基因Ⅱ、Ⅶ型二价新城疫病毒样颗粒的制备和鉴定
|
摘要
将基因Ⅶ型新城疫NA-1株F、HN基因的胞外域与基因Ⅱ型新城疫Lasota株F、HN基因的胞内域及跨膜域进行融合,并命名为rNA-1 F/HN。利用昆虫杆状病毒表达系统构建含有新城疫LaSota株M、F、HN等3个结构基因及rNA-1F、rNA-1 HN基因的重组杆状病毒rBV-LaSota M、rBV-LaSota F、rBV-rNA-1 F、rBV-LaSota HN、rBV-rNA-1 HN。将几种重组杆状病毒共感染Sf9细胞96h后收取上清,经超速离心及蔗糖密度离心纯化病毒样粒子(virus-like particles,VLPs),并利用Western blot方法和透射电子显微镜技术检测,结果显示目的蛋白均正确表达且组装成与野生型NDV形态大小相似的VLPs。结果表明:成功制备了新城疫标准疫苗株(LaSota株)及流行强毒株(NA-1株)二价NDV VLPs,为当前新城疫的防控提供新策略。
Newcastle disease(ND)is a highly contagious infectious disease which seriously endangering the poultry industry.Vaccine immunization is the main way to prevent Newcastle disease based on attenuated vaccine and inactivated vaccine,but it can not meet the purification requirements put forward by the《National Newcastle Disease Prevention and Control Guidance(2017-2020)》.Virus-like particles(VLPs)are hollow particles assembled from the structural proteins of the virus and do not contain viral nucleic acids which can be as a new candidate vaccine.In this experiment,the extracellular domain of Fand HN gene of typeⅦ Newcastle disease NA-1 gene was fused with the intracellular and transmembrane domains of Fand HN ofⅡtype Newcastle disease LaSota strain and named as rNA-1 F/HN.Five recombinant baculoviruses of rBV-Lasota M,rBV-Lasota F,rBV-rNA-1 F,rBV-LaSota HN and rBV-rNA-1 HN were constructed by the insect baculovirus expression system.The several recombinant baculoviruses were co-infected with Sf9 cells for 96 h,and the supernatant was collected.The VLPs were purified by sucrose density centrifugation and detected by Western blot and transmission electron microscopy.The results showed that the target proteins were correctly expressed and assembled into virions with similar size to wild NDV which indicating that the bivalent NDV VLPs were successfully prepared and provided a new strategy for the prevention and control of Newcastle disease.
Newcastle disease(ND)is a highly contagious infectious disease which seriously endangering the poultry industry.Vaccine immunization is the main way to prevent Newcastle disease based on attenuated vaccine and inactivated vaccine,but it can not meet the purification requirements put forward by the《National Newcastle Disease Prevention and Control Guidance(2017-2020)》.Virus-like particles(VLPs)are hollow particles assembled from the structural proteins of the virus and do not contain viral nucleic acids which can be as a new candidate vaccine.In this experiment,the extracellular domain of Fand HN gene of typeⅦ Newcastle disease NA-1 gene was fused with the intracellular and transmembrane domains of Fand HN ofⅡtype Newcastle disease LaSota strain and named as rNA-1 F/HN.Five recombinant baculoviruses of rBV-Lasota M,rBV-Lasota F,rBV-rNA-1 F,rBV-LaSota HN and rBV-rNA-1 HN were constructed by the insect baculovirus expression system.The several recombinant baculoviruses were co-infected with Sf9 cells for 96 h,and the supernatant was collected.The VLPs were purified by sucrose density centrifugation and detected by Western blot and transmission electron microscopy.The results showed that the target proteins were correctly expressed and assembled into virions with similar size to wild NDV which indicating that the bivalent NDV VLPs were successfully prepared and provided a new strategy for the prevention and control of Newcastle disease.
引文
[1]贺奋义.新城疫病毒分子生物学研究进展[J].畜牧兽医杂志,2013,32(5):35-39.
[2]中华人民共和国农业部兽医局.国家新城疫防治指导意见(2017-2020年)[J].农村实用技术,2017(7):8-9.
[3]MCGINNES L W,PANTUA H,LALIBERTE J P,et al.Assembly and biological and immunological properties of Newcastle disease virus-like particles[J].JVirol,2010,84(9):4513-4523.
[4]NOAD R,ROY P.Virus-like particles as immunogens[J].Trends Microbiol,2003,11(9):438-444.
[5]O′NEAL C M,CRAWFORD S E,ESTES M K,et al.Rotavirus VLPs administered mucosally induce protective immunity[J].J Virol,1997,71(11):8707-8717.
[6]MANOLOVA V,FLACE A,BAUER M,et al.Nanoparticles target distinct dendritic cell populations according to their size[J].Eur J Immunol,2008,38(5):1404-1413.
[7]BUONAGURO L,TAGLIAMONTE M,TORNE-SELLO M L,et al.Developments in virus-like particle-based vaccines for infectious diseases and cancer[J].Expert Rev Vaccines,2011,10(11):1569-1583.
[8]常旭东.鸡新城疫病毒性脑炎的中枢神经系统基因表达谱的研究[D].陕西杨凌:西北农林科技大学,2015.
[9]DE MARTEL C,FERLAY J,FRANCESCHI S,et al.Global burden of cancers attributable to infections in2008:a review and synthetic analysis[J].Lancet Oncol,2012,13(6):607-615.
[10]EINSTEIN M H,BARON M,LEVIN M J,et al.Comparative immunogenicity and safety of human papillomavirus(HPV)-16/18 vaccine and HPV-6/11/16/18vaccine:follow-up from months 12-24in a Phase III randomized study of healthy women aged18-45years[J].Human Vaccines,2011,7(12):1343-1358.
[11]赵晓云,乔绪稳,陈瑾,等.利用E.coli表达猪圆环病毒2型Cap蛋白生产病毒样颗粒疫苗[J].中国农业科学,2015,48(5):976-986.
[12]BOSIO C M,MOORE B D,WARFIELD K L,et al.Ebola and Marburg virus-like particles activate human myeloid dendritic cells[J].Virology,2004,326(2):280-287.
[13]GRILLBERGER L,KREIL T R,NASR S,et al.E-merging trends in plasma-free manufacturing of recombinant protein therapeutics expressed in mammalian cells[J].Biotechnol J,2010,4(2):186-201.
[14]MARTELLI P,FERRARI L,MORGANTI M,et al.One dose of a porcine circovirus 2subunit vaccine induces humoral and cell-mediated immunity and protects against porcine circovirus-associated disease under field conditions[J].Vet Microbiol,2011,149(3/4):339-351.
[15]SKOUNTZOU I,QUAN F S,GANGADHARA S,et al.Incorporation of glycosylphosphatidylinositol-anchored granulocyte macrophage colony-stimulating factor or CD40ligand enhances immunogenicity of chimeric simian immunodeficiency virus-like particles[J].J Virol,2007,81(3):1083-1094.
[2]中华人民共和国农业部兽医局.国家新城疫防治指导意见(2017-2020年)[J].农村实用技术,2017(7):8-9.
[3]MCGINNES L W,PANTUA H,LALIBERTE J P,et al.Assembly and biological and immunological properties of Newcastle disease virus-like particles[J].JVirol,2010,84(9):4513-4523.
[4]NOAD R,ROY P.Virus-like particles as immunogens[J].Trends Microbiol,2003,11(9):438-444.
[5]O′NEAL C M,CRAWFORD S E,ESTES M K,et al.Rotavirus VLPs administered mucosally induce protective immunity[J].J Virol,1997,71(11):8707-8717.
[6]MANOLOVA V,FLACE A,BAUER M,et al.Nanoparticles target distinct dendritic cell populations according to their size[J].Eur J Immunol,2008,38(5):1404-1413.
[7]BUONAGURO L,TAGLIAMONTE M,TORNE-SELLO M L,et al.Developments in virus-like particle-based vaccines for infectious diseases and cancer[J].Expert Rev Vaccines,2011,10(11):1569-1583.
[8]常旭东.鸡新城疫病毒性脑炎的中枢神经系统基因表达谱的研究[D].陕西杨凌:西北农林科技大学,2015.
[9]DE MARTEL C,FERLAY J,FRANCESCHI S,et al.Global burden of cancers attributable to infections in2008:a review and synthetic analysis[J].Lancet Oncol,2012,13(6):607-615.
[10]EINSTEIN M H,BARON M,LEVIN M J,et al.Comparative immunogenicity and safety of human papillomavirus(HPV)-16/18 vaccine and HPV-6/11/16/18vaccine:follow-up from months 12-24in a Phase III randomized study of healthy women aged18-45years[J].Human Vaccines,2011,7(12):1343-1358.
[11]赵晓云,乔绪稳,陈瑾,等.利用E.coli表达猪圆环病毒2型Cap蛋白生产病毒样颗粒疫苗[J].中国农业科学,2015,48(5):976-986.
[12]BOSIO C M,MOORE B D,WARFIELD K L,et al.Ebola and Marburg virus-like particles activate human myeloid dendritic cells[J].Virology,2004,326(2):280-287.
[13]GRILLBERGER L,KREIL T R,NASR S,et al.E-merging trends in plasma-free manufacturing of recombinant protein therapeutics expressed in mammalian cells[J].Biotechnol J,2010,4(2):186-201.
[14]MARTELLI P,FERRARI L,MORGANTI M,et al.One dose of a porcine circovirus 2subunit vaccine induces humoral and cell-mediated immunity and protects against porcine circovirus-associated disease under field conditions[J].Vet Microbiol,2011,149(3/4):339-351.
[15]SKOUNTZOU I,QUAN F S,GANGADHARA S,et al.Incorporation of glycosylphosphatidylinositol-anchored granulocyte macrophage colony-stimulating factor or CD40ligand enhances immunogenicity of chimeric simian immunodeficiency virus-like particles[J].J Virol,2007,81(3):1083-1094.