家蚕核型多角体病毒表面展示猪流行性腹泻病毒S1蛋白及其黏膜免疫原性研究
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摘要
猪流行性腹泻病毒(porcine epidemic diarrhea virus,PEDV)可以通过呼吸道感染在猪群中传播,提示呼吸道粘膜也许可以作为疫苗免疫接种的位点。PEDV通过表面的纤突蛋白S识别受体并侵入宿主细胞,该蛋白质S1区(1～735 aa)是PEDV主要的中和位点以及受体结合域,参与病毒吸附入侵宿主细胞。利用家蚕核型多角体病毒在BmN细胞中表达融合蛋白S1-eGFP并展示于病毒表面,Western blot鉴定融合蛋白分子质量约为150 kD。以重组杆状病毒为免疫原雾化免疫BALB/c小鼠,结果表明免疫16 d后小鼠血清中S1特异性IgG抗体效价可达1∶3 200,血清和肺气管润洗液中都能检测到高水平的S1特异性sIgA,说明引起了较强的黏膜免疫反应;脾淋巴细胞增殖结果进一步表明重组杆状病毒雾化免疫可以有效地诱导小鼠产生细胞免疫。研究结果初步表明,表面展示S1的重组杆状病毒可以作为PEDV黏膜免疫候选疫苗,具有进一步开发研究的价值。
Porcine epidemic diarrhea virus(PEDV) can be transmitted in pig farms through respiratory tract infection, suggesting that respiratory tract mucosa may serve as a site for vaccination. PEDV recognizes the receptor through the surface spike protein S and invades the host cell. S1 region(1-735 aa) of this protein contains the main neutralizing epitopes and receptor binding domain of PEDV, which is closely related to virus antigenicity and adsorption invasion. The fusion protein S1-eGFP was expressed in BmN cells by silkworm baculovirus and displayed on the surface of the virus, and Western blot analysis indicated that the molecular mass of the protein was about 150 kD. The recombinant virus as immunogen was used to vaccinate BALB/c mice by nebulization. Our data showed that the titers of S1 specific IgG in mouse serum got up to 1∶ 3 200 on the 16 th day after vaccination, and a relatively high level of S1 specific sIgA could be detected in both serum and alveolar lavage fluid from immunized mice, sugges-ting that some extent of mucosal immune response was elicited to PEDV. Further spleen lymphocyte proliferation showed that recombinant baculovirus nebulization immunization could effectively induce mice to produce cellular immunity. The results of this study preliminarily indicated that the recombinant baculovirus displaying S1 could be used as a candidate vaccine for mucosal immune against PEDV infection, which has the value for further development and research.
Porcine epidemic diarrhea virus(PEDV) can be transmitted in pig farms through respiratory tract infection, suggesting that respiratory tract mucosa may serve as a site for vaccination. PEDV recognizes the receptor through the surface spike protein S and invades the host cell. S1 region(1-735 aa) of this protein contains the main neutralizing epitopes and receptor binding domain of PEDV, which is closely related to virus antigenicity and adsorption invasion. The fusion protein S1-eGFP was expressed in BmN cells by silkworm baculovirus and displayed on the surface of the virus, and Western blot analysis indicated that the molecular mass of the protein was about 150 kD. The recombinant virus as immunogen was used to vaccinate BALB/c mice by nebulization. Our data showed that the titers of S1 specific IgG in mouse serum got up to 1∶ 3 200 on the 16 th day after vaccination, and a relatively high level of S1 specific sIgA could be detected in both serum and alveolar lavage fluid from immunized mice, sugges-ting that some extent of mucosal immune response was elicited to PEDV. Further spleen lymphocyte proliferation showed that recombinant baculovirus nebulization immunization could effectively induce mice to produce cellular immunity. The results of this study preliminarily indicated that the recombinant baculovirus displaying S1 could be used as a candidate vaccine for mucosal immune against PEDV infection, which has the value for further development and research.
引文
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[5] CHOUDHURY B,DASTJERDI A,DOYLE N,et al.From the field to the lab:an European view on the global spread of PEDV[J].Virus Res,2016,226:40-49
[6] LEE C.Porcine epidemic diarrhea virus:an emerging and re-emerging epizootic swine virus[J/OL].Virol J,2015,12:193[2019-02-15].https://doi.org/10.1186/s12985-016-0465-y
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[9] LI J,JIN Z,GAO Y,et al.Development of the full-length cDNA clones of two porcine epidemic diarrhea disease virus isolates with different virulence[J/OL].2017,12(3):e0173998[2019-02-15].https://www.ncbi.nlm.nih.gov/pubmed/28301551.DOI:10.1371/journal.pone.0173998
[10] LI W,VAN KUPPEVELD F J M,HE Q,et al.Cellular entry of the porcine epidemic diarrhea virus[J].Virus Res,2016,226:117-127
[11] LI C,LI W,LUCIO DE ESESARTE E,et al.Cell attachment domains of the porcine epidemic diarrhea virus spike protein are key targets of neutralizing antibodies[J/OL].J Virol,2017,91(12):e00273-17[2019-02-15].https://www.ncbi.nlm.nih.gov/pubmed/28381581.DOI:10.1128/JVI.00273-17
[12] MAKADIYA N,BROWNLIE R,VAN DEN HURK J,et al.S1 domain of the porcine epidemic diarrhea virus spike protein as a vaccine antigen[J/OL].Virol J,2016,13:57[2019-02-15].https://doi.org/10.1186/s12985-016-0512-8
[13] LIN S Y,CHUNG Y C,HU Y C.Update on baculovirus as an expression and/or delivery vehicle for vaccine antigens[J].Expert Rev Vaccines,2014,13(12):1501-1521
[14] LI Y,WU Q,HUANG L,et al.An alternative pathway of enteric PEDV dissemination from nasal cavity to intestinal mucosa in swine[J/OL].Nat Commun,2018,9(1):3811[2019-02-15].https://doi.org/10.1038/s41467-018-06056-w
[15] 史翠萍,严婷婷,崔康乐,等.表面展示猪流行性腹泻病毒纤突蛋白S1的重组杆状病毒及其免疫效果研究[J].蚕业科学,2016,42(6):1041-1048
[16] FENG Q,LIU Y,QU X,et al.Baculovirus surface display of SARS coronavirus (SARS-CoV) spike protein and immunogenicity of the displayed protein in mice models[J].DNA Cell Biol,2006,25(12):668-673
[17] KAIKKONEN M U,RATY J K,AIRENNE K J,et al.Truncated vesicular stomatitis virus G protein improves baculovirus transduction efficiency in vitro and in vivo[J].Gene Ther,2006,13(4):304-312
[18] ROBISON C S,WHITT M A.The membrane-proximal stem region of vesicular stomatitis virus G protein confers efficient virus assembly[J].J Virol,2000,74(5):2239-2246
[19] CZERKINSKY C,HOLMGREN J.Mucosal delivery routes for optimal immunization:targeting immunity to the right tissues[J].Curr Top Microbiol Immunol,2012,354:1-18
[20] GAYET R,BIOLEY G,ROCHEREAU N,et al.Vaccination against Salmonella infection:the mucosal way[J/OL].Microbiol Mol Biol Rev,2017,81(3):e00007-00017[2019-02-15].https://www.ncbi.nlm.nih.gov/pubmed/28615285.DOI:10.1128/MMBR.00007-17
[21] RIESE P,SAKTHIVEL P,TRITTEL S,et al.Intranasal formula-tions:promising strategy to deliver vaccines[J].Expert Opin Drug Deliv,2014,11(10):1619-1634
[22] PIGNY F,LASSUS A,TERRETTAZ J,et al.Intranasal vaccination with salmonella-derived serodominant secreted effector protein B associated with gas-filled microbubbles partially protects against gut infection in mice[J].J Infect Dis,2016,214(3):438-446
[2] SUN R Q,CAI R J,CHEN Y Q,et al.Outbreak of porcine epidemic diarrhea in suckling piglets,China[J].Emerg Infect Dis,2012,18(1):161-163
[3] HANKE D,POHLMANN A,SAUTER-LOUIS C,et al.Porcine epidemic diarrhea in Europe:in-detail analyses of disease dynamics and molecular epidemiology[J/OL].Viruses,2017,9(7):e177[2019-02-15].https://www.ncbi.nlm.nih.gov/pubmed/28684708.DOI:10.3390/v9070177
[4] HUANG Y W,DICKERMAN A W,PINEYRO P,et al.Origin,evolution,and genotyping of emergent porcine epidemic diarrhea virus strains in the United States[J/OL].mBio,2013,4(5):e0073713[2019-02-15].https://mbio.asm.org/cgi/doi/10.1128/mBio.00737-13
[5] CHOUDHURY B,DASTJERDI A,DOYLE N,et al.From the field to the lab:an European view on the global spread of PEDV[J].Virus Res,2016,226:40-49
[6] LEE C.Porcine epidemic diarrhea virus:an emerging and re-emerging epizootic swine virus[J/OL].Virol J,2015,12:193[2019-02-15].https://doi.org/10.1186/s12985-016-0465-y
[7] REEVES D E.The application of biotechnical and epidemiologic tools for pig health[J].Anim Biotechnol,2006,17(2):177-187
[8] RIBBENS S,DEWULF J,KOENEN F,et al.A survey on biosecurity and management practices in Belgian pig herds[J].Prev Vet Med,2008,83(3/4):228-241
[9] LI J,JIN Z,GAO Y,et al.Development of the full-length cDNA clones of two porcine epidemic diarrhea disease virus isolates with different virulence[J/OL].2017,12(3):e0173998[2019-02-15].https://www.ncbi.nlm.nih.gov/pubmed/28301551.DOI:10.1371/journal.pone.0173998
[10] LI W,VAN KUPPEVELD F J M,HE Q,et al.Cellular entry of the porcine epidemic diarrhea virus[J].Virus Res,2016,226:117-127
[11] LI C,LI W,LUCIO DE ESESARTE E,et al.Cell attachment domains of the porcine epidemic diarrhea virus spike protein are key targets of neutralizing antibodies[J/OL].J Virol,2017,91(12):e00273-17[2019-02-15].https://www.ncbi.nlm.nih.gov/pubmed/28381581.DOI:10.1128/JVI.00273-17
[12] MAKADIYA N,BROWNLIE R,VAN DEN HURK J,et al.S1 domain of the porcine epidemic diarrhea virus spike protein as a vaccine antigen[J/OL].Virol J,2016,13:57[2019-02-15].https://doi.org/10.1186/s12985-016-0512-8
[13] LIN S Y,CHUNG Y C,HU Y C.Update on baculovirus as an expression and/or delivery vehicle for vaccine antigens[J].Expert Rev Vaccines,2014,13(12):1501-1521
[14] LI Y,WU Q,HUANG L,et al.An alternative pathway of enteric PEDV dissemination from nasal cavity to intestinal mucosa in swine[J/OL].Nat Commun,2018,9(1):3811[2019-02-15].https://doi.org/10.1038/s41467-018-06056-w
[15] 史翠萍,严婷婷,崔康乐,等.表面展示猪流行性腹泻病毒纤突蛋白S1的重组杆状病毒及其免疫效果研究[J].蚕业科学,2016,42(6):1041-1048
[16] FENG Q,LIU Y,QU X,et al.Baculovirus surface display of SARS coronavirus (SARS-CoV) spike protein and immunogenicity of the displayed protein in mice models[J].DNA Cell Biol,2006,25(12):668-673
[17] KAIKKONEN M U,RATY J K,AIRENNE K J,et al.Truncated vesicular stomatitis virus G protein improves baculovirus transduction efficiency in vitro and in vivo[J].Gene Ther,2006,13(4):304-312
[18] ROBISON C S,WHITT M A.The membrane-proximal stem region of vesicular stomatitis virus G protein confers efficient virus assembly[J].J Virol,2000,74(5):2239-2246
[19] CZERKINSKY C,HOLMGREN J.Mucosal delivery routes for optimal immunization:targeting immunity to the right tissues[J].Curr Top Microbiol Immunol,2012,354:1-18
[20] GAYET R,BIOLEY G,ROCHEREAU N,et al.Vaccination against Salmonella infection:the mucosal way[J/OL].Microbiol Mol Biol Rev,2017,81(3):e00007-00017[2019-02-15].https://www.ncbi.nlm.nih.gov/pubmed/28615285.DOI:10.1128/MMBR.00007-17
[21] RIESE P,SAKTHIVEL P,TRITTEL S,et al.Intranasal formula-tions:promising strategy to deliver vaccines[J].Expert Opin Drug Deliv,2014,11(10):1619-1634
[22] PIGNY F,LASSUS A,TERRETTAZ J,et al.Intranasal vaccination with salmonella-derived serodominant secreted effector protein B associated with gas-filled microbubbles partially protects against gut infection in mice[J].J Infect Dis,2016,214(3):438-446