MDGPV PT株E-box缺失感染性克隆的构建及生物学特性分析
|
摘要
为获得含有缺失突变体病毒全基因组的番鸭小鹅瘟病毒(MDGPV)PT株缺失病毒并分析其生物学特性,本试验以含有MDGPV PT株全长感染性DNA克隆的质粒pMDGPVPT为模板,通过重叠延伸PCR方法将5′末端倒置重复序列(5′-ITR)第287位E盒基序(E-box)缺失,获得感染性重组质粒pPT△E287。pPT△E287经卵黄囊接种转染9日龄番鸭胚后拯救出缺失病毒rPT△E287。生物学特性试验显示,rPT△E287感染番鸭胚后能够引起特征性病变,与其亲本病毒株PT相比,rPT△E287的毒价及其在番鸭胚中增殖能力有所下降。本试验为进一步了解5′-ITR中E-box的缺失与MDGPV致病力之间的关系奠定基础。
In order to construct recombinant muscovy duck-origin goose parvovirus(MDGPV) that contained the whole MDGPV(PT strain) genome but with E-box motif deleted and analyze its biological characteristics,the plasmid pMDGPVPT containing full-length infectious DNA clone of MDGPV PT strain was used as the template.Overlap extension PCR was utilized to delete the E-box motif at nucleotide position 287 on 5′ inverted terminal repeats(5′-ITR),thus the recombinant plasmid pPT△E287 was obtained.To accomplish this,the 9-day-old muscovy duck embryos were transfected with the deletion mutant via yolk sac inoculation,the deletion virus rPT△E287 was rescued.The experiments of biological characteristics indicated that rPT△E287 was still able to lead to muscovy duck embryo dwarf.Compared with its parental virus strain PT,the virulence of rPT△E287 and its proliferation ability in muscovy duck embryos was attenuated.This study laid the foundation for the further studies on the relationship between the E-box deletion in 5′-ITR and pathogenicity in MDGPV.
In order to construct recombinant muscovy duck-origin goose parvovirus(MDGPV) that contained the whole MDGPV(PT strain) genome but with E-box motif deleted and analyze its biological characteristics,the plasmid pMDGPVPT containing full-length infectious DNA clone of MDGPV PT strain was used as the template.Overlap extension PCR was utilized to delete the E-box motif at nucleotide position 287 on 5′ inverted terminal repeats(5′-ITR),thus the recombinant plasmid pPT△E287 was obtained.To accomplish this,the 9-day-old muscovy duck embryos were transfected with the deletion mutant via yolk sac inoculation,the deletion virus rPT△E287 was rescued.The experiments of biological characteristics indicated that rPT△E287 was still able to lead to muscovy duck embryo dwarf.Compared with its parental virus strain PT,the virulence of rPT△E287 and its proliferation ability in muscovy duck embryos was attenuated.This study laid the foundation for the further studies on the relationship between the E-box deletion in 5′-ITR and pathogenicity in MDGPV.
引文
[1] 宋爽,高旭,于清洋,等.鹅细小病毒VP3基因与鹅γ干扰素基因在重组禽痘病毒中的共表达[J].中国兽医学报,2017,37(5):811-814.
[2] 程晓霞,陈少莺,朱小丽,等.番鸭小鹅瘟病毒的分离与鉴定[J].福建农业学报,2008,23(4):355-358.
[3] 陈少莺,胡奇林,程晓霞,等.鹅细小病毒弱毒株选育的研究[J].中国预防兽医学报,2002,24(4):286-288.
[4] GLáVITS R,ZOLNAI A,SZABó E,et al.Comparative pathological studies on domestic geese (Anser anser domestica) and Muscovy ducks (Cairina moschata) experimentally infected with parvovirus strains of goose and Muscovy duck origin[J].Acta Vet Hung,2005,53(1):73-89.
[5] ZADORI Z,ERDEI J,KISARY J.Characteristics of the genome of goose parvovirus[J].Avian Pathol,1994,23(2):359-364.
[6] ZáDORI Z,STEFANCSIK R,RAUCH T,et al.Analysis of the complete nucleotide sequences of goose and muscovy duck parvoviruses indicates common ancestral origin with adeno-associated virus 2[J].Virology,1995,212(2):562-573.
[7] GU Z,PLAZA S,PERROS M,et al.NF-Y controls transcription of the minute virus of mice P4 promoter through interaction with an unusual binding site[J].J Virol,1995,69(1):239-246.
[8] WANG J,DUAN J,ZHU L,et al.Sequencing and generation of an infectious clone of the pathogenic goose parvovirus strain LH[J].Arch Virol,2015,160(3):711-718.
[9] 程晓霞,陈仕龙,陈少莺,等.番鸭细小病毒和鹅细小病毒的抗原相关性研究[J].福建农业学报,2013,28(9):869-871.
[10] XIAO S,CHEN S,CHENG X,et al.The newly emerging duck-origin goose parvovirus in China exhibits a wide range of pathogenicity to main domesticated waterfowl[J].Vet Microbiol,2017,203:252-256.
[11] ZHI N,ZáDORI Z,BROWN K E,et al.Construction and sequencing of an infection clone of the human parvovirus B19[J].Virology,2004,318(1):142-152.
[12] WANG X S,PONNAZHAGAN S,SRIVASTAVA A.Rescue and replication of adeno-associated virus type 2 as well as vector DNA sequences from recombinant plasmids containing deletions in the viral inverted terminal repeats:selective encapsidation of viral genomes in progeny virions[J].J Virol,1996,70(3):1668-1677.
[13] CHE X,BERARDUCCI B,SOMMER M,et al.The ubiquitous cellular transcriptional factor USF targets the varicella-zoster virus open reading frame 10 promoter and determines virulence in human skin xenografts in SCIDhu mice in vivo[J].J Virol,2007,81(7):3229-3239.
[14] CHEN C,GUO L,SHI M,et al.Modulation of IFN-γ receptor 1 expression by AP-2α influences IFN-γ sensitivity of cancer cells[J].Am J Pathol,2012,180(2):661-671.
[15] KISARY J.Interaction in replication between goose parvovirus strain B and duck plague herpesvirus[J].Arch Virol,1979,59(1/2):81-88.
[2] 程晓霞,陈少莺,朱小丽,等.番鸭小鹅瘟病毒的分离与鉴定[J].福建农业学报,2008,23(4):355-358.
[3] 陈少莺,胡奇林,程晓霞,等.鹅细小病毒弱毒株选育的研究[J].中国预防兽医学报,2002,24(4):286-288.
[4] GLáVITS R,ZOLNAI A,SZABó E,et al.Comparative pathological studies on domestic geese (Anser anser domestica) and Muscovy ducks (Cairina moschata) experimentally infected with parvovirus strains of goose and Muscovy duck origin[J].Acta Vet Hung,2005,53(1):73-89.
[5] ZADORI Z,ERDEI J,KISARY J.Characteristics of the genome of goose parvovirus[J].Avian Pathol,1994,23(2):359-364.
[6] ZáDORI Z,STEFANCSIK R,RAUCH T,et al.Analysis of the complete nucleotide sequences of goose and muscovy duck parvoviruses indicates common ancestral origin with adeno-associated virus 2[J].Virology,1995,212(2):562-573.
[7] GU Z,PLAZA S,PERROS M,et al.NF-Y controls transcription of the minute virus of mice P4 promoter through interaction with an unusual binding site[J].J Virol,1995,69(1):239-246.
[8] WANG J,DUAN J,ZHU L,et al.Sequencing and generation of an infectious clone of the pathogenic goose parvovirus strain LH[J].Arch Virol,2015,160(3):711-718.
[9] 程晓霞,陈仕龙,陈少莺,等.番鸭细小病毒和鹅细小病毒的抗原相关性研究[J].福建农业学报,2013,28(9):869-871.
[10] XIAO S,CHEN S,CHENG X,et al.The newly emerging duck-origin goose parvovirus in China exhibits a wide range of pathogenicity to main domesticated waterfowl[J].Vet Microbiol,2017,203:252-256.
[11] ZHI N,ZáDORI Z,BROWN K E,et al.Construction and sequencing of an infection clone of the human parvovirus B19[J].Virology,2004,318(1):142-152.
[12] WANG X S,PONNAZHAGAN S,SRIVASTAVA A.Rescue and replication of adeno-associated virus type 2 as well as vector DNA sequences from recombinant plasmids containing deletions in the viral inverted terminal repeats:selective encapsidation of viral genomes in progeny virions[J].J Virol,1996,70(3):1668-1677.
[13] CHE X,BERARDUCCI B,SOMMER M,et al.The ubiquitous cellular transcriptional factor USF targets the varicella-zoster virus open reading frame 10 promoter and determines virulence in human skin xenografts in SCIDhu mice in vivo[J].J Virol,2007,81(7):3229-3239.
[14] CHEN C,GUO L,SHI M,et al.Modulation of IFN-γ receptor 1 expression by AP-2α influences IFN-γ sensitivity of cancer cells[J].Am J Pathol,2012,180(2):661-671.
[15] KISARY J.Interaction in replication between goose parvovirus strain B and duck plague herpesvirus[J].Arch Virol,1979,59(1/2):81-88.