携带绿色荧光蛋白基因重组猫泛白细胞减少症病毒的拯救

doi:10.16431/j.cnki.1671-7236.2020.06.025

摘要/Abstract

摘要： 本研究旨在构建携带绿色荧光蛋白（green fluorescent protein，GFP）的重组猫泛白细胞减少症病毒（feline panleukopenia virus，FPV），以便更快速有效的检测中和抗体。参照GenBank中GFP基因序列设计特异性引物，应用PCR技术扩增获得携带AgeⅠ和NheⅠ酶切位点的GFP基因，并对pFPV质粒进行定点突变，从而获得AgeⅠ和Nhe Ⅰ酶切位点，然后用AgeⅠ和Nhe Ⅰ同时双酶切pFPV和GFP基因，经4 ℃过夜连接、转化，成功构建携带GFP基因的重组猫泛白细胞减少症病毒质粒pFPV-GFP，应用脂质体转染法将重组质粒pFPV-GFP导入猫肾细胞（F81），利用GFP独特的发光机制可在荧光显微镜下对标记的重组病毒rFPV-GFP进行细胞内活体观察，利用Western blotting鉴定重组病毒rFPV-GFP中GFP的表达情况。结果显示，重组质粒pFPV-GFP转染F81细胞24 h时可观察到绿色荧光蛋白，且随着转染时间的延长观察到的绿色荧光蛋白数量增多，说明重组质粒pFPV-GFP成功转染到F81细胞中，Western blotting鉴定出GFP在重组病毒rFPV-GFP中稳定表达。本研究通过在pFPV上成功插入GFP并拯救出病毒，说明pFPV上可以插入外源基因，为其他外源基因在pFPV上的插入奠定基础，同时GFP的插入为FPV的研究提供了更加有利的研究工具，为FPV的基础研究以及中和抗体的快速检测奠定基础。

关键词: 猫泛白细胞减少症病毒(FPV); 绿色荧光蛋白(GFP); 重组病毒; 拯救

Abstract: The purpose of this study was to construct a recombinant feline panleukopenia virus (FPV) carrying green fluorescent protein (GFP) in order to detect neutralizing antibody more quickly and effectively.GFP specific primers were designed by referring to the GFP gene sequence in GenBank,and the GFP gene carrying AgeⅠ and NheⅠ digestion sites was amplified by PCR,and plasmid pFPV preserved in laboratory was subjected to site-directed mutagenesis to obtain the Age Ⅰ and Nhe Ⅰ restriction sites.pFPV and GFP genes were double-digested with Age Ⅰ and Nhe Ⅰ,and ligated at 4 ℃ overnight and transformed to obtain the recombinant plasmid pFPV-GFP.The recombinant plasmid pFPV-GFP was successfully transfected into feline kidney cells (F81) by Lipofection 3000.Using the unique luminescence mechanism of GFP,the labeled recombinant virus rFPV-GFP could be observed in vitro under fluorescence microscope,and Western blotting was used to identify the expression of GFP in the recombinant virus rFPV-GFP.The results showed that green fluorescent protein could be observed when the recombinant plasmid pFPV-GFP was transfected into F81 cells for 24 hours,and with the extension of transfection time,the amount of green fluorescent protein was increased,indicating that the recombinant plasmid pFPV-GFP was successfully transfected into F81 cells and GFP was stably expressed in the recombinant virus rFPV-GFP.In this study,GFP was successfully inserted into pFPV and rescued recombinant virus,indicating that foreign genes could be inserted into pFPV,laying a foundation for the insertion of other foreign genes into pFPV.At the same time,GFP insertion provided a more favorable research tool for the research of FPV,which laid the foundation for the basic research of FPV and the rapid detection of neutralization antibodies.

Key words: feline panleukopenia virus (FPV); green fluorescent protein (GFP); recombinant virus; rescue

中图分类号:

S852.65

殷娟斌, 周亚花, 殷相平, 张志东. 携带绿色荧光蛋白基因重组猫泛白细胞减少症病毒的拯救[J]. 中国畜牧兽医, 2020, 47(6): 1853-1860.

YIN Juanbin, ZHOU Yahua, YIN Xiangping, ZHANG Zhidong. Rescue of Recombinant Feline Panleukopenia Virus Carrying GFP Gene[J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(6): 1853-1860.

参考文献

[1] WANG F,WEI Y,ZHU C,et al.Novel parvovirus sublineage in the family of parvoviridae[J].Virus Genes,2010,41(2):305-308.
[2] 程楠.猫泛白细胞减少症病毒反向遗传学操作系统的建立[D].北京:中国农业科学院,2016. CHENG N.Establishment of feline panleukopenia virus revers genetic system[D].Beijing:Chinese Academy of Agricultural Sciences Dissertation,2016.(in Chinese)
[3] VANESSA R B.Feline panleukopenia:A re-emergent disease[J].The Veterinary Clinics of North America:Small Animal Practice,2019,49(4):651-670.
[4] 刘翠,殷相平.携带GFP基因的重组狂犬病病毒的拯救及其生物学特性研究[J].中国畜牧兽医,2018,45(7):1965-1971. LIU C,YIN X P.Rescue and biological characteristics analysis of recombinant rabies virus containing GFP gene[J].China Aniaml Husbandry & Veterinary Medicine,2018,45(7):1965-1971.(in Chinese)
[5] CRAMERI A,WHITEHORN E A,TATE E,et al.Improved green fluorescent protein by molecular evolution using DNA shuffling[J].Nature Biotechnology,1996,14(3):315-319.
[6] BALE S S,KWON S J,SHAH D A,et al.A GFP complementation system for monitoring and directing nanomaterial mediated protein delivery to human cellular organelles[J].Biotechnology & Bioengineering,2010,107(6):1040-1047.
[7] PINAUD F,DAHAN M.Targeting and imaging single biomolecules in living cells by complementation-activated light microscopy with split-fluorescent proteins[J].Proceedings of the National Academy of Sciences of the United States of America,2011,108(24):9735-9736.
[8] KAMIYAMA D,SEKINE S,BARSI-RHYNE B,et al.Versatile protein tagging in cells with split fluorescent protein[J].Nature Communications,2016,7:11046.
[9] KIM Y E,KIM Y N,KIM J A,et al.Green fluorescent protein nanopolygons as monodisperse supramolecular assemblies of functional proteins with defined valency[J].Nature Communications,2015,6:7134.
[10] 卫巧林.嵌合犬细小病毒VP2基因重组狂犬病毒的拯救与鉴定[D].北京:中国农业科学院,2016. WEI Q L.The rescue and identification of recombinant rabies virus containing VP2 gene of canine parvovirus[D].Beijing:Chinese Academy of Agricultural Sciences,2016.(in Chinese)
[11] BARKER I K,POVEY R C,VOIGT D R.Response of mink,skunk,red fox and raccoon to inoculation with mink virus enteritis,feline panleukopenia and canine parvovirus and prevalence of antibody to parvovirus in wild carnivores in Ontario[J].Canadian Journal of Comparative Medicine,1983,47(2):188-197.
[12] 赵新泰,吴祥甫,李载平,等.貂肠炎病毒基因的分子克隆和结构研究[J].病毒学报,1991,7(3):235-240. ZHAO X T,WU X F,LI Z P,et al.Cloning and characterization of mink ent eritis virus genome[J].Chinese Journal of Virology,1991,7(3):235-240.(in Chinese)
[13] 李刚,蔡宝祥,张振兴.猫泛白细胞减少症病毒的分离与鉴定[J].病毒学报,1985,4:60-64. LI G,CAI B X,ZHANG Z X.Isolation and identification of feline panleukopenia virus[J].Chinese Journal of Virology,1985,4:60-64.(in Chinese)
[14] 邢建民.猫细小病毒病的病原分离鉴定及其病理形态学的初步观察[D].吉林:吉林大学,2010. XING J M.Isolation and identification of feline parvovirus and its pathological observation[D].Jilin:Jilin University,2010.(in Chinese)
[15] HUEFFER K,PARRISH C R.Parvovirus host range,cell tropism and evolution[J].Current Opinion in Microbiology, 2003,6(4):392-398.
[16] FRANZO G,TUCCIARONE C M,CECCHINATO M,et al.Canine parvovirus type 2(CPV-2) and feline panleukopenia virus (FPV) codon bias analysis reveals a progressive adaptation to the new niche after the host jump[J].Molecular Phylogenetics & Evolution,2017,114:82-89.
[17] BARKER I K,POVEY R C,VOIGT D R.Response of mink,skunk,red fox and raccoon to inoculation with mink virus enteritis,feline panleukopenia and canine parvovirus and prevalence of antibody to parvovirus in wild carnivores in Ontario[J].Canadian Journal of Comparative Medicine,1983,47(2):188.
[18] 刘琪.猫泛白细胞减少症病毒的分离鉴定及VP2蛋白优势抗原区的原核表达[D].北京:中国农业科学院,2019. LIU Q.Isolation,identification of feline panleukopenia virus and prokaryotic expression of the dominant antigentic region of VP2 protein[D].Beijing:Chinese Academy of Agricultural Sciences,2019.(in Chinese)
[19] FLETRCHER K C,EUGSTER A K,SCHMIDT R E,et al.Parvovirus infection in maned wolves[J].Journal of the American Veterinary Medical Association,1979,175(9):897-900.
[20] CLEGG S R,COYNE K P,DAWSON S,et al.Canine parvovirus in asymptomatic feline carriers[J].Veterinary Microbiology,2012,157(1-2):78-85.
[21] DECARO N,BUONAVOGLIA C.Canine parvovirus——A review of epidemiological and diagnostic aspects,with emphasis on type 2C[J].Veterinary Microbiology, 2012,155(1):1-12.
[22] IKEDA Y,MOCHIZUKI M,NAITO R,et al.Predominance of canine parvovirus (CPV) in unvaccinated cat populations and emergence of new antigenic types of CPVs in cats[J].Virology,2000,278(1):13-19.
[23] BATTILANI M,BASSANI M,FORTI D,et al.Analysis of the evolution of feline parvovirus (FPV)[J].Veterinary Research Communications,2006,30:223-226.
[24] DECARO N,BUONAVOGLIA D,DESARIO C,et al.Characterisation of canine parvovirus strains isolated from cats with feline panleukopenia[J].Research in Veterinary Science,2010,89(2):275-278.
[25] MUKHOPADHYAY H K,NOOKALA M,THANGAMANI N R,et al.Molecular characterisation of parvoviruses from domestic cats reveals emergence of newer variants in India[J].Journal of Feline Medicine & Surgery, 2016,19(8):846-852
[26] 刘琪,史利军,袁维峰,等.猫泛白细胞减少症病毒北京株NS1基因克隆与生物信息学分析[J].中国畜牧兽医,2018,45(12):30-39. LIU Q,SHI L J,YUAN W F,et al.Cloning and bioinformatic analysis of NS1 gene of feline parvovirus in Beijing[J].China Aniaml Husbandry & Veterinary Medicine,2018,45(12):30-39.(in Chinese)
[27] PARRISH C R.Mapping specific functions in the capsid structure of canine parvovirus and feline panleukopenia virus using infectious plasmid clones[J].Virology,1991,183(1):195-205.