农业生物技术学报2025，Vol.33Issue(1)：228-236,9.DOI:10.3969/j.issn.1674-7968.2025.01.021
非洲猪瘟病毒p34蛋白重组纳米颗粒的制备及免疫原性评价
Preparation and Immunogenicity Evaluation of Recombinant Nanoparticles of African swine fever virus p34 Protein
摘要
Abstract
Ferritin nanoparticles are widely used in vaccine research due to their surface that can display multiple antigens,which can better promote cell uptake and antigen presentation,and thereby enhance immune efficacy.This study developed ferritin-based nanoparticles carrying the African swine fever virus(ASFV)p34 protein and evaluated its immunogenicity,aiming to provide an reference for the research on nanoparticle vaccines against ASFV.Initially,the gene sequences encoding the p34 protein and Spytag(ST)were cascaded and inserted into the pET-28a(+)vector to create the pET-p34-ST recombinant plasmid.The gene sequences encoding Spycatcher(SC)and ferritin(F)were cascaded and then inserted into the pET-28a(+)vector to produce the pET-SC-F recombinant plasmid.Both plasmids were expressed in Escherichia coli upon induction.Subsequently,after purifying the 2 recombinant proteins p34-ST and SC-F by affinity chromatography,they were conjugated in vitro to obtain the recombinant nanoparticle F-p34.The F-p34 was identified and morphologically characterized by SDS-PAGE,Western blot,dynamic light scattering(DLS)and transmission electron microscopy(TEM),and finally,the immunogenicity was evaluated by immunizing BALB/c mice(Mus musculus).The results showed that in this study,the prokaryotic expression vectors pET-28a-p34-ST and pET-28a-SC-F were successfully designed and constructed.Through expression,purification and coupling in vitro,recombinant nanoparticle F-p34,which was conjugated with ASFV p34 protein,was successfully prepared,and it could specifically react with ASFV positive serum.In vitro the characterization by DLS and TEM showed that the particle size of F-p34 was approximately 21 nm,and it had a uniform nanoparticle structure.After immunizing mice with F-p34,a specific immune response could be elicited in the body within 7 d.The antibody titer reached the highest level at 42 d and was significantly higher than that of the p34 immunization group(P＜0.05).The proliferation level of spleen lymphocytes in the F-p34 immunization group and the secretion levels of cytokines interleukin 4(IL-4),IL-10,IL-2 and interferon(IFN-γ)in serum were significantly higher than those in the p34 immunization group(P＜0.05).In conclusion,this research successfully prepared nanoparticle antigen carrying the ASFV p34 protein with good immunogenicity,expanding the research perspectives of subunit vaccines for African swine fever.关键词
非洲猪瘟病毒(ASFV)/p34蛋白/铁蛋白纳米颗粒/偶联/免疫原性Key words
African swine fever virus(ASFV)/p34 protein/Ferritin nanoparticles/Couple/Immunogenicity分类
农业科技引用本文复制引用
张荣,杨进才,安兰芳,魏彦明,茹毅,蒋成辉,张越,赵冬梅,郝荣增,李亚军,郑海学,刘学荣..非洲猪瘟病毒p34蛋白重组纳米颗粒的制备及免疫原性评价[J].农业生物技术学报,2025,33(1):228-236,9.基金项目
国家重点研发计划(2021YFD1801302-3) （2021YFD1801302-3）
甘肃省自然科学基金重点项目(23YFNA0011) （23YFNA0011）
中央高校基本科研业务费专项(lzujbky-2022-ct02) （lzujbky-2022-ct02）
兰州市青年科技人才创新项目(2023-QN-3) （2023-QN-3）
中央级公益性科研院所基本科研业务费专项(CAAS-ZDRW202409) （CAAS-ZDRW202409）
国家生猪产业技术体系(CARS-35) （CARS-35）
甘肃省科技重大专项(23ZDKA0002) （23ZDKA0002）
