大熊猫犬瘟热病毒H基因重组山羊痘病毒的构建和鉴定
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摘要
本研究参照GenBank上已公布的大熊猫犬瘟热病毒(CDV)H编码的基因序列,对其抗原保守结构和识别区域进行修饰,将CDV保护性抗原H基因克隆到pMDTK-pEL载体中,然后运用酶切、连接将表达盒PELH构建到pTK-Eg载体中,从而获得重组转移载体质粒pTK-H-Eg。将重组转移载体质粒pTK-H-Eg与GTPV AV41株共转染BHK细胞,使其在细胞内发生同源重组,获得重组CDV的H基因的山羊痘病毒,然后在Vero细胞上加压筛选,利用gpt培养基筛选到稳定表达EGFP的重组病毒。通过PCR、免疫荧光以及Western Blot鉴定,证明CDVH基因成功构建到GTPV基因组中,并且在细胞中获得了正确表达。本研究获得了表达CDVH基因重组山羊痘病毒,并命名为vpTK-H-Eg。
In this study,the H gene sequence of the canine distemper virus(CDV)in giant panda was modified and synthesized based on the published coding sequences on GenBank.First the gene transfer vector pMDTK-PEL was constructed.The H gene from the CDV was cloned into the transfer vector pMDTKPEL through digestion of restriction enzyme,H Gene Expression Cassettes were inserted into the vector pTK-Eg,and we obtained the recombinant transfer vector plasmid pTK-H-Eg.The recombinant transfer vector plasmid was transfected into BHK cells that had been infected with the goatpox virus,through homologous recombination recombinant goatpox virus was generated.The recombinant goatpox virus containing the H gene of the goatpox virus cultured in Vero cells underwent pressure selection by adding mycophenolic acid to the culture.After purification and polymerase chain reaction,the immunofluorescence test and Western Blot demonstrated that the H gene of the CDV had been inserted into the goatpox virus and correctly expressed the H protein of the CDV in cells.We obtained the recombinant GTPV AV41 and name it"vpTK-H-Eg".
In this study,the H gene sequence of the canine distemper virus(CDV)in giant panda was modified and synthesized based on the published coding sequences on GenBank.First the gene transfer vector pMDTK-PEL was constructed.The H gene from the CDV was cloned into the transfer vector pMDTKPEL through digestion of restriction enzyme,H Gene Expression Cassettes were inserted into the vector pTK-Eg,and we obtained the recombinant transfer vector plasmid pTK-H-Eg.The recombinant transfer vector plasmid was transfected into BHK cells that had been infected with the goatpox virus,through homologous recombination recombinant goatpox virus was generated.The recombinant goatpox virus containing the H gene of the goatpox virus cultured in Vero cells underwent pressure selection by adding mycophenolic acid to the culture.After purification and polymerase chain reaction,the immunofluorescence test and Western Blot demonstrated that the H gene of the CDV had been inserted into the goatpox virus and correctly expressed the H protein of the CDV in cells.We obtained the recombinant GTPV AV41 and name it"vpTK-H-Eg".
引文
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[11]单芬,吴其锐,绉洁建,花福有,陈武.虎源犬瘟热病毒的分离鉴定及H基因的序列分析[J].野生动物学报,2018,39(1):035-040.
[12]Na Zhao,Meng Li,Jing Lou,Supen Wang,Shelan Liu,Shan Wang,Wenting Lyu,Lin Chen,Wen Su,Hua Ding,Hongxuan he.Impacts of canine distemper virus infection on the giant panda population from the perspective of gut microbiota[J].Sci Rep,2017,7:39954,1-10.
[13]Carmichael L E.Canine viral vaccines at a turning point-apersonal perspective[J].Adv Vet Med,1999;41:289-307
[14]Wolff J A,Budker V.The mechanism of naked DNA uptake and expression[J].Adv Genet,2005,54:3-20.
[15]Stephensen C B,Welter J,Thaker S R,Taylor J,Paoletti E.Canine distemper virus(CDV)infection of ferrets as a model for testing Morbillivirus vaccine strategies:NYVAC-and ALVAC-based CDV recombinants protect against symptomatic infection[J].J Virol,1997,71(2):1506-1513.
[16]Naldini L,Blomer U,Gallay P,Ory D,Mulliqan R,Gage F H,Verma I M,Trono D.In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector[J].Science,1996,272(5259):263-267.
[17]Kafri T,Blomer U,Peterson D A,Gage F H,Verma I M.Sustained expression of genes delivered directly into liver and muscle by lentiviral vectors[J].Nat Genet,1997,17(3):314-317.
[18]曲林茂,表达小反刍兽疫病毒H、F蛋白的重组山羊痘病毒的构建及免疫原性的研究[D].南京农业大学,2006.
[19]程小文,胡森,王喜军,鲍恩东,步志高.表达外源绿荧光蛋白重组山羊痘病毒的构建及纯化[J].农业生物技术学报,2006,14(5):798-802.
[20]郑敏,梁晟,郑彦梓,兰彬,韦显凯,苏娇秀,郑列丰,李常挺.TK基因重组缺陷山羊痘病毒构建及其生物学特性鉴定[J].南方农业学报,2013,44(9):1552-1557.
[21]Falkner F G,Moss B.Transient dominant selection of recombinant vaccinia viruses[J].J Virol,1990,64(6):3108-3111.
[2]Fauquet C M,Mayo M A,Maniloff J,Desselberger U,Ball L A.Virus Taxonomy:VIIIth Report of the International Committee on Taxonomy of Viruses[M].San Diego:Elsevier Academic Press,2005.
[3]何洪彬,夏咸柱.犬瘟热的诊断与免疫研究进展[J].动物医学进展,2001,22(1):12-14.
[4]王磊,王照,李天松,刘玉秀,冯娜,王胜乐,许薇薇,姜雪,高玉伟,王铁成,杨松涛,夏咸柱.小熊猫源犬瘟热病毒全基因序列的克隆及序列分析[J].中国畜牧兽医,2011,38(11):77-82.
[5]Deem S L,Spelman L H,Yates R A,Montali R J.Canine distemper in terrestrial carnivores:a review[J].J Zoo Wildl Med,2000,31(4):441-451.
[6]Yoshikawa Y,Ochikubo F,Matsubara Y,Tsuruoka H,Ishii M,Shirota K,Nomura Y,Sugiyama M,Yamanouchi K.Natural infection with canine distemper virus in a Japanese monkey(Macaca fuscata)[J].Vet Microbiol,1989,20(30):193-205.
[7]张晓明,单芬,陈武,彭仕明,李少基,李婉萍,黄勉.小熊猫犬瘟热病毒的分离鉴定及分子特点[J].畜牧与兽医,2013,45(10):60-63.
[8]Moss B.Vaccinia virus:a tool for research and vaccine development[J].Science,1991,252(5013),1662-1667.
[9]Goebel S J,Johnson G P,Perkus M E,Davis S W,Winslow J P,Paoletti E.The complete DNA seduence of vaccinia virus[J].Virology,1990,179(1):247-266.
[10]Moss B.Replicating and host-restricted non-replicating vaccine development[J].Dev Biol Stand,1994,82:55-63.
[11]单芬,吴其锐,绉洁建,花福有,陈武.虎源犬瘟热病毒的分离鉴定及H基因的序列分析[J].野生动物学报,2018,39(1):035-040.
[12]Na Zhao,Meng Li,Jing Lou,Supen Wang,Shelan Liu,Shan Wang,Wenting Lyu,Lin Chen,Wen Su,Hua Ding,Hongxuan he.Impacts of canine distemper virus infection on the giant panda population from the perspective of gut microbiota[J].Sci Rep,2017,7:39954,1-10.
[13]Carmichael L E.Canine viral vaccines at a turning point-apersonal perspective[J].Adv Vet Med,1999;41:289-307
[14]Wolff J A,Budker V.The mechanism of naked DNA uptake and expression[J].Adv Genet,2005,54:3-20.
[15]Stephensen C B,Welter J,Thaker S R,Taylor J,Paoletti E.Canine distemper virus(CDV)infection of ferrets as a model for testing Morbillivirus vaccine strategies:NYVAC-and ALVAC-based CDV recombinants protect against symptomatic infection[J].J Virol,1997,71(2):1506-1513.
[16]Naldini L,Blomer U,Gallay P,Ory D,Mulliqan R,Gage F H,Verma I M,Trono D.In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector[J].Science,1996,272(5259):263-267.
[17]Kafri T,Blomer U,Peterson D A,Gage F H,Verma I M.Sustained expression of genes delivered directly into liver and muscle by lentiviral vectors[J].Nat Genet,1997,17(3):314-317.
[18]曲林茂,表达小反刍兽疫病毒H、F蛋白的重组山羊痘病毒的构建及免疫原性的研究[D].南京农业大学,2006.
[19]程小文,胡森,王喜军,鲍恩东,步志高.表达外源绿荧光蛋白重组山羊痘病毒的构建及纯化[J].农业生物技术学报,2006,14(5):798-802.
[20]郑敏,梁晟,郑彦梓,兰彬,韦显凯,苏娇秀,郑列丰,李常挺.TK基因重组缺陷山羊痘病毒构建及其生物学特性鉴定[J].南方农业学报,2013,44(9):1552-1557.
[21]Falkner F G,Moss B.Transient dominant selection of recombinant vaccinia viruses[J].J Virol,1990,64(6):3108-3111.