共表达H5亚型AIV HA基因和IBDV VP2基因重组DEV的构建及免疫效力评价
|
摘要
禽流感(Avian influenza,AI)和传染性法氏囊病(Infectious bursal disease,IBD)严重危害家禽养殖业,疫苗免疫是防治的主要手段之一。为探索以鸭瘟病毒(Duck enteritis virus,DEV)(又称鸭病毒性肠炎病毒)为载体,研制在鸡(Gallus gallus)中使用的AI-IBD二联疫苗的可行性,本研究利用overlap PCR及Gateway LR克隆技术,扩增出血凝素(hemagglutinin,HA)-内部核糖体进入位点(internal ribosome entry site,IRES)-病毒蛋白2(virus protein 2,VP2)和VP2-IRES-HA基因片段,并将其克隆入DEV多片段拯救系统T-us78Kan ccd B粘粒短独特区7(unique short region 7,US7)和US8位点之间,利用该系统成功拯救出两株共表达H5亚型禽流感病毒(Avian influenza virus,AIV)HA基因和传染性法氏囊病病毒(Infectious bursal disease virus,IBDV)VP2基因的重组病毒r DEV-HA/VP2和r DEV-VP2/HA,并对其进行了初步免疫效果评价。经系统体外实验表明,病毒传至20代,外源基因均能在重组病毒中稳定遗传和表达,且不影响载体病毒的增殖。但免疫效力实验显示,重组病毒以103半数组织培养感染剂量(tissue culture infective dose,TCID50)、104TCID50、105TCID50免疫鸡后14 d,r DEV-HA/VP2最高仅诱导对同源AIV强毒70%的免疫保护和对超强IBDV(very virulent IBDV,vv IBDV)30%的免疫保护,部分鸡中能检测到血凝抑制(hemagglutination inhibition,HI)抗体,但整体水平较低,IBD抗体未检测到;r DEV-VP2/HA最高仅诱导对vv IBDV 40%的免疫保护,不能诱导对同源AIV强毒的免疫保护,HI抗体和IBD抗体均未检测到。结果显示,以IRES串联基因构建鸭瘟载体禽流感传染性法氏囊病二联疫苗的策略不可行。本研究为以DEV为载体构建在鸡中使用的多联疫苗的研究提供参考资料。
Avain influenza(AI) and Infectious bursal disease(IBD) are harmful to poultry industry, and vaccination is one of the most effective method of controling these two diseases.In order to explore the feasibility of Duck enteritis virus(DEV)vector combined vaccine against AI and IBD that were used in chicken(Gallus gallus),two recombinant viruses that co-expressed hemagglutinin(HA)gene of H5 type Avian influenza virus(AIV)and virus protein 2(VP2)gene of Infectious bursal disease virus(IBDV)were constructed,and experiments were done to evaluate the immune efficacy of the recombinant viruses.HA gene from A/Chicken/Guizhou/4/2013(H5N1)(GZ/4)and VP2 gene from IBDV(HLJ-0504)were connected by internal ribosome entry site(IRES)sequence in different orders,HA-IRES-VP2 and VP2-IRES-HA.The two gene fragments were then inserted between unique short region 7(US7)and US8 genes of DEV genome in fosmid T-us78Kan ccd B which is overlapping fosmid DNAs rescue system.Using this system,the target gene between the US7 and US8 genes of the DEV genome were successfully inserted,and the two recombinant viruses r DEV-HA/VP2 and r DEV-VP2/HA were successfully constructed.The in vitro experiments showed that the inserted gene could be stably inherited and express without affecting replication of virus vector in cells.In order to evaluate the protective efficacy induced by recombinant virus in specific pathogen free(SPF)chicken,groups of ten 3-week-old SPF chicken were immunized with 103tissue culture infective dose(TCID50),104TCID50,105TCID50recombinant virus,and challenged with 100 median lethal dose(LD50)GZ/4or 100 LD50very virulent IBDV(vv IBDV)at 14 days post vaccination.Recombinant DEV with only HA gene of GZ/4 inserting between US7 and US8 genes(r DEV H5-8)and commercial IBDV vaccine Gt strain were immunized as control.The result showed that chicken which were immunized with 103TCID50,104TCID50,105TCID50r DEV-HA/VP2 induced low level of hemagglutination inhibition(HI)antibody,and no IBD antibody could be detected.The protective efficiency against H5 AIV was lower than 70%,and the protective efficiency against vv IBDV was lower than 30%,respectively.While,chicken which were immunized with 103TCID50,104TCID50,105TCID50r DEV-VP2/HA could not induce HI or IBD antibody.The protective efficiency against IBDV was lower than 40%,and the protective efficiency against H5 AIV was 0.However,chicken which were immunized with r DEV H5-8 or Gt strain could induce 100%protection against H5 AIV or vv IBDV.In conclusion,the two recombinant viruses r DEV-HA/VP2 and r DEV-VP2/HA could not induce complete protection against AIV or IBDV,and the strategy of using IRES to connect HA and VP2 genes to construct the DEV vector vaccine against AI and IBD was not feasible.This study provides a reference for the construction of DEV vectored multi vaccine in chicken
Avain influenza(AI) and Infectious bursal disease(IBD) are harmful to poultry industry, and vaccination is one of the most effective method of controling these two diseases.In order to explore the feasibility of Duck enteritis virus(DEV)vector combined vaccine against AI and IBD that were used in chicken(Gallus gallus),two recombinant viruses that co-expressed hemagglutinin(HA)gene of H5 type Avian influenza virus(AIV)and virus protein 2(VP2)gene of Infectious bursal disease virus(IBDV)were constructed,and experiments were done to evaluate the immune efficacy of the recombinant viruses.HA gene from A/Chicken/Guizhou/4/2013(H5N1)(GZ/4)and VP2 gene from IBDV(HLJ-0504)were connected by internal ribosome entry site(IRES)sequence in different orders,HA-IRES-VP2 and VP2-IRES-HA.The two gene fragments were then inserted between unique short region 7(US7)and US8 genes of DEV genome in fosmid T-us78Kan ccd B which is overlapping fosmid DNAs rescue system.Using this system,the target gene between the US7 and US8 genes of the DEV genome were successfully inserted,and the two recombinant viruses r DEV-HA/VP2 and r DEV-VP2/HA were successfully constructed.The in vitro experiments showed that the inserted gene could be stably inherited and express without affecting replication of virus vector in cells.In order to evaluate the protective efficacy induced by recombinant virus in specific pathogen free(SPF)chicken,groups of ten 3-week-old SPF chicken were immunized with 103tissue culture infective dose(TCID50),104TCID50,105TCID50recombinant virus,and challenged with 100 median lethal dose(LD50)GZ/4or 100 LD50very virulent IBDV(vv IBDV)at 14 days post vaccination.Recombinant DEV with only HA gene of GZ/4 inserting between US7 and US8 genes(r DEV H5-8)and commercial IBDV vaccine Gt strain were immunized as control.The result showed that chicken which were immunized with 103TCID50,104TCID50,105TCID50r DEV-HA/VP2 induced low level of hemagglutination inhibition(HI)antibody,and no IBD antibody could be detected.The protective efficiency against H5 AIV was lower than 70%,and the protective efficiency against vv IBDV was lower than 30%,respectively.While,chicken which were immunized with 103TCID50,104TCID50,105TCID50r DEV-VP2/HA could not induce HI or IBD antibody.The protective efficiency against IBDV was lower than 40%,and the protective efficiency against H5 AIV was 0.However,chicken which were immunized with r DEV H5-8 or Gt strain could induce 100%protection against H5 AIV or vv IBDV.In conclusion,the two recombinant viruses r DEV-HA/VP2 and r DEV-VP2/HA could not induce complete protection against AIV or IBDV,and the strategy of using IRES to connect HA and VP2 genes to construct the DEV vector vaccine against AI and IBD was not feasible.This study provides a reference for the construction of DEV vectored multi vaccine in chicken
引文
陈柳,余斌,倪征,等.2016.表达小鹅瘟病毒VP2蛋白重组鸭瘟病毒的构建及其生物学特性[J].中国农业科学49(14):2813-2821.(Chen L,Yu B,Ni Z,et al.2016Construction and characterization of a recombinan Duck enteritis virus expressing VP2 gene of Goose parvovi rus[J].Scientia Agricultura Sinica,49(14):2813-2821.)
陈普成.2009.鸭瘟病毒部分基因组序列的克隆及重组病毒转移载体的构建[D].硕士学位论文,中国农业科学院导师:田国彬.(Chen P C.2009.Cloning and analysi of partial genomic sequence of Duck enteritis virus and construction of transfer vector for recombinant virus[D]Thesis for M.S.,Chinese Academy of Agricultural Sci ences,Supervisor:Tian G B.)
胡潇,高轩,王明杰,等.2008.表达H5N1亚型禽流感病毒HA和NA基因的重组鸭瘟病毒的构建[J].中国动物检疫,25(05):22-25.(Xiao H,Gao X,Wang M J,et al2008.Construction of recombinant Duck plague virus ex pressing HA and NA gene of H5N1 subtype Avian influ enza virus[J].China Animal Health Inspection,25(05)22-25.)
祁小乐,高立,吴关,等.2011.鸡传染性法氏囊病病毒VP2基因突变对病毒体外复制的影响[J].畜牧兽医学报42(11):1577-1583.(Qi X L,Gao L,Wu G,et al2011.Influence of the mutations of VP2 on the replica tion efficiency of infectious bursal diease virus in vitro[J].Acta Veterinaria et Zootechnica Sinica,42(11)1577-1583.)
王玉龙.2015.表达IBV主要结构蛋白重组DEV的构建及其免疫保护性评价[D].硕士学位论文,甘肃农业大学,导师:刘胜旺,胡永浩.(Wang Y L.2015.Construc tion and evaluation on the immunoprotectivity of recom binant Duck enteritis virus expressing the major structur al proteins of infectious bronchitis virus[D].Thesis fo M.S.,Gansu Agricultural University,Supervisor:Liu SW,Hu Y H.)
于建立,王雷,魏波,等.2016.鸡传染性法氏囊病疫苗应用现状与研究进展[J].安徽农业科学,44(1):91-92,155(Yu J L,Wang L,Wei B,et al.2016.Application statu and research progress of infectious bursal disease vac cine in chicken[J].Journal of Anhui Agriculture,44(1)91-92,155.)
张瀚元,左青山,李明义,等.2010.表达I型鸭甲肝病毒VP1基因的重组鸭瘟病毒的构建[J].中国动物检疫,27(06):28-31.(Zhang H Y,Zuo Q S,Li M Y,et al.2010Construction of recombinant Duck plague virus express ing vp1 Gene of type-I Duck hepatitis virus[J].Chin Animal Health Inspection,25(05):22-25.)
Hermann M,Egbert M,Nicolas E,et al.2012.Current statu of vaccines against infectious bursal disease[J].Avian Pathology,41(2):133-139.
Li C,Bu Z,Chen H.2014.Avian influenza vaccines against H5N1'bird flu'[J].Trends in Biotechnology,32(3):147-156.
Liu J X,Chen P C,Jiang Y P,et al.2011.A Duck enteritis vi rus-vectored bivalent live vaccine provides fast and com plete protection against H5N1 Avian influenza virus in fection in ducks[J].Journal of Virology,85(21):1098910998.
Liu X,Wei S,Liu Y,et al.2013a.Recombinant Duck enteriti virus expressing the HA gene from goose H5 subtype Avi an influenza virus[J].Vaccine,31(50):5953-5959.
Liu J X,Chen P C,Jiang Y P,et al.2013b.Recombinant Duc enteritis virus works as a single-dose vaccine in broiler providing rapid protection against H5N1 influenza infec tion[J].Antiviral Research,97:329-333.
Lozano G,Martínez-Salas E.2015.Structural insights into vi ral IRES-dependent translation mechanisms[J].Curren Opinion in Virology,12:113-120.
Neumann G,Chen H,Gao G F,et al.2010.H5N1 influenz viruses:Outbreaks and biological properties[J].Cell Re search,20(1):51-61.
Peng Y S,Li X D,Zhu H B,et al.2017.Continual antigeni diversification in china leads to global antigenic com plexity of avian influenza H5N1 viruses[J].Scientifi Reports,7:43566.
Prandini F,Simon B,Jung A,et al.2016.Comparison of infec tious bursal disease live vaccines and a HVT-IBD vecto vaccine and their effects on the immune system of com mercial layer pullets[J].Avian Pathology,45(1):114125.
Zou Z,Hu Y,Liu Z,et al.2015.Efficient strategy for con structing duck enteritis virus-based live attenuated vac cine against homologous and heterologous H5N1 avian influenza virus and Duck enteritis virus infection[J].Vet erinary Research,46(1):1-15.
陈普成.2009.鸭瘟病毒部分基因组序列的克隆及重组病毒转移载体的构建[D].硕士学位论文,中国农业科学院导师:田国彬.(Chen P C.2009.Cloning and analysi of partial genomic sequence of Duck enteritis virus and construction of transfer vector for recombinant virus[D]Thesis for M.S.,Chinese Academy of Agricultural Sci ences,Supervisor:Tian G B.)
胡潇,高轩,王明杰,等.2008.表达H5N1亚型禽流感病毒HA和NA基因的重组鸭瘟病毒的构建[J].中国动物检疫,25(05):22-25.(Xiao H,Gao X,Wang M J,et al2008.Construction of recombinant Duck plague virus ex pressing HA and NA gene of H5N1 subtype Avian influ enza virus[J].China Animal Health Inspection,25(05)22-25.)
祁小乐,高立,吴关,等.2011.鸡传染性法氏囊病病毒VP2基因突变对病毒体外复制的影响[J].畜牧兽医学报42(11):1577-1583.(Qi X L,Gao L,Wu G,et al2011.Influence of the mutations of VP2 on the replica tion efficiency of infectious bursal diease virus in vitro[J].Acta Veterinaria et Zootechnica Sinica,42(11)1577-1583.)
王玉龙.2015.表达IBV主要结构蛋白重组DEV的构建及其免疫保护性评价[D].硕士学位论文,甘肃农业大学,导师:刘胜旺,胡永浩.(Wang Y L.2015.Construc tion and evaluation on the immunoprotectivity of recom binant Duck enteritis virus expressing the major structur al proteins of infectious bronchitis virus[D].Thesis fo M.S.,Gansu Agricultural University,Supervisor:Liu SW,Hu Y H.)
于建立,王雷,魏波,等.2016.鸡传染性法氏囊病疫苗应用现状与研究进展[J].安徽农业科学,44(1):91-92,155(Yu J L,Wang L,Wei B,et al.2016.Application statu and research progress of infectious bursal disease vac cine in chicken[J].Journal of Anhui Agriculture,44(1)91-92,155.)
张瀚元,左青山,李明义,等.2010.表达I型鸭甲肝病毒VP1基因的重组鸭瘟病毒的构建[J].中国动物检疫,27(06):28-31.(Zhang H Y,Zuo Q S,Li M Y,et al.2010Construction of recombinant Duck plague virus express ing vp1 Gene of type-I Duck hepatitis virus[J].Chin Animal Health Inspection,25(05):22-25.)
Hermann M,Egbert M,Nicolas E,et al.2012.Current statu of vaccines against infectious bursal disease[J].Avian Pathology,41(2):133-139.
Li C,Bu Z,Chen H.2014.Avian influenza vaccines against H5N1'bird flu'[J].Trends in Biotechnology,32(3):147-156.
Liu J X,Chen P C,Jiang Y P,et al.2011.A Duck enteritis vi rus-vectored bivalent live vaccine provides fast and com plete protection against H5N1 Avian influenza virus in fection in ducks[J].Journal of Virology,85(21):1098910998.
Liu X,Wei S,Liu Y,et al.2013a.Recombinant Duck enteriti virus expressing the HA gene from goose H5 subtype Avi an influenza virus[J].Vaccine,31(50):5953-5959.
Liu J X,Chen P C,Jiang Y P,et al.2013b.Recombinant Duc enteritis virus works as a single-dose vaccine in broiler providing rapid protection against H5N1 influenza infec tion[J].Antiviral Research,97:329-333.
Lozano G,Martínez-Salas E.2015.Structural insights into vi ral IRES-dependent translation mechanisms[J].Curren Opinion in Virology,12:113-120.
Neumann G,Chen H,Gao G F,et al.2010.H5N1 influenz viruses:Outbreaks and biological properties[J].Cell Re search,20(1):51-61.
Peng Y S,Li X D,Zhu H B,et al.2017.Continual antigeni diversification in china leads to global antigenic com plexity of avian influenza H5N1 viruses[J].Scientifi Reports,7:43566.
Prandini F,Simon B,Jung A,et al.2016.Comparison of infec tious bursal disease live vaccines and a HVT-IBD vecto vaccine and their effects on the immune system of com mercial layer pullets[J].Avian Pathology,45(1):114125.
Zou Z,Hu Y,Liu Z,et al.2015.Efficient strategy for con structing duck enteritis virus-based live attenuated vac cine against homologous and heterologous H5N1 avian influenza virus and Duck enteritis virus infection[J].Vet erinary Research,46(1):1-15.