病病毒株rPRVTJ-delgE/gI/TK-E2-Cap在制备预防猪 圆环病毒所导致疾病疫苗中的用途;
(4)重组伪狂犬病病毒株rPRVTJ-delgE/gI/TK-E2-Cap在制备预防猪 伪狂犬病和猪瘟病疫苗中的用途;
(5)重组伪狂犬病病毒株rPRVTJ-delgE/gI/TK-E2-Cap在制备预防猪 伪狂犬病和猪圆环病毒所导致疾病疫苗中的用途;
(6)重组伪狂犬病病毒株rPRVTJ-delgE/gI/TK-E2-Cap在制备预防猪 瘟病和猪圆环病毒所导致疾病疫苗中的用途;
(7)重组伪狂犬病病毒株rPRVTJ-delgE/gI/TK-E2-Cap在制备预防猪 伪狂犬病、猪瘟病和猪圆环病毒所导致疾病疫苗中的用途。
本发明整体技术方案详述
本发明基于PRV TJ株fosmid拯救系统的缺失/插入构建PRV重组 毒。由于同源重组等传统方法是通过一系列空斑纯化获得纯化的重组病 毒,所以被认为是非常低效和费力。虽然BAC修饰是操作疱疹病毒的一 个高效工具,但它可能要耗费数月并且可能由于存在BAC载体序列导致 基因和表型改变。fosmid文库平台是构建重组病毒的高效工具,,该系统 中只包含病毒基因组DNA片段,与限制性酶处理产生片段相比,采取无 缝基因修饰得到的片段没有任何间隔。因此,为了增强基因操作的效率与 质量,来快速构建重组PRVs,本发明使用forsmid文库平台操作。
大量研究表明基因删除导致PRV复制能力降低,在鼻腔上皮组织 中,毒力是直接与病毒的增殖能力有关(Cong,X.;Lei,J.L.;Xia,S.L.;Wang, Y.M.;Li,Y.;Li,S.;Luo,Y.;Sun,Y.;Qiu,H.J.,Pathogenicity and immunogenicity of a gE/gI/TK gene-deletedpseudorabies virus variant in susceptible animals.Veterinary Microbiology2016,182,170-177.)。病毒复 制能力降低导致低水平的抗原表达和基因缺失突变体的免疫原性损伤 (Swenson,S.L.;McMillen,J.;Hill,H.T.,Evaluation of the safety andefficacy of a thymidine kinase,inverted repeat,gI,and gpX gene-deletedpseudorabies vaccine. Journal of Veterinary Diagnostic Investigation 1993,5,(3),341-346.)。与亲本毒 相比,理想的疫苗载体必须安全且外源基因插入不能干扰病毒免疫原性和 生长性能。
本发明通过Red/ET重组介导的fosmid基因操纵系统,对TK和gE/gI 基因部分缺失,Cap与gG(US4)融合,在US9插入了E2表达框,构建了 一株表达猪瘟病毒E2蛋白和猪圆环病毒Cap蛋白的重组伪狂犬病病毒株 rPRVTJ-delgE/gI/TK-E2-Cap。
试验结果表明,rPRVTJ-delgE/gI/TK-E2-Cap在体外的复制和生长动 力学与亲本病毒rPRVTJ-delgE/gI/TK相当,而且外源基因的插入不影响 rPRVTJ-delgE/gI/TK-E2-Cap的遗传稳定性。
用rPRVTJ-delgE/GI/TK-E2-Cap免疫猪,可诱导出较强的PRV特异 性体液免疫应答,未显示出任何PRV特异性的临床症状。
本发明所提供的基于fosmid系统构建了的重组病毒株 rPRVTJ-delgE/gI/TK-E2-Cap,实现了PRV基因组的无缝修饰,有效平衡 了重组病毒的免疫原性和毒力,该重组病毒株在体外表达CSFV E2蛋白 和PCV 2的Cap蛋白,遗传稳定,缺失/插入基因不影响重组病毒的表型。 兔和猪接种后,未发现临床症状,因此,进一步研究 rPRVTJ-delgE/gI/TK-E2-Cap的优化设计和构建作为候选疫苗是可行的。
附图说明
图1构建重组病毒rPRVTJ-delgE/gI/TK-E2-Cap的路线图;A.PRV-TJ 的基因组以及源自PRV-TJ毒株的5种fosmid粘粒;单字母代表fosmid 的名称和数字代表每个fosmid片段所在PRV-TJ毒株基因组中的位置; B.表示Fosmid-f缺失TK基因的修饰过程的示意图;C.Fosmid-s缺失gE 和gI基因的修饰过程的示意图;D.中间体Fosmid-s-ΔgE/gI的US9基因后 插入E2基因的修饰过程示意图;E.中间体Fosmid-s-ΔgE/gI-US9E2的US4 基因内插入Cap基因的修饰程序示意图;F.用于拯救 rPRVTJ-delgE/gI/TK-E2-Cap重组病毒的Fosmids组成。
图2重组病毒rPRVTJ-delgE/gI/TK-E2-Cap的拯救与鉴定;A.不同 PRV毒株在Vero或PK-15细胞的细胞病变(CPE);其中Vero细胞上的 病变是由fosmid粘粒组合物转染形成,用缺失一种fosmid的fosmid组合 转染组为阴性对照。PK-15细胞上的病变是由收集上述Vero细胞培养上 清液形成;B.重组病毒的透射电子显微镜照片,其中rPRVTJ-delgE/gI/TK和亲代病毒PRVTJ作为阳性对照;C.针对F1和F2代重组病毒靶基因TK、 gE/gI、US9(E2基因)和US4的PCR分析其中PRVTJ基因组DNA作为 阴性对照,无DNA模板作为空白对照。
图3重组病毒rPRVTJ-delgE/gI/TK-E2-Cap的E2和Cap表达的鉴定; A.E2和Cap的表达的IFA鉴定;抗E2 MAb HQ06和抗Cap MAb 36A9 作为一抗,FITC的山羊抗小鼠IgG为二抗显色;B.E2和Cap的表达的 Western印迹法检测;MAb HQ06和抗Cap Mab 36A9为一抗,IRDye 800CW标记的山羊抗小鼠二抗(Li-Cor)显色,GAPDH为内参。
图4重组病毒重组病毒rPRVTJ-delgE/gI/TK-E2-Cap的的复制动力学 和稳定性分析;A.一步生长曲线;B.噬斑测定;C.F5、F10、F15和F20 代重组病毒靶基因TK,gE/gI,E2和Cap基因的PCR鉴定;D.F5、F10、 F15和F20代重组病毒重组病毒E2和Cap表达的IFA抗E2单抗HQ06 和抗Cap-Mab 36A9作为一抗,FITC的山羊抗小鼠IgG作为二抗。
图5免疫后家兔各种器官大体病理学检测。
具体实施方式
以下结合具体实施例来进一步描述本发明,本发明的优点和特点将 会随着描述而更为清楚。但这些实施例仅是范例性的,并不对本发明的范 围构成任何限制。本领域技术人员应该理解的是,在不偏离本发明的精神 和范围下可以对本发明的细节和形式进行修改或替换,但这些修改和替换 均落入本发明的保护范围内。
试验例1重组伪狂犬病病毒株rPRVTJ-delgE/gI/TK-E2-Cap的拯救、鉴 定及有关性能测定
1材料和方法
1.1主要试剂
PRV变异株TJ株(PRV-TJ)由本实验室分离、鉴定和保存;非洲绿 猴肾细胞(Vero细胞)、猪肾细胞(PK-15细胞)由本实验室保存,用含 有10%胎牛血清(FBS)的DMEM培养基(Gibco)于37℃、5%CO2培 养箱中培养;大肠杆菌DH5α感受态细胞由本实验室制备和保存;pOK12 载体质粒购买于Novagene公司,pMD18-T SimpleVector质粒购于宝生物 (TaKaRa)公司;FITC标记的羊抗猪IgG荧光二抗购买于Sigma公司。
KpnⅠ、NcoⅠ和PstⅠ限制性内切酶购买于TaKaRa公司;低熔点琼 脂糖购自Invitrogen公司(UltraPureTM LMP Agarose,16520-050),所 有抗生素与L-阿拉伯糖(Sigma A-3256)均购买于Sigma公司;质粒小提 试剂盒购买于Epigenetics公司(ZR BACDNA Miniprep Kit,D4049); 质粒中提试剂盒购买于Qiagen公司(QIAfilter PlasmidMidi Kit,12243);X-tremeGENE HPDNA转染试剂购自Roche公司(Cat.No.06366546001)。所有引物均由擎科生物合成(表1)。
1.2构建重组E2表达质粒
E2基因以CSFV石门株基因组为模板,经PCR扩增获得;人类巨细 胞病毒(hCMV)启动子和PolyA序列以质粒pEGFP-N1为模板,经PCR 扩增获得;CMV、E2和PolyA基因片段经over-lap PCR扩增获得;CMV、 E2和PolyA片段的扩增引物中引入EcoR I和Xba I限制位点,经酶切连 接到pOK-12的相应位点,经测序确定正确的克隆命名为 pOK12-CMV-E2-PolyA。
1.3利用Red/ET技术缺失PRV TJ fosmid粘粒中TK和gE/gI基因
gE/gI和TK基因缺失的PRV毒株具有良好的免疫原性,以三基因缺 失弱毒株为载体,首先需要对PRV TJ株的fosmid系统中含TK和gE/gI 基因的粘粒进行缺失突变,详细的构建方案如图(图1A-C)所示。以缺 失TK基因为例,首先将fosmid-f和Red/ET质粒共同电转DH10B感受态 细胞,将转化后的菌100μL涂布于含氯霉素(Cm+)和四环素(Tet+)双 抗LB中,在30℃避光培养30-40h;挑取单克隆制备电转感受态细胞 DH10B-fosmid-f-Red/ET,PCR扩增含有TK基因两侧各50bp同源重组臂 的rpsL-neo基因片段,并将rpsL-neo基因片段电激转化至感受态细胞 DH10B-fosmid-f-Red/ET,转化后的菌100μL涂布于含氯霉素(Cm+)、 四环素(Tet+)和卡那霉素(Kan+)的三抗性平板上,在30三避光培养 30-40h,挑取单克隆进行PCR鉴定和测序,确定rpsL-neo已成功替换TK 基因,获得重组菌DH10B-fosmid-f-rpsL;将重组DH10B-fosmid-f-rpsL电 转至感受态细胞,PCR扩增含有TK基因缺失的两侧各50bp同源重组臂 的ΔTK片段;将ΔTK片段电激转化感受态细胞DH10b-fosmid-f-rpsL,转 化后的菌100uL涂布于含氯霉素(Cm+)、链霉素(Str+)的双抗性平板 上,在37双中避光培养18-24h,挑取单克隆进行PCR鉴定和测序,确 定ΔTK已成功替换rpsL-neo基因,获得重组菌DH10B-fosmid-f-ΔTK。以 缺失TK基因相似的方法缺失gE/gI基因,获得重组菌 DH10B-fosmid-s-ΔgE/gI。
1.4利用Red/ET技术在PRF TJ fosmid粘粒中插入E2和Cap基因
本试验在fosmid-s-ΔgE/gI粘粒的基础上将CSFV E2基因独立表达框 插入US9基因后,同时将Cap基因与US4基因的C末端融合(图1D和 E)。以质粒pOK12-CMV-E2-PolyA为模板,PCR扩增E2独立表达框的 基因片段,并在引物中引入50bp同源重组臂。PCV2的Cap经密码子优 化合成后,直接PCR扩增获得含50bp同源重组臂的基因片段。利用 Red/ET技术以TK基因缺失相同的方法将E2和Cap基插入 fosmid-s-ΔgE/gI粘粒的相应位置,经测序确定正确的克隆命名为 fosmid-s-ΔgE/gI-E2-Cap。
表1构建和鉴定重组病毒rPRVTJ-delgE/gI/TK-E2-Cap所用引物
1.5病毒的拯救
按照X-tremeGENE HP DNA转染试剂说明书,将纯化好的粘粒组合 fosmid-a、fosmid-f-ΔTK、fosmid-o、fosmid-q和fosmid-s-ΔgE/gI-E2-Cap 共转染至密度为90%的10cm细胞培养板Vero细胞中,每种粘粒的转染 剂量为2μg(图1F)。待细胞病变后反复冻融细胞,收获病毒,将拯救 的病毒命名为rPRVTJ-ΔTK/gE/gI-E2-Cap。
1.6重组病毒rPRVTJ-ΔTK/gE/gI-E2-Cap基因组的PCR鉴定
将拯救的病毒于-80℃/37℃条件下反复冻融后,在PK-15细胞上连续 传20代,提取每个毒株第20代病毒的DNA,应用特异性引物扩增TK、 gB/gE、Cap和E2基因片段,并对扩增片段进行测序分析。
1.7间接免疫荧光试验(IFA)和蛋白质免疫印迹(WB)
将拯救病毒接种密度为90%的PK-15细胞,48h后用抗PRV的多克 隆抗体进行IFA实验。具体操作如下:弃掉培养液,用PBS清洗细胞2 遍,然后用冷无水乙醇固定细胞,30min后加入猪源抗PRV抗体,在室 温下作用2h后用PBS洗涤细胞5次,并加入100倍稀释的FITC标记羊 抗猪IgG,于室温条件下避光作用1h,用PBS洗涤细胞5次,5min/次, 在倒置荧光显微镜下观察并拍照。
将拯救病毒接种密度为90%的PK-15细胞,48h后弃掉细胞培养液, 用PBS洗细胞两次,应用含蛋白裂解液(10mM HEPES,pH 7.4,150mM KCl,3mM MgCl2,1%NP-40)裂解细胞,提取细胞总蛋白,SDS-PAGE 电泳分离后,电转到硝酸纤维膜上,抗E2、Cap和GAPDH一抗孵育37℃ 孵育1h,IRDye 800CW标记的山羊抗小鼠二抗(Li-Cor)显色。
1.8重组病毒rPRVTJ-ΔTK/gE/gI-E2-Cap病毒粒子的电镜观察
将收获的rePRV-TJ病毒粒子3,000r/min离心10min,取上清液置于 新的1.5mL离心管中,13,000r/min离心10min。样品经2%磷钨酸负染 后,病毒粒子的形态通过透射电子显微镜检测。
1.9一步生长曲线
将病毒以感染复数(Multiplicity of infection,MOI)为10的剂量接 种于生长至单层的PK-15细胞,并在感染后0、4、8、12、16、20、24、 28、32、36、48、60和72h收获病毒。将不同时间点收获的病毒分别用 IFA测定病毒滴度,并绘制出病毒的生长曲线。
1.10重组病毒rPRVTJ-ΔTK/gE/gI-E2-Cap免疫家兔和猪
根据中国农业科学院哈尔滨兽医研究所《实验动物的护理和使用指 南》进行动物实验。将通过酶联免疫吸附试验(ELISA)和PCR证实无 CSFV、PRV和PCV2病原的35只6周龄家兔用于疫苗评估实验。所有家 兔随机分为7组,每组5只(表2)。A至C组分别肌肉注射(im),接 种剂量分别为107、106和105TCID50的rPRVTJ-delgE/gI/TK-E2-Cap;D 和E组感染剂量分别为105TCID50的rPRVTJ-delgE/gI/TK(或DMEM;F 组和G组在另一支笼子中分别接种一个剂量的C株疫苗和一个剂量的 PCV2疫苗。第一次免疫三周后,分别用与第一次免疫相同的疫苗和剂量 加强免疫。免疫6周后,从家兔身上取出各种组织样本,包括心脏、肝脏、 肺、肾脏和脾脏,并进行PCR和病理检测。
表2家兔免疫实验方案
15头6周龄的猪瘟、伪狂犬病及圆环病毒病抗原及抗体均为阴性的 仔猪随机分成三组,每组5只(表3)。A组接种106TCID50重组病毒 rPRVTJ-delgE/gI/TK-E2-Cap,B组接种106TCID50重组病毒 rPRVTJ-delgE/gI/TK,C组免疫DMEM。首次免疫后三周以相同剂量进行二免。
表3猪免疫实验设计
2.试验结果
2.1rPRVTJ-delgE/gI/TK-E2-Cap的拯救及鉴定
拯救rPRVTJ-delgE/gI/TK-E2-Cap共需要5个fosmid质粒,其中包括 fosmid-f-ΔTK、fosmid-s-ΔgE/gI、fosmid-s-ΔgE/gI-US9E2-US4Cap和两个 包含其余PRVTJ基因组的fosmid质粒。用X-treme GENE HP DNA转染 试剂分别将2μg的5个fosmid共转染到Vero细胞中用于拯救病毒。在转 染后的72-96h可观察到PRV感染形成的典型病变,并且将拯救出的病毒 感染PK-15细胞后仅24h就能观察到明显的细胞病变(图2A)。在电镜 下观察到rPRVTJ-delgE/gI/TK-E2-Cap与亲本毒具有相似的形态(图2B)。 同时,为了进一步确定E2和Cap基因准确的插入到了PRVTJ基因组相 应的位置,我们提取rPRVTJ-delgE/gI/TK-E2-Cap的基因组并用PCR扩增 相应的目的片段后进行测序鉴定(图2C)。
2.2验证E2和Cap蛋白的表达
首先,采用间接免疫荧光实验来鉴定rPRVTJ-delgE/gI/TK-E2-Cap感染 细胞的E2和Cap蛋白表达情况,结果表明E2和Cap蛋白均正常表达(图 3A)。此外,蛋白质免疫印迹的结果表明E2和Cap蛋白均大量表达(图 3B)。以上实验结果均验证了E2和Cap蛋白均能在rPRVTJ-delgE/gI/TK-E2-Cap感染的细胞中表达。
2.3rPRVTJ-delgE/gI/TK-E2-Cap生长动力学及病毒稳定性的鉴定
rPRVTJ-delgE/gI/TK-E2-Cap的生长动力学和空斑的形态和大小均与rPRVTJ-delgE/gI/TK相似,但二者的生长动力学和空斑大小都与亲本毒存 在明显差异(图4A和B),主要表现在病毒复制能力有所降低。为了鉴 定rPRVTJ-delgE/gI/TK-E2-Cap的稳定性,对其进行连续传代直至20代, 并对不同代次病毒的基因组进行目的基因扩增和测序,发现插入的E2和 Cap基因均稳定存在于PRVTJ基因组中(图4C)。同时也用间接免疫荧 光实验来进一步验证了其稳定表达(图4D)。
2.4rPRVTJ-delgE/gI/TK-E2-Cap在家兔上的安全性评估
用107、106和105TCID50剂量的rPRVTJ-delgE/gI/TK-E2-Cap对家兔 进行免疫,免疫后的家兔均未表现出任何伪狂犬病典型的临床特征。对免 疫后的家兔进行剖检后,各个脏器均未发现组织病理变化(图5),同时 对家兔各组织进行PRV核酸检测结果均为阴性。
2.5在家兔和猪上评价rPRVTJ-delgE/gI/TK-E2-Cap的免疫原性
以107TCID50剂量的rPRVTJ-delgE/gI/TK-E2-Cap免疫家兔后,能在 免疫后7d检测到PRV gB蛋白的特异性抗体,而以106TCID50和105TCID50剂量的rPRVTJ-delgE/gI/TK-E2-Cap免疫的家兔均在免疫后14d伪狂犬病 病毒gB抗体转阳(表4),但以上所有实验组的家兔血清中均未检测到 gE特异性的抗体。
除此之外,家猪的免疫实验表明,仅以106TCID50剂量的 rPRVTJ-delgE/gI/TK-E2-Cap免疫猪后,就能在免疫后7天检测到伪狂犬 病病毒gB蛋白的特异性抗体,并且其抗体水平与106TCID50剂量的 rPRVTJ-delgE/gI/TK免疫猪产生的抗体水平相当,同样在所有实验组中均 检测不到gE抗体的产生(表5)。阻断ELISA的结果表明, rPRVTJ-delgE/gI/TK-E2-Cap免疫后的家兔和猪均不能诱导产生E2和Cap 相应的抗体。
表4家兔血清中gB抗体水平
a与rPRVTJ-delgE/gI/TK-E2-cap的差异(107or 105TCID50)(P<0.05)
b与rPRVTJ-delgE/gI/TK-E2-cap的差异(107or 106TCID50)(P<0.05)
“—”没有检测.
表5猪血清中gB抗体水平
序列表
<110> 中国农业科学院哈尔滨兽医研究所(中国动物卫生与流行病学中心哈尔滨分中心)
<120> 表达猪瘟病毒E2蛋白和猪圆环病毒Cap蛋白的重组伪狂犬病病毒株及其构建方法和用途
<130> HLJ-2001-191022A
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<170> SIPOSequenceListing 1.0
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<211> 74
<212> DNA
<213> Artifical sequence
<400> 1
cgtgatctcc tcgccgcccg ggggcacggc ggcggcgagg aggcgcgccg ggcctggtga 60
tgatggcggg atcg 74
<210> 2
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<212> DNA
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cggcgcgcgc ccccagtcgt cgcgccagcg gcgccccgag ctcaggtagc tcagaagaac 60
tcgtcaagaa ggcg 74
<210> 3
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<400> 3
cgtgatctcc tcgccgcccg ggggcacggc ggcggcgagg aggcgcgccg agtcgcgcag 60
ctggcacagc 70
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cggcgcgcgc ccccagtcgt cgcgccagcg gcgccccgag ctcaggtagc gcgacgtgtt 60
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<212> DNA
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ccgcgatcgc gatcaccgc 19
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gcccacgcgt gcacctcgag 20
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cgcctgaggg gggcgaaggg gtatcgcctc ctgggcggtc ccgcggacgc ggcctggtga 60
tgatggcggg atcg 74
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<211> 74
<212> DNA
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tcggcggccg ggttcgagac gctcgtcggg acgggggcgc tggggtcaaa tcagaagaac 60
tcgtcaagaa ggcg 74
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<212> DNA
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<212> DNA
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gtgatcgtcg gcacgggcac 20
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<212> DNA
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ctgccggcgt cccacgcgg 19
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<212> DNA
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<212> DNA
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<212> DNA
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<212> DNA
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<210> 20
<211> 74
<212> DNA
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<212> DNA
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<212> DNA
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Claims (10)
1.一株表达猪瘟病毒E2蛋白和猪圆环病毒Cap蛋白的重组伪狂犬病病毒株,其特征在于,该重组伪狂犬病病毒株以PRV TJ株基因组为骨架,在PRV TJ株基因组中插入CSFV E2基因和PCV2 Cap基因。
2.按照权利要求1所述的重组伪狂犬病病毒株,其特征在于,该重组伪狂犬病病毒株以缺失了TK和gE/gI基因的PRV TJ株基因组为骨架,将CSFV E2基因插入PRV TJ株基因组US9基因,另外,将猪圆环病毒Cap基因与PRV TJ株gG(US4)基因的C末端融合。
3.按照权利要求1或2所述的重组伪狂犬病病毒株,其特征在于,其微生物保藏编号为:CGMCC No.18220。
4.一种构建权利要求1-3任何一项所述重组伪狂犬病病毒株的方法,包括:
(1)将PRV TJ株的fosmid系统中含TK和gE/gI基因的粘粒进行缺失突变;
(2)将猪圆环病毒Cap蛋白基因与PRV TJ株的fosmid系统中gG(US4)基因融合,在PRVTJ株的US9基因中插入猪瘟病毒E2蛋白基因表达框;
(3)将fosmid质粒转染细胞,拯救病毒,即得。
5.按照权利要求4所述的方法,其特征在于,步骤(3)中将fosmid-f-ΔTK、fosmid-s-ΔgE/gI、fosmid-s-ΔgE/gI-US9E2-US4Cap和两个包含其余PRVTJ基因组的fosmid质粒共转染Vero细胞,拯救病毒。
6.权利要求1-3任何一项所述重组伪狂犬病病毒株在制备预防伪狂犬病疫苗、猪圆环病毒所导致疾病疫苗或猪瘟病疫苗中的用途。
7.权利要求1-3任何一项所述重组伪狂犬病病毒株在制备预防伪狂犬病和猪瘟病疫苗中的用途。
8.权利要求1-3任何一项所述重组伪狂犬病病毒株在制备预防伪狂犬病和猪圆环病毒所导致的疾病疫苗中的用途。
9.权利要求1-3任何一项所述重组伪狂犬病病毒株在制备预防猪瘟病和猪圆环病毒所导致的疾病疫苗中的用途。
10.权利要求1-3任何一项所述重组伪狂犬病病毒株在制备预防伪狂犬病、猪瘟病和猪圆环病毒所导致的疾病疫苗中的用途。
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