V5标记的猪圆环病毒2型Cap蛋白重组杆状病毒表达载体的构建
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摘要
试验旨在利用真核表达系统构建V5标记的猪圆环病毒2型(PCV2)Cap蛋白的表达载体。采用基因工程技术将V5标签引入到PCV2 ORF2基因C末端,并将标记基因定向克隆入pFastBacHTA载体,将pFastBacHTA-ORF2-V5重组转移载体转化大肠杆菌DH10Bac感受态细胞,使用脂质体介导法将鉴定后的重组杆状病毒质粒转染于Sf9细胞中;运用间接免疫荧光试验(IFA)、SDS-PAGE和Western blotting试验对Sf9细胞中V5标记Cap蛋白的表达进行验证。结果显示,本试验成功将V5标签引入到PCV2 ORF2基因末端,pFastBacHTAORF2-V5重组转移载体转化大肠杆菌DH10Bac感受态细胞后获得了Bacmid-ORF2-V5重组杆状病毒质粒;IFA、SDS-PAGE和Western blotting试验结果显示,在Sf9细胞中能检测到重组杆状病毒V5标记的PCV2Cap蛋白,具有良好的反应原性,说明本试验成功获得一株rAcMNPV Cap-V5重组杆状病毒。试验结果为PCV2标记亚单位疫苗的研制提供一定的理论依据。
The aim of the experiment was to construct the expression vector of V5-labeled porcine circovirus type 2(PCV2)Cap protein using eukaryotic expression system.The V5 tag was introduced into the end of the PCV2 ORF2 gene using genetic engineering,and the tagged gene was cloned into the pFastBacHTA vector.Recombinant transfer vector pFastBacHTA-ORF2-V5 was transformed into E.coli DH10 Bac.The identified recombinant bacmid was transfected into Sf9 cells using liposome-mediated method;Indirect immunofluorescence assay(IFA),SDS-PAGE and Western blotting were used to test the expression of V5-labeled Cap protein in Sf9 cells.The results showed that this experiment successfully introduced the V5 tag to the end of the PCV2 ORF2 gene.After the recombinant transfer vector pFastBacHTA-ORF2-V5 transformed into E.coli DH10 Bac,Bacmid-ORF2-V5 recombinant bacmid was obtained.The result of IFA,SDSPAGE and Western blotting showed that it could be expressed in Sf9 cells.The recombinant baculovirus V5-labeled PCV2 Cap protein was detected to have good reactogenicity,indicating that this experiment successfully obtained a rAcMNPV-Cap-V5 recombinant baculovirus.The test resultsprovided a theoretical basis for the development of PCV2 marker subunit vaccine.
The aim of the experiment was to construct the expression vector of V5-labeled porcine circovirus type 2(PCV2)Cap protein using eukaryotic expression system.The V5 tag was introduced into the end of the PCV2 ORF2 gene using genetic engineering,and the tagged gene was cloned into the pFastBacHTA vector.Recombinant transfer vector pFastBacHTA-ORF2-V5 was transformed into E.coli DH10 Bac.The identified recombinant bacmid was transfected into Sf9 cells using liposome-mediated method;Indirect immunofluorescence assay(IFA),SDS-PAGE and Western blotting were used to test the expression of V5-labeled Cap protein in Sf9 cells.The results showed that this experiment successfully introduced the V5 tag to the end of the PCV2 ORF2 gene.After the recombinant transfer vector pFastBacHTA-ORF2-V5 transformed into E.coli DH10 Bac,Bacmid-ORF2-V5 recombinant bacmid was obtained.The result of IFA,SDSPAGE and Western blotting showed that it could be expressed in Sf9 cells.The recombinant baculovirus V5-labeled PCV2 Cap protein was detected to have good reactogenicity,indicating that this experiment successfully obtained a rAcMNPV-Cap-V5 recombinant baculovirus.The test resultsprovided a theoretical basis for the development of PCV2 marker subunit vaccine.
引文
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[13]刘春菊,任炜杰,王志亮.杆状病毒表达系统在兽用亚单位疫苗领域应用的研究进展[J].中国畜牧兽医,2013,40(9):101-105.LIU C J,REN W J,WANG Z L.Research progress on application of baculovirus expression system in veterinary subunit vaccine field[J].China Animal Husbandry&Veterinary Medicine,2013,40(9):101-105.(in Chinese)
[14]史英力.以凡纳滨对虾β-actin基因启动子为元件的重组昆虫杆状病毒表达系统的建立[D].青岛:中国科学院研究生院(海洋研究所),2016.SHI Y L.Establishment of a recombinant baculovirus expression system under the control ofβ-actin promoter from the pacific white shrimp,Litopenaeus vannamei[D].Qingdao:University of Chinese Academy of Sciences(Institute of Oceanology),2016.(in Chinese)
[15]何庆东.猪圆环病毒2型ORF2基因在昆虫细胞中的表达及其免疫特性研究[D].南京:南京农业大学,2013.HE Q D.Expression and immunogenicity of the recombinant capsid protein of porcine circovirus type2in insect cells[D].Nanjing:Nanjing Agricultural University,2013.(in Chinese)
[16]朱鹏,张小飞,徐延伟,等.猪圆环病毒2型ORF2基因在Sf9细胞中表达及免疫原性研究[J].畜牧与兽医,2015,47(2):85-88.ZHU P,ZHANG X F,XU Y W,et al.Study on porcine circovirus virus type 2 ORF2gene expression in Sf9cells and immunogenicity[J].Animal Husbandry&Veterinary Medicine,2015,47(2):85-88.(in Chinese)
[17]许信刚,张宽,王志昇,等.PRRSV GP3蛋白和PCV-2Cap蛋白在杆状病毒囊膜表面的共展示及展示蛋白的小鼠免疫试验[J].中国兽医科学,2011,41(1):53-59.XU X G,ZHANG K,WANG Z S,et al.Baculovirus surface display of GP3 envelope glycoprotein of PRRSV and Cap protein of PCV-2and immunogenicity of the displayed proteins in mouse model[J].Chinese Veterinary Science,2011,41(1):53-59.(in Chinese)
[18]郭慧娟,樊翠,张英,等.猪圆环病毒2型CAP蛋白在flashBAC杆状病毒表达系统中的表达与鉴定[J].中国畜牧兽医,2016,43(7):1876-1883.GUO H J,FAN C,ZHANG Y,et al.Expression and identification of porcine circovirus type 2 capsid protein using flashBAC baculovirus expression system[J].China Animal Husbandry&Veterinary Medicine,2016,43(7):1876-1883.(in Chinese)
[19]张晓,尹秀凤,徐凯,等.猪圆环病毒2型Cap蛋白在杆状系统中的表达与纯化[J].中国动物传染病学报,2015,23(4):53-56.ZHANG X,YIN X F,XU K,et al.Expression and purification of capsid protein of porcine circovirus type 2by baculouirus[J].Chinese Journal of Animal Infectious Diseases,2015,23(4):53-56.(in Chinese)
[20]MUNRO S,PELHAM H R.Use of peptide tagging to detect proteins expressed from cloned genes:Deletion mapping functional domains of Drosophila hsp 70[J].Embo Journal,1984,3(13):3087-3093.
[21]BEACH N M,SMITH S M,RAMAMOORTHY S,et al.Chimeric porcine circoviruses(PCV)containing amino acid epitope tags in the C terminus of the capsid gene are infectious and elicit both anti-epitope tag antibodies and anti-PCV type 2neutralizing antibodies in pigs[J].Journal of Virology,2011,85(9):4591-4595.
[22]BATY D U,SOUTHERN J A,RANDALL R E.Sequence comparison between the haemagglutininneuraminidase genes of simian,canine and human isolates of simian virus 5[J].Journal of General Virology,1991,72(12):3103-3107.
[23]黄立平.猪圆环病毒2型抗原分型、构建中和表位分析及其V5表位标记重组毒株的构建[D].北京:中国农业科学院,2012.HUANG L P.Antigenic subtyping and conformational neutralizing epitopes analysis of procine circovirus type 2(PCV2)and construction of recombinant PCV2with V5epitope tag[D].Beijing:Chinese Academy of Agricultural Sciences,2012.(in Chinese)
[2]赵燕,喻正军.猪圆环病毒2型基因组DNA的结构与功能研究进展[J].中国畜牧兽医,2016,43(5):1176-1181.ZHAO Y,YU Z J.Advances on structure and function of genomic DNA of porcine circovirus type 2[J].China Animal Husbandry&Veterinary Medicine,2016,43(5):1176-1181.(in Chinese)
[3]王宪文.猪圆环病毒2型疫苗研究进展[J].中国畜牧兽医,2014,41(4):252-255.WANG X W.Research progress on porcine circovirus type 2vaccine[J].China Animal Husbandry&Veterinary Medicine,2014,41(4):252-255.(in Chinese)
[4]GEX N,WANG F,GUO X,et al.Porcine circovirus type 2and its associated diseases in China[J].Virus Research,2012,164(1-2):100-106.
[5]夏应菊,訾占超,蔡林,等.猪细环病毒和猪圆环病毒2型混合感染状况的调查[J].畜牧兽医学报,2012,43(3):424-430.XIA Y J,ZI Z C,CAI L,et al.The prevalence of torque teno sus virus and porcine circovirus 2co-infection in pigs[J].Chinese Journal of Animal and Veterinary Sciences,2012,43(3):424-430.(in Chinese)
[6]胡帅,周庆安,李军,等.2013—2014年广西主要猪病毒性传染病流行病学调查[J].中国畜牧兽医,2016,43(6):1618-1623.HU S,ZHOU Q A,LI J,et al.Epidemiological research of viral infectious diseases in Guangxi during2013to 2014[J].China Animal Husbandry&Veterinary Medicine,2016,43(6):1618-1623.(in Chinese)
[7]韦显凯,蒋晓霞,苏姣秀,等.2014年广西部分地区常见猪群疫病感染情况监测分析[J].中国畜牧兽医,2016,43(4):1079-1085.WEI X K,JIANG X X,SU J X,et al.Monitoring analysis of common swine disease in partial areas of Guangxi in 2014[J].China Animal Husbandry&Veterinary Medicine,2016,43(4):1079-1085.(in Chinese)
[8]赵晨辰,王敏,刘长辉,等.猪圆环病毒2型分子流行病学及疫苗研究进展[J].中国兽药杂志,2015,49(2):63-69.ZHAO C C,WANG M,LIU C H,et al.An update insights to the molecular epidemiology and vaccine of porcine circovirus type 2[J].Chinese Journal of Veterinary Drug,2015,49(2):63-69.(in Chinese)
[9]刘钊.贵州省猪圆环病毒2型的分离鉴定与遗传变异分析[D].贵阳:贵州大学,2016.LIU Z.Isolation and identification of porcine circovirus type 2in Guizhou province and genetic variation analysis of the isolated strains[D].Guizhou:Guizhou University,2016.(in Chinese)
[10]SARI D,GUPTA K,RAJ D B,et al.The multibac baculovirus/insect cell expression vector system for producing complex protein biologics[J].Advances in Experimental Medicine&Biology,2016,896:199-215.
[11]SMITH M D.Production of human beta interferon in insect cell infected with baculovirus express vector[J].Molecular and Cellular Biology,1983,3(12):2156-2165.
[12]MAEDA S,KAWAI T,OBINATA M,et al.Production of human alpha-interferon in silkworm using a baculovirus vector[J].Nature,1985,315(6020):592-594.
[13]刘春菊,任炜杰,王志亮.杆状病毒表达系统在兽用亚单位疫苗领域应用的研究进展[J].中国畜牧兽医,2013,40(9):101-105.LIU C J,REN W J,WANG Z L.Research progress on application of baculovirus expression system in veterinary subunit vaccine field[J].China Animal Husbandry&Veterinary Medicine,2013,40(9):101-105.(in Chinese)
[14]史英力.以凡纳滨对虾β-actin基因启动子为元件的重组昆虫杆状病毒表达系统的建立[D].青岛:中国科学院研究生院(海洋研究所),2016.SHI Y L.Establishment of a recombinant baculovirus expression system under the control ofβ-actin promoter from the pacific white shrimp,Litopenaeus vannamei[D].Qingdao:University of Chinese Academy of Sciences(Institute of Oceanology),2016.(in Chinese)
[15]何庆东.猪圆环病毒2型ORF2基因在昆虫细胞中的表达及其免疫特性研究[D].南京:南京农业大学,2013.HE Q D.Expression and immunogenicity of the recombinant capsid protein of porcine circovirus type2in insect cells[D].Nanjing:Nanjing Agricultural University,2013.(in Chinese)
[16]朱鹏,张小飞,徐延伟,等.猪圆环病毒2型ORF2基因在Sf9细胞中表达及免疫原性研究[J].畜牧与兽医,2015,47(2):85-88.ZHU P,ZHANG X F,XU Y W,et al.Study on porcine circovirus virus type 2 ORF2gene expression in Sf9cells and immunogenicity[J].Animal Husbandry&Veterinary Medicine,2015,47(2):85-88.(in Chinese)
[17]许信刚,张宽,王志昇,等.PRRSV GP3蛋白和PCV-2Cap蛋白在杆状病毒囊膜表面的共展示及展示蛋白的小鼠免疫试验[J].中国兽医科学,2011,41(1):53-59.XU X G,ZHANG K,WANG Z S,et al.Baculovirus surface display of GP3 envelope glycoprotein of PRRSV and Cap protein of PCV-2and immunogenicity of the displayed proteins in mouse model[J].Chinese Veterinary Science,2011,41(1):53-59.(in Chinese)
[18]郭慧娟,樊翠,张英,等.猪圆环病毒2型CAP蛋白在flashBAC杆状病毒表达系统中的表达与鉴定[J].中国畜牧兽医,2016,43(7):1876-1883.GUO H J,FAN C,ZHANG Y,et al.Expression and identification of porcine circovirus type 2 capsid protein using flashBAC baculovirus expression system[J].China Animal Husbandry&Veterinary Medicine,2016,43(7):1876-1883.(in Chinese)
[19]张晓,尹秀凤,徐凯,等.猪圆环病毒2型Cap蛋白在杆状系统中的表达与纯化[J].中国动物传染病学报,2015,23(4):53-56.ZHANG X,YIN X F,XU K,et al.Expression and purification of capsid protein of porcine circovirus type 2by baculouirus[J].Chinese Journal of Animal Infectious Diseases,2015,23(4):53-56.(in Chinese)
[20]MUNRO S,PELHAM H R.Use of peptide tagging to detect proteins expressed from cloned genes:Deletion mapping functional domains of Drosophila hsp 70[J].Embo Journal,1984,3(13):3087-3093.
[21]BEACH N M,SMITH S M,RAMAMOORTHY S,et al.Chimeric porcine circoviruses(PCV)containing amino acid epitope tags in the C terminus of the capsid gene are infectious and elicit both anti-epitope tag antibodies and anti-PCV type 2neutralizing antibodies in pigs[J].Journal of Virology,2011,85(9):4591-4595.
[22]BATY D U,SOUTHERN J A,RANDALL R E.Sequence comparison between the haemagglutininneuraminidase genes of simian,canine and human isolates of simian virus 5[J].Journal of General Virology,1991,72(12):3103-3107.
[23]黄立平.猪圆环病毒2型抗原分型、构建中和表位分析及其V5表位标记重组毒株的构建[D].北京:中国农业科学院,2012.HUANG L P.Antigenic subtyping and conformational neutralizing epitopes analysis of procine circovirus type 2(PCV2)and construction of recombinant PCV2with V5epitope tag[D].Beijing:Chinese Academy of Agricultural Sciences,2012.(in Chinese)