乙型脑炎病毒囊膜蛋白I176R位点突变株的生物学特性比较研究  

Comparative Study on the Biological Characteristics of Japanese encephalitis virus Envelope Protein I176R Site Mutant Strain

作　　者：唐冕耀 赵勤 石艳萍[1,2,3] 曹玉琴 文翼平 伍锐[1,2,3] 杜森焱 黄小波[1,2,3] 颜其贵 曹三杰[1,2,3] TANG Mian-Yao;ZHAO Qin;SHI Yan-Ping;CAO Yu-Qin;WEN Yi-Ping;WU Rui;DU Sen-Yan;HUANG Xiao-Bo;YAN Qi-Gui;CAO San-Jie(College of Veterinary Medicine/Research Center for Swine Diseases,Sichuan Agricultural University,Chengdu 611130,China;National Demonstration Center for Experimental Animal Education,Sichuan Agricultural University,Chengdu 611130,China;Sichuan Science-Observation Experimental Station of Veterinary Drugs and Veterinary Diagnostic Technique,Ministry of Agriculture and Rural Affairs,Chengdu 61130,China)

机构地区：[1]四川农业大学动物医学院/猪病研究中心,成都611130 [2]四川农业大学国家级动物类实验教学示范中心,成都611130 [3]农业农村部兽用药物与兽医诊断技术四川科学观测站,成都611130

出　　处：《农业生物技术学报》2024年第10期2391-2401,共11页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(31972722)。

摘　　要：乙型脑炎病毒(Japanese encephalitis virus,JEV)是一种危害严重的人畜共患病毒,囊膜蛋白(envelope protein,E)对JEV的神经毒力起关键作用。前期研究发现,重组乙型脑炎病毒(recombinant Japanese encephalitis virus,rJEV)囊膜蛋白I176R位点突变可致弱其神经毒力。为鉴定rJEV-EI176R位点突变后其生物学特性的变化,本研究克隆分析了E176位点突变重组病毒rJEV-EI176R的E基因序列和E蛋白的生物信息学特征;利用qPCR、蚀斑试验和间接免疫荧光(indirect immunofluorescence assay,IFA)等技术测定rJEV-EI176R的生长曲线、对多种细胞的吸附差异和rJEV-EI176R诱导小鼠小胶质细胞系(mouse microglia cell line,BV-2)细胞炎性水平差异。结果显示,rJEV-EI176R的E蛋白带正电荷的残基增加1个,等电点升高0.37,不稳定指数升高0.39;E蛋白的α螺旋、β折叠、延伸链以及无规则卷曲的数量及占比均发生了改变,同时E176位点氨基酸与附近氨基酸有了不同的连接;rJEV-EI176R在BV-2细胞上的生长曲线没有显著变化且能够感染不同来源的神经细胞和肾细胞;rJEV-EI176R的吸附能力显著提高(P<0.05)但蚀斑大小变化不显著;rJEV-EI176R接种BV-2细胞后,在12和24 h均引起肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、白细胞介素-6(interleukin-6,IL-6)、干扰素γ诱导蛋白-10(interferongamma-induced protein-10,IP-10)的水平显著低于亲本株(P<0.05)。本研究丰富了JEV毒株E蛋白的生物学特性研究,为后期深入研究JEV减毒机制和疫苗研发提供基础资料。Japanese encephalitis virus(JEV)is a serious zoonotic virus,and the envelope protein(E)plays a key role in the neurovirulence of JEV.Previous studies have found that the mutation of EI176R site of recombinant Japanese encephalitis virus(rJEV-EI176R)can weaken its neurovirulence.In order to identify the biological characteristics of rJEV-EI176R,the E gene sequence of rJEV-EI176R was cloned and analyzed,and the bioinformatics characteristics of E protein were analyzed.The growth curve of rJEV-EI176R,the adsorption difference of various cells and the inflammatory level of mouse microglia cell line(BV-2)induced by rJEV-EI176R were determined by qPCR,plaque assay and indirect immunofluorescence assay(IFA).The results showed that the positive charge residue of E protein of rJEV-EI176R increased by 1,the isoelectric point increased by 0.37,and the instability index increased by 0.39.The number and proportion ofα-helix,β-sheet,extended strand and random coil of E protein changed,and the amino acid at E176 site had different connections with nearby amino acids.The growth curve of rJEV-EI176R on BV-2 cells did not change significantly and could infect nerve cells and renal cells from different sources.The adsorption capacity of rJEV-EI176R was significantly improved(P<0.05).After inoculation of BV-2 cells with rJEV-EI176R,the levels of tumor necrosis factor-α(TNF-α),interleukin-6(IL-6)and interferon-γ-induced protein-10(IP-10)were significantly lower than those of the parental strain at 12 and 24 h(P<0.05).This study enriched the biological characteristics of E protein of JEV strain,and provides basic data for further study of JEV attenuated mechanism and vaccine development.

关 键 词：乙型脑炎病毒(JEV) 囊膜蛋白(E) I176R位点突变 神经毒力致弱 生物学特性 

分 类 号：S855.3[农业科学—临床兽医学]