O型口蹄疫病毒样颗粒编码基因的真核表达载体构建及表达
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摘要
为了构建O型口蹄疫病毒(FMDV)病毒样颗粒编码基因的真核表达载体,将FMDV结构蛋白VP0、VP1、VP3的基因分别克隆到真核表达载体pVAX1中,再分别通过带有BsmBⅠ、EcoRⅠ和SalⅠ酶切位点的引物,分别对重组质粒pVAX-VP0、pVAX-VP1和pVAX-VP3扩增后进行酶切,用T4连接酶连接得到重组的真核表达载体pVAX-VP0VP1VP3。对重组真核表达载体pVAX-VP0VP1VP3进行PCR鉴定并测序。用脂质体将重组真核表达载体pVAX-VP0VP1VP3转染BHK-21细胞,用间接免疫荧光试验(IFA)及Western blot检测重组真核表达载体pVAX-VP0VP1VP3在细胞中的表达情况。结果表明,成功构建了重组真核表达载体pVAX-VP0VP1VP3,并在BHK-21细胞中得到表达。
To construct and express the eukaryotic expression vector of serotype O foot-and-mouth disease virus-like particles encoding gene,VP0,VP1 and VP3genes were cloned into the eukaryotic expression vector pVAX1 through the known FMDV structural protein gene sequences in this paper.Then,the recombinant plasmids pVAX-VP0,pVAX-VP1,pVAX-VP3 were amplified by using primers with BsmB Ⅰ,EcoR Ⅰ and Sal Ⅰ restriction sites respectively,the recombinant eukaryotic expression vector pVAXVP0VP1VP3 was obtained by ligating with T4 ligase.The recombinant eukaryotic expression vector pVAX-VP0VP1VP3 was identified by PCR and sent to Suzhou Jinzhizhi Biotechnology Co.,Ltd.for sequencing.The recombinant eukaryotic expression vector pVAX-VP0VP1VP3 was transfected into BHK cells by lipofectin transfection method and the expression of recombinant eukaryotic expression vector pVAX-VP0VP1VP3 in cells was detected by indirect immunofluorescence assay test(IFAT)and Western blot.The results showed that the recombinant eukaryotic expression vector pVAX-VP0VP1VP3 was constructed and expressed successfully in BHK-21 cells.
To construct and express the eukaryotic expression vector of serotype O foot-and-mouth disease virus-like particles encoding gene,VP0,VP1 and VP3genes were cloned into the eukaryotic expression vector pVAX1 through the known FMDV structural protein gene sequences in this paper.Then,the recombinant plasmids pVAX-VP0,pVAX-VP1,pVAX-VP3 were amplified by using primers with BsmB Ⅰ,EcoR Ⅰ and Sal Ⅰ restriction sites respectively,the recombinant eukaryotic expression vector pVAXVP0VP1VP3 was obtained by ligating with T4 ligase.The recombinant eukaryotic expression vector pVAX-VP0VP1VP3 was identified by PCR and sent to Suzhou Jinzhizhi Biotechnology Co.,Ltd.for sequencing.The recombinant eukaryotic expression vector pVAX-VP0VP1VP3 was transfected into BHK cells by lipofectin transfection method and the expression of recombinant eukaryotic expression vector pVAX-VP0VP1VP3 in cells was detected by indirect immunofluorescence assay test(IFAT)and Western blot.The results showed that the recombinant eukaryotic expression vector pVAX-VP0VP1VP3 was constructed and expressed successfully in BHK-21 cells.
引文
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[10] Borca,Pacheco,Holinka,et al.Role of arginine-56within the structural protein VP3of foot-and-mouth disease virus(FMDV)O1Campos in virus virulence[J].Virology,2012,422(1):37-45.
[11]Fuenmayor J,Gòdia F,Cervera L.Production of virus-like particles for vaccines[J].N Biotechnol,2017,39(PtB):174-180.
[12] Rodriguez L L,Gay C G.Development of vaccines toward the global control and eradication of foot-and-mouth disease[J].Expert Rev Vac,2011,10(3):377-387.
[13] Kushnir N,Streatfield S J,Yusibov V.Virus-like particles as a highly efficient vaccine platform:diversity of targets and production systems and advances in clinical development[J].Vaccine,2012,31(1):58-83.
[14] Jennings G T,Bachmann M F.The coming of age of virus-like particle vaccines[J].Biol Chem,2008,389(5):521-536.
[15] Deml L,Wild J,Wagner R.Virus-like particles:a novel tool for the induction and monitoring of both T-helper and cytotoxic T-lymphocyte activity[J].Meth Mol Med,2004,94(1):33-57.
[16] Falk M M,Grigera P R,Bergmann I E,et al.Foot-and-mouth disease virus protease 3Cinduces specific proteolytic cleavage of host cell histone H3[J].J Virol,1990,64(2):748-756.
[2] Grubman M J,Baxt B.Foot-and-mouth disease[J].Clin Microbiol Rev,2004,17(2):465-493.
[3] Jamal S M,Belsham G J.Foot-and-mouth disease:past,present and future[J].Vet Res,2013,44(1):116.
[4] Mohana Subramanian B,Madhanmohan M,Sriraman R,et al.Development of foot-and-mouth disease virus(FMDV)serotype O virus-like-particles(VLPs)vaccine and evaluation of its potency[J].Antiviral Res,2012,96(3):288-295.
[5] Guo H,Liu Z,Sun S,et al.Immune response in guinea pigs vaccinated with DNA vaccine of foot-and-mouth disease virus O/China99[J].Vaccine,2005,23(25):3236-3242.
[6] Li D,Wei J,Yang F,et al.Foot-and-mouth disease virus structural protein VP3degrades Janus kinase 1to inhibit IFN-gamma signal transduction pathways[J].Cell Cycle,2016,15(6):850-860.
[7] Guo H C,Sun S Q,Jin Y,et al.Foot-and-mouth disease viruslike particles produced by a SUMO fusion protein system in Escherichia coli induce potent protective immune responses in guinea pigs,swine and cattle[J].Vet Res,2013,44:48.https://doi.org/10.1186/1297-9716-44-48.
[8] Veerapen V P,Van A Z,Wigdorovitz A,et al.Novel expression of immunogenic foot-and-mouth disease virus-like particles in Nicotiana benthamiana[J].Virus Res,2017,244:213-217.
[9] Strohmaier K,Franze R,Adam K H.Location and characterization of the antigenic portion of the FMDV immunizing protein[J].J Gen Virol,1982,59(Pt2):295-306.
[10] Borca,Pacheco,Holinka,et al.Role of arginine-56within the structural protein VP3of foot-and-mouth disease virus(FMDV)O1Campos in virus virulence[J].Virology,2012,422(1):37-45.
[11]Fuenmayor J,Gòdia F,Cervera L.Production of virus-like particles for vaccines[J].N Biotechnol,2017,39(PtB):174-180.
[12] Rodriguez L L,Gay C G.Development of vaccines toward the global control and eradication of foot-and-mouth disease[J].Expert Rev Vac,2011,10(3):377-387.
[13] Kushnir N,Streatfield S J,Yusibov V.Virus-like particles as a highly efficient vaccine platform:diversity of targets and production systems and advances in clinical development[J].Vaccine,2012,31(1):58-83.
[14] Jennings G T,Bachmann M F.The coming of age of virus-like particle vaccines[J].Biol Chem,2008,389(5):521-536.
[15] Deml L,Wild J,Wagner R.Virus-like particles:a novel tool for the induction and monitoring of both T-helper and cytotoxic T-lymphocyte activity[J].Meth Mol Med,2004,94(1):33-57.
[16] Falk M M,Grigera P R,Bergmann I E,et al.Foot-and-mouth disease virus protease 3Cinduces specific proteolytic cleavage of host cell histone H3[J].J Virol,1990,64(2):748-756.