大肠埃希菌外膜囊泡递呈猪瘟病毒E2蛋白的表达及其免疫效果评价
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摘要
为探讨大肠埃希菌外膜囊泡(outer membrane vesicles,OMVs)作为猪瘟病毒(classical swine fever virus,CSFV)E2蛋白的递呈载体所产生的免疫效应,将切除跨膜区后的CSFV E2基因连接到p BAD18-Cm-Cly A的C-端,转化E.coli DH5α,用0.2%阿拉伯糖诱导表达后,收集含E2蛋白的OMVs免疫家兔。免疫印迹表明,该OMVs可特异性地与组氨酸标签单抗结合。间接ELISA结果表明,重组OMVs可诱导兔体产生高水平的Ig G抗体。免疫后的家兔能很好地防御高剂量CSFV活疫苗攻击。说明大肠埃希菌OMVs递呈CSFV E2蛋白具有较好的免疫原性,为研究OMVs递呈CSFV E2蛋白疫苗奠定了基础。
In order to investigate the immune effect of E. coli outer membrane vesicles(OMVs) as presentation vector of classical swine fever virus E2 protein,the classical swine fever virus E2 gene was ligated to the C-terminus of p BAD18-Cm-Cly A after excision of the transmembrane region,and then transformed into E. coli DH5α. Inducted with 0. 2% arabinose,the OMVs containing the E2 protein were collected and the rabbits were immunized. Western blotting showed that the OMVs could specifically bind to CSFV antibody. Indirect ELISA showed that the recombinant OMVs could induce high level of Ig G antibody in rabbits. After immunization,rabbits could well protect the live CSFV challenge. All these results indicated that E. coli CSFV E2 protein had good immunogenicity,which laid the foundation for the research of OMVs presentation of classical swine fever virus E2 protein vaccine.
In order to investigate the immune effect of E. coli outer membrane vesicles(OMVs) as presentation vector of classical swine fever virus E2 protein,the classical swine fever virus E2 gene was ligated to the C-terminus of p BAD18-Cm-Cly A after excision of the transmembrane region,and then transformed into E. coli DH5α. Inducted with 0. 2% arabinose,the OMVs containing the E2 protein were collected and the rabbits were immunized. Western blotting showed that the OMVs could specifically bind to CSFV antibody. Indirect ELISA showed that the recombinant OMVs could induce high level of Ig G antibody in rabbits. After immunization,rabbits could well protect the live CSFV challenge. All these results indicated that E. coli CSFV E2 protein had good immunogenicity,which laid the foundation for the research of OMVs presentation of classical swine fever virus E2 protein vaccine.
引文
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[19]韩成钢.密码子优化的猪瘟病毒E2基因DNA疫苗的构建及其免疫效力评价[D].哈尔滨:中国农业科学院,2007.HAN C G.Construction of codon-optimized classical swine fever virus E2 DNA vaccine and its immunogenicity evaluation[D].Harbin:Chinese Academy of Agricultural Sciences,2007.(in Chinese with English abstract)
[2]龙虎,姚伦广,王姗姗,等.杆状病毒多基因表达系统介导的轮状病毒样颗粒表达与组装[J].南方医科大学学报,2010,30(7):1491-1495.LONG H,YAO L G,WANG S S,et al.Expression and assembly of rotavirus-like particles in insect cells mediated by recombinant Bombyx mori Multi Bac[J].Journal of Southern Medical University,2010,30(7):1491-1495.(in Chinese with English abstract)
[3]MOENNIG V.Introduction to classical swine fever:virus,disease and control policy[J].Veterinary Microbiology,2000,73(2/3):93-102.
[4]LIN M,LIN F,MALLORY M,et al.Deletions of structural glycoprotein E2 of classical swine fever virus strain alfort/187resolve a linear epitope of monoclonal antibody WH303 and the mini[J].Journal of Virology,2000,74(24):11619-11625.
[5]CLAVIJO A,LIN M,RIVA J,et al.Development of a competitive ELISA using a truncated E2 recombinant protein as antigen for detection of antibodies to classical swine fever virus[J].Research in Veterinary Science,2001,70(1):1-7.
[6]KATO H,TAKEUCHI O,AKIRA S.Cell type specific involvement of RIG-I in antiviral responses[J].Immunity,2005,23(1):19-28.
[7]LEE E Y,CHOI D S,KIM K P,et al.Proteomics in gramnegative bacterial outer membrane vesicles[J].Mass Spectrometry Reviews,2008,27(6):535-555.
[8]KAPARAKISLIASKOS M,FERRERO R L.Immune modulation by bacterial outer membrane vesicles[J].Nature Reviews Immunology,2015,15(6):375-387.
[9]KUEHN M J,KESTY N C.Bacterial outer membrane vesicles and the host-pathogen interaction[J].Genes&Development,2005,19(22):2645-2655.
[10]LUIJKX T,DIJKEN H V,ELS C V,et al.Heterologous prime-boost strategy to overcome weak immunogenicity of two serosubtypes in hexavalent Neisseria meningitidis outer membrane vesicle vaccine[J].Vaccine,2006,24(10):1569-1577.
[11]PETERSEN H,NIEVES W,RUSSELLLODRIGUE K,et al.Evaluation of a Burkholderia pseudomallei outer membrane vesicle vaccine in nonhuman primates[J].Procedia in Vaccinology,2014,8(8):38-42.
[12]NIEVES W,PETERSEN H,JUDY B M,et al.A Burkholderia pseudomallei outer membrane vesicle vaccine provides protection against lethal sepsis[J].Clinical&Vaccine Immunology,2014,21(5):747-754.
[13]CHEN D J,OSTERRIEDER N,METZGER S M,et al.Delivery of foreign antigens by engineered outer membrane vesicle vaccines[J].Proceedings of the National Academy of Sciences of the United States of America,2010,107(7):3099-3104.
[14]FAHIE M,ROMANO F B,CHISHOLM C,et al.A nonclassical assembly pathway of Escherichia coli pore-forming toxin cytolysin A[J].Journal of Biological Chemistry,2013,288(43):31042-31051.
[15]KIM J Y,DOODY A M,CHEN D J,et al.Engineered bacterial outer membrane vesicles with enhanced functionality[J].Journal of Molecular Biology,2008,380(1):51-66.
[16]王世杰,黄惟巍,舒聪妍,等.新型抗原递送载体:细胞溶素A融合目的蛋白整合的重组外膜囊泡的构建与鉴定[J].中国生物化学与分子生物学报,2016,32(11):1272-1278.WANG S J,HUANG W W,SHU C Y,et al.A novel antigen delivery vector:construction and identification of recombinant outer membrane vesicles with integration of target protein by cytolysin A[J].Chinese Journal of Biochemistry and Molecular Biology,2016,32(11):1272-1278.(in Chinese with English abstract)
[17]孙元,祁巧芬,梁冰冰,等.表达猪瘟病毒E2蛋白的重组腺病毒的构建及其在兔体内的免疫原性分析[J].生物工程学报,2008,24(10):1734-1739.SUN Y,QI Q F,LIANG B B,et al.Construction of recombinant adenovirus expressing classical swine fever virus E2protein and analysis of its immunogenicity in rabbits[J].Chinese Journal of Biotechnology,2008,24(10):1734-1739.(in Chinese with English abstract)
[18]李文良,毛立,杨蕾蕾,等.猪瘟病毒糖基化E2蛋白和E0蛋白的协同免疫保护作用[J].江苏农业学报,2015,31(2):357-361.LI W L,MAO L,YANG L L,et al.Synergistic immunoprotection of swine fever virus glycosylated E2 protein and E0protein[J].Jiangsu Journal of Agricultural Sciences,2015,31(2):357-361.(in Chinese with English abstract)
[19]韩成钢.密码子优化的猪瘟病毒E2基因DNA疫苗的构建及其免疫效力评价[D].哈尔滨:中国农业科学院,2007.HAN C G.Construction of codon-optimized classical swine fever virus E2 DNA vaccine and its immunogenicity evaluation[D].Harbin:Chinese Academy of Agricultural Sciences,2007.(in Chinese with English abstract)