摘要
目的旨在研究新型冠状病毒Omicron(BA.1、BA.2)N蛋白的免疫学功能,并评估不同新冠病毒突变株N蛋白的免疫原性差异。方法运用序列比对确定Omicron(BA.1、BA.2)N蛋白相对新冠病毒原型株(Wuhan-Hu-1)的突变位点,以pET-28a-N-Wuhan-Hu-1质粒为模板经单点突变或同源重组构建pET-28a-BA.1/BA.2-N。利用原核系统分别表达上述三种N蛋白,经Ni-NTA亲和层析柱纯化后免疫小鼠。运用间接ELISA和Western blot法检测多克隆抗体的效价和反应性以及免疫小鼠脾脏细胞中白细胞介素1β(IL-1β)和γ干扰素(IFN-γ)的表达水平。结果成功利用构建的原核表达质粒在E.coli BL21(DE3)中表达了Wuhan-Hu-1 N、BA.1 N和BA.2 N蛋白,表达条件为37℃,4 h。间接ELISA试验测得三种N蛋白制备的多克隆抗体效价均为1∶51200。三种N蛋白均可使IFN-γ和IL-1β两种细胞因子表达升高,但是Omicron N蛋白较Wuhan-Hu-1 N蛋白激活两种细胞因子的效果更为明显。结论研究获得了三种新冠病毒N蛋白及多克隆抗体,并证实了N蛋白发生氨基酸位点的突变会对其免疫原性产生影响。这为开发针对不同新冠变异株N蛋白的快速诊断方法提供了依据。
Objective The study aims to investigate the immunological functions of the nucleocapsid(N)protein of the novel coronavirus Omicron(BA.1,BA.2)and evaluate the differences among different N proteins of mutant strains in immunogenicity.Methods By aligning sequences,the mutation sites of the Omicron(BA.1,BA.2)N protein relative to prototype strain of the novel coronavirus(Wuhan-Hu-1)were determined.The pET-28a-N-Wuhan-Hu-1 plasmid was used as template to construct pET-28a-BA.1/BA.2-N through single point mutation or homologous recombination.The three kinds of N protein were expressed in prokaryotic system,purified through Ni-NTA affinity chromatography,and then immunized into mice.The titer and reactivity of the polyclonal antibody,as well as the expression level of IL-1βand IFN-γin mouse spleen cells,were detected using indirect ELISA and Western blot assay.Results The constructed prokaryotic expression plasmids were successfully used to express the Wuhan-Hu-1 N,BA.1 N,and BA.2 N proteins in E.coli BL21(DE3)at 37℃for 4 hours.The indirect ELISA test showed that the titers of polyclonal antibody prepared by three N proteins were all 1∶51200.All three N proteins can increase the expression of IFN-γand IL-1βcytokines,but the effect of Omicron N protein in activing two cytokines was more obvious than that of Wuhan-Hu-1 N protein.Conclusion The study obtained three new coronavirus N proteins and polyclonal antibodies,and confirmed that mutations in the amino acid sites of the N protein can affect its immunogenicity.This provides a basis for developing rapid diagnostic methods targeting N protein of different novel coronavirus variants.
作者
屠泽文
王全胜
刘仕国
刘浩森
曾春燕
谢娟娟
李名志
李精彩
王敏
翁诗琦
康路妹
孔令保
TU Zewen;WANG Quansheng;LIU Shiguo;LIU Haosen;ZENG Chunyan;XIE Juanjuan;LI Mingzhi;LI Jingcai;WANG Min;WENG Shiqi;KANG Lumei;KONG Lingbao(College of Biological Science and Engineering,Jiangxi Agricultural University,Nanchang 330045;Nanchang Key Laboratory of Animal Virus and Genetic Engineering,Nanchang 330045;Department of Clinical Laboratory,The Second Affiliated Hospital of Nanchang University,Nanchang 330006;College of Animal Science and Technology,Jiangxi Agricultural University,Nanchang 330045;Center of Laboratory Animal Science and Technology,Nanchang University,Nanchang 330031,China)
出处
《细胞与分子免疫学杂志》
北大核心
2025年第8期735-743,共9页
Chinese Journal of Cellular and Molecular Immunology
基金
国家自然科学基金(32360908)
江西省自然科学基金重点项目(20224ACB205004)
南昌市动物病毒与基因工程重点实验室项目(31860038)。
