农业生物技术学报2025，Vol.33Issue(4)：879-887,9.DOI:10.3969/j.issn.1674-7968.2025.04.016
新城疫病毒基因Ⅶ型血凝素-神经氨酸酶蛋白在水稻细胞中的表达及免疫原性
Expression and Immunogenicity of Newcastle disease virus Gene Ⅶ Hemagglutinin-neuraminidase(HN)Protein in Rice(Oryza sativa)Cells
摘要
Abstract
Newcastle disease is an acute,febrile and highly contagious disease caused by Newcastle disease virus(NDV),in which genotype Ⅶ is widespread.Hemagglutinin-neuraminidase protein(HN)is a structural protein of viruses and an important immunogenic protein for host cell receptors to recognize and promote cell membrane fusion.Rice(Oryza sativa)cells are a promising economic and effective bioreactor platform for the production of biological drugs.The rice α-amylase 3D system contains an inducer promoter,which has strong activation activity under the condition of sugar starvation and can express foreign proteins efficiently and at a high level.In this study,NDV gene Ⅶ HN sequence was screened from NCBI,HN gene was optimized according to rice codon preference,and the recombinant plant expression vector pCAMBIA1300-HN was constructed.HN gene was transferred into rice callus by Agrobacterium-mediated method,and the positive callus was identified by hygromycin screening and PCR.HN protein expression was induced in the positive callus,and then identified by Western blot.HN protein was purified by SP cationic chromatography and Hiloard 75 pg gel chromatography,and emulsified with Al(OH)3 and 50V adjuvant at doses of 5 μg and 10 μg,respectively.BALB/c female mice(Mus musculus)aged 6 weeks were immunized,the titer of antibody was detected by hemagglutination inhibition(HI)test and neutralizing test(NT),and the level of interleukin-2(IL-2)after 2 weeks of immunization was detected.The results showed that the recombinant plant expression vector pCAMBIA1300-HN was successfully constructed,and 85 positive calluses were identified.HN protein was successfully expressed and purified in rice cells.After immunizing mice,the antibody levels of each group except PBS group achieved immune protection(HI≥24),and neutralized antibodies were produced,which had virus neutralization effect.In this study,HN protein of Newcastle disease was expressed in rice cells,and a purification method of HN protein was established.After immunizing animals with HN protein at low dose,the immune effect was good.This study provides a reference for the preparation of Newcastle disease subunit vaccine in the future.关键词
新城疫病毒(NDV)/血凝素-神经氨酸酶蛋白(HN)/水稻细胞/纯化/免疫试验Key words
Newcastle disease virus(NDV)/Hemagglutinin-neuraminidase protein(HN)/Rice cells/Purification/Immunological assay分类
农业科技引用本文复制引用
史倩倩,陈金炫,陈恒耀,寇少康,楚红燕,张磊,潘世源,张二芹,张改平..新城疫病毒基因Ⅶ型血凝素-神经氨酸酶蛋白在水稻细胞中的表达及免疫原性[J].农业生物技术学报,2025,33(4):879-887,9.基金项目
河南省科技攻关项目(222102110210) （222102110210）
河南省重大科技专项(221100110600) （221100110600）
