猪流感病毒新型病毒样颗粒的构建及鉴定  

Construction and Identification of Novel Virus-like Particles of Swine influenza virus

作　　者：周雨彤 张梦佳 谯雯月 郎一飞 颜其贵[1] 李文涛[2,3] 赵珊[1] ZHOU Yu-Tong;ZHANG Meng-Jia;QIAO Wen-Yue;LANG Yi-Fei;YAN Qi-Gui;LI Wen-Tao;ZHAO Shan(College of Veterinary Medicine,Sichuan Agricultural University,Chengdu 611130,China;College of Veterinary Medicine,Huazhong Agricultural University,Wuhan 430070,China;National Key Laboratory of Agricultural Microbial Resource Exploration and Utilization,Huazhong Agricultural University,Wuhan 430070,China)

机构地区：[1]四川农业大学动物医学院,成都611130 [2]华中农业大学动物医学学院,武汉430070 [3]华中农业大学农业微生物资源发掘与利用全国重点实验室,武汉430070

出　　处：《农业生物技术学报》2025年第3期697-708,共12页Journal of Agricultural Biotechnology

基　　金：四川省大学生创新创业训练计划(S202310626073);四川省自然科学基金(2022NSFSC1692);四川省留学回国人员科技活动项目择优资助。

摘　　要：猪流感(swine influenza,SI)是由猪流感病毒(Swine influenza virus,SIV)引起的一种猪(Sus scrofa)急性传染病。近年来,我国主要流行H1N1、H1N2以及H3N23种SIV亚型。本研究制备H1和H3亚型SIV的病毒样颗粒(virus-like particles,VLP),并深入鉴定其形态结构及生物学活性。合成编码H1或H3亚型SIV囊膜蛋白—血凝素(hemagglutinin,HA)的基因并连接至表达载体,获得重组质粒并转染至HEK-293T细胞进行表达。同时,合成编码源自超嗜热菌(Aquifex aeolicus)的2,4-二羟基蝶啶合酶(lumazine synthase,LS)的基因,在序列N端引入信号肽及链球菌(Streptococcus)蛋白G(streptococcal protein G,p G)中的免疫球蛋白结合域序列构建pG-LS重组质粒,并通过大肠杆菌(Escherichia coli)和HEK-293T细胞表达系统分别进行表达。将2种蛋白络合后利用透射电镜观察重组蛋白形态以及能否形成VLP;利用动态光散射法测定络合蛋白的平均粒径;利用SDS-PAGE、Western blot和血凝试验对络合蛋白进行生物学活性分析。SDS-PAGE结果表明重组蛋白H1-Fc和H3-Fc均得到正确表达;同时,Western blot及SDS-PAGE分析结果显示原核和真核表达均可得到p G-LS纳米颗粒,且真核表达纯度更高,并用于后续pG-LS-HA病毒样颗粒的构建。将2种蛋白络合后的电镜结果表明,两种病毒样颗粒均呈球形,其直径约为80 nm。对该颗粒的进一步免疫学鉴定及粒径测量结果均证实了病毒样颗粒的成功构建。同时,血凝实验结果表明,仅HA-Fc蛋白存在时未出现血凝,而由该蛋白制备的病毒样颗粒亲和力相比单体蛋白呈指数级增长,可高效凝集不同物种来源的红细胞。本研究成功构建了pG-LS纳米颗粒以及由2种蛋白络合而成的pGLS-HA病毒样颗粒,可实现简单、高效的HA蛋白表面展示;2种p G-LS-HA均具有稳定的性状及生物学活性,具备大规模投产的应用潜力。本研究制备的H1和H3亚型HA均可有效被展示并组成VLP,为将来使用�Swine influenza(SI)is an acute infectious disease of pigs(Sus scrofa)caused by Swine influenza virus(SIV).In recent years,3 SIV subtypes,namely H1N1,H1N2 and H3N2,are most prevalent in China.This study aimed to develop virus-like particles(VLP)of SIV H1 and H3 subtype and determine their morphological structure and biological characteristics.Genes encoding the SIV surface protein—hemagglutinin(HA)of H1 and H3 subtypes were synthesized and ligated unto the expression vector.The recombinant plasmids was obtained and transfected to HEK-293T cells for protein expression.Meanwhile,the gene encoding the 2,4-dihydroxyprotenine synthase(LS)derived from the super pyretic Aquifex aeolicus was synthesized,and both a signaling peptide and immunoglobulin-bin-binding domain sequence in streptococcal protein G(pG)were introduced at the N-terminus of the sequence.The pG-LS recombinant plasmids were constructed and expressed separately through Escherichia coli and HEK-293T cell expression systems.After the conjugation of the 2 proteins,the morphology and the formation of VLP could be observed by transmission electron microscopy.Average particle diameters of the conjugated proteins were measured with dynamic light scattering method while the biological properties of the complex protein were analyzed by SDS-PAGE,Western blot and hemagglutination assay.SDS-PAGE results indicated that both recombinant proteins(H1-Fc and H3-Fc)were correctly expressed;at the same time,Western blot showed that both prokaryotic and eukaryotic systems resulted in successful expression of pG-LS nanoparticles.Together with SDS-PAGE analysis,it was shown that pG-LS expressed in HEK-293T cells had higher purity,and henceforth used for the subsequent construction of pG-LS-HA virus-like particles.Electron microscopy analysis of the conjugated proteins showed that the pG-LS-HA forms spherical virus-like particles,with a diameter around 80 nm.Further immunological analysis and particle diameter measurement both showed the successful construction of VLPs.The re

关 键 词：猪流感病毒(SIV) 血凝素(HA) 病毒样颗粒 多价展示 生物学活性 

分 类 号：S855.3[农业科学—临床兽医学]