猪繁殖与呼吸障碍综合征病毒GP5蛋白羧基端的噬菌体展示及其诱导仔猪产生中和抗体水平
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摘要
为了发现并验证猪繁殖与呼吸障碍综合征病毒(PRRSV)保护性抗原GP5蛋白羧基端新的中和性表位,本研究运用生物信息学软件预测PRRSV GP5蛋白羧基端的抗原性,全基因合成GP5蛋白羧基端168～198位氨基酸的编码序列,构建了重组噬菌体M13-GP5aa168～198,以临床阳性血清进行Western blot分析重组噬菌体表面展示的GP5aa168～198多肽的免疫反应原性。以1.0×10~(13) PFU/mL重组噬菌体混合氢氧化铝佐剂制备免疫抗原,按照灭活疫苗免疫程序肌肉注射免疫PRRSV抗体阴性断乳仔猪2次,间隔2周,分别于末次免疫后的第2,4和6周采血,分离血清,以临床分离PRRSV强毒株进行中和试验。结果显示:重组噬菌体表面展示的GP5aa168～198多肽具有免疫反应原性,免疫仔猪后,能够诱导仔猪产生较高水平的中和性抗体,为构建PRRSV GP5新型多表位疫苗的开发奠定了基础。
To find and validate the new neutralizing epitope at the carboxy terminal of protective antigen GP5 protein from porcine reproductive and respiratory syndrome virus(PRRSV),the antigenicity of the carboxy terminus of PRRSV GP5 protein was predicted by bioinformatics software,the coding sequence of amino acid 168-198 at the carboxy terminus of GP5 protein was synthesized,then the recombinant phage M13-GP5 168-198 was constructed.The immunoreactivity of the GP5 168-198 polypeptide displayed on the surface of the recombinant phage was analyzed by Western blot using clinically positive serum.The immunized antigen,prepared by 1.0×10~(13) PFU/mL recombinant phage mixed with aluminum hydroxide adjuvant was injected intramuscularly into the PRRSV antibody negative weaned piglets according to inactivated vaccine immunization program,twice at intervals of 2 weeks,and at the 2,4,6 th week after the last immunization,blood was collected respectively,serum was separated for neutralization test with the clinical isolated virulent strain of PRRSV.The results showed that,the GP5 aa168-198 polypeptide that displayed on the surface of recombinant phage analyzed by Western blot with clinical positive serum was immunogenic,after immunization of piglets,it could induce a higher level of neutralizing antibodies in piglets.The results indicate that recombinant phage lay a foundation for the development of a novel PRRSV GP5 multi-epitope vaccine.
To find and validate the new neutralizing epitope at the carboxy terminal of protective antigen GP5 protein from porcine reproductive and respiratory syndrome virus(PRRSV),the antigenicity of the carboxy terminus of PRRSV GP5 protein was predicted by bioinformatics software,the coding sequence of amino acid 168-198 at the carboxy terminus of GP5 protein was synthesized,then the recombinant phage M13-GP5 168-198 was constructed.The immunoreactivity of the GP5 168-198 polypeptide displayed on the surface of the recombinant phage was analyzed by Western blot using clinically positive serum.The immunized antigen,prepared by 1.0×10~(13) PFU/mL recombinant phage mixed with aluminum hydroxide adjuvant was injected intramuscularly into the PRRSV antibody negative weaned piglets according to inactivated vaccine immunization program,twice at intervals of 2 weeks,and at the 2,4,6 th week after the last immunization,blood was collected respectively,serum was separated for neutralization test with the clinical isolated virulent strain of PRRSV.The results showed that,the GP5 aa168-198 polypeptide that displayed on the surface of recombinant phage analyzed by Western blot with clinical positive serum was immunogenic,after immunization of piglets,it could induce a higher level of neutralizing antibodies in piglets.The results indicate that recombinant phage lay a foundation for the development of a novel PRRSV GP5 multi-epitope vaccine.
引文
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[12] PLAGEMANN P G.The primary GP5 neutralization epitope of North American isolates of porcine reproductive and respiratory syndrome virus[J].Vet Immunol Immunopathol,2004,102(3):263-275.
[13] REN J Q,SUN W C,LU H J,et al.Construction and immunogenicity of a DNA vaccine coexpressing GP3 and GP5 of genotype-I porcine reproductive and respiratory syndrome virus[J].BMC Vet Res,2014,10:128.
[14] NAM H M,CHAE K S,SONG Y J,et al.Immune responses in mice vaccinated with virus-like particles composed of the GP5 and M proteins of porcine reproductive and respiratory syndrome virus[J].Arch Virol,2013,158(6):1275-1285.
[15] LI W,JOSHI M D,SINGHANIA S,et al.Peptide vaccine:progress and challenges[J].Vaccines (Basel),2014,2(3):515-536.
[16] WANG Y,GUO J,QIAO S,et al.GP5 Protein-based ELISA for the detection of PRRSV antibodies[J].Pol J Vet Sci,2016,19(3):495-501.
[17] 杨柳,沈克飞,郑华,等.噬菌体展示颗粒疫苗的研究进展[J].中国生物制品学杂志,2013,26(4):569-573.
[18] AGHEBATI-MALEKI L,BAKHSHINEJAD B,BARADARAN B,et al.Phage display as a promising approach for vaccine development[J].J Biomed Sci,2016,23(1):66.
[2] 曹素芳.靶向HP-PRRSV ORF7基因的siRNA重组伪狂病病毒的体外复制抑制效果[J].中国兽医学报,2018,38(11):2044-2050.
[3] 赵孟孟,张雅,谢莎,等.猪2′,5′寡腺苷酸合成酶OAS1a基因克隆与序列分析及其在Marc-145细胞上对PRRSV复制的影响[J].中国兽医学报,2018,38(1):25-32.
[4] 郭玉堃,郭婉莹,明胜利,等.猪繁殖与呼吸综合征病毒GP5/M蛋白可溶性表达及免疫原性分析[J].中国兽医学报,2018,38(4):609-617.
[5] RENUKARADHYA G J,MENG X J,CALVERT J G,et al.Inactivated and subunit vaccines against porcine reproductive and respiratory syndrome:current status and future direction[J].Vaccine,2015,33(27):3065-3072.
[6] RENUKARADHYA G J,MENG X J,CALVERT J G,et al.Live porcine reproductive and respiratory syndrome virus vaccines:Current status and future direction[J].Vaccine,2015,33(33):4069-4080.
[7] ZHAO Z,QIN Y,LAI Z,et al.Microbial ecology of swine farms and PRRS vaccine vaccination strategies[J].VET Microbiol,2012,155(2-4):247-256.
[8] KIMMAN T G,CORNELISSEN L A,MOORMANN R J,et al.Challenges for porcine reproductive and respiratory syndrome virus (PRRSV) vaccinology[J].Vaccine,2009,27(28):3704-3718.
[9] LI B,XIAO S,WANG Y,et al.Immunogenicity of the highly pathogenic porcine reproductive and respiratory syndrome virus GP5 protein encoded by a synthetic ORF5 gene[J].Vaccine,2009,27(13):1957-1963.
[10] POPESCU L N,TRIBLE B R,CHEN N,et al.GP5 of porcine reproductive and respiratory syndrome virus (PRRSV) as a target for homologous and broadly neutralizing antibodies[J].VET MICROBIOL,2017,209:90-96.
[11] OSTROWSKI M,GALEOTA J A,JAR A M,et al.Identification of neutralizing and nonneutralizing epitopes in the porcine reproductive and respiratory syndrome virus GP5 ectodomain[J].J Virol,2002,76(9):4241-4250.
[12] PLAGEMANN P G.The primary GP5 neutralization epitope of North American isolates of porcine reproductive and respiratory syndrome virus[J].Vet Immunol Immunopathol,2004,102(3):263-275.
[13] REN J Q,SUN W C,LU H J,et al.Construction and immunogenicity of a DNA vaccine coexpressing GP3 and GP5 of genotype-I porcine reproductive and respiratory syndrome virus[J].BMC Vet Res,2014,10:128.
[14] NAM H M,CHAE K S,SONG Y J,et al.Immune responses in mice vaccinated with virus-like particles composed of the GP5 and M proteins of porcine reproductive and respiratory syndrome virus[J].Arch Virol,2013,158(6):1275-1285.
[15] LI W,JOSHI M D,SINGHANIA S,et al.Peptide vaccine:progress and challenges[J].Vaccines (Basel),2014,2(3):515-536.
[16] WANG Y,GUO J,QIAO S,et al.GP5 Protein-based ELISA for the detection of PRRSV antibodies[J].Pol J Vet Sci,2016,19(3):495-501.
[17] 杨柳,沈克飞,郑华,等.噬菌体展示颗粒疫苗的研究进展[J].中国生物制品学杂志,2013,26(4):569-573.
[18] AGHEBATI-MALEKI L,BAKHSHINEJAD B,BARADARAN B,et al.Phage display as a promising approach for vaccine development[J].J Biomed Sci,2016,23(1):66.