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 GeneⅦHemagglutinin-neuraminidase(HN)Protein in Rice(Oryza sativa)Cells

作　　者：史倩倩 陈金炫 陈恒耀 寇少康 楚红燕 张磊[1] 潘世源 张二芹 张改平[1,2,3,4] SHI Qian-Qian;CHEN Jin-Xuan;CHEN Heng-Yao;KOU Shao-Kang;CHU Hong-Yan;ZHANG Lei;PAN Shi-Yuan;ZHANG Er-Qin;ZHANG Gai-Ping(College of Veterinary Medicine/International Joint Research Center of National Animal Immunology,Henan Agricultural University,Zhengzhou 450046,China;Longhu Laboratory of Advanced Immunology,Zhengzhou 450046,China;College of Modern Agriculture,Peking University,Beijing 100871,China;Key Laboratory of Animal Immunology,Henan Academy of Agricultural Sciences,Zhengzhou 450002,China)

机构地区：[1]河南农业大学动物医学院/国家动物免疫学国际联合研究中心,郑州450046 [2]龙湖现代免疫实验室,郑州450046 [3]北京大学现代农学院,北京100871 [4]河南省农业科学院动物免疫学重点实验室,郑州450002

出　　处：《农业生物技术学报》2025年第4期879-887,共9页Journal of Agricultural Biotechnology

基　　金：河南省科技攻关项目(222102110210);河南省重大科技专项(221100110600)。

摘　　要：新城疫是新城疫病毒(Newcastle disease virus,NDV)引起的一种急性、热性、高度接触性传染病,其中基因Ⅶ型病毒流行广泛。血凝素-神经氨酸酶蛋白(hemagglutinin-neuraminidase protein,HN)是病毒的结构蛋白,也是宿主细胞受体识别和促进细胞膜融合的重要免疫原性蛋白。水稻(Oryza sativa)细胞是生产生物药物的一个有前景的经济有效的生物反应器平台,水稻α-淀粉酶3D系统包含一个诱导启动子,在糖饥饿条件下具有很强的激活活性,可以高效、高水平地表达外源蛋白。本研究从NCBI上筛选NDV基因Ⅶ型HN氨基酸序列,根据水稻密码子偏爱性对HN基因进行优化,构建了重组植物表达载体pCAMBIA1300-HN;利用根癌农杆菌(Agrobacterium tumefaciens)介导法将HN基因转入到水稻愈伤组织中,经潮霉素筛选和PCR鉴定阳性愈伤,在阳性愈伤中诱导表达HN蛋白,通过Western blot鉴定。利用SP阳离子层析和Hiloard 75 pg凝胶层析纯化HN蛋白,并以5和10μg剂量分别与Al(OH)_(3)和50V佐剂乳化,免疫6周龄BALB/c雌鼠(Mus musculus),通过血凝抑制(hemagglutination inhibition,HI)实验和中和实验(neutralizing test,NT)检测抗体的效价,并检测二次免疫6周后白细胞介素-2(interleukin-2,IL-2)水平。结果显示,成功构建了重组植物表达载体pCAMBIA1300-HN,并鉴定出85个阳性愈伤,HN蛋白在水稻细胞中成功表达和纯化。免疫小鼠后,除PBS组,每组产生的抗体水平达到免疫保护效果(HI≥24),能够产生中和抗体,具有病毒中和作用。本研究在水稻细胞中表达了新城疫HN蛋白,建立了HN蛋白纯化方法,低剂量HN蛋白免疫动物后,具有良好的免疫效果,为今后新城疫亚单位疫苗的研究提供了参考依据。Newcastle disease is an acute,febrile and highly contagious disease caused by Newcastle disease virus(NDV),in which genotypeⅦis 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 riceα-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 geneⅦHN sequence was screened from NCBI,HN gene was optimized according to rice codon preference,and the recombinant plant expression vector p CAMBIA1300-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 5μg and 10μg,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

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 

分 类 号：S858.3[农业科学—临床兽医学]