非洲猪瘟病毒多基因家族MGF360-13L基因功能的初步研究  

Preliminary Study on the Function of MGF360-13L Gene of African Swine Fever Virus Multigene Family

作　　者：陈达年 马旭升 代军飞 汪洋 李茜 白衡 毛甜甜 刘永生 丁龙 陈昊翰 陈思言 饶宇飞 贾宁[1] 张杰[2,3] 郑海学 刘湘涛[2] CHEN Danian;MA Xusheng;DAI Junfei;WANG Yang;LI Qian;BAI Heng;MAO Tiantian;LIU Yongsheng;DING Long;CHEN Haohan;CHEN Siyan;RAO Yufei;JIA Ning;ZHANG Jie;ZHENG Haixue;LIU Xiangtao(College of Veterinary Medicine,Gansu Agricultural University,Lanzhou 730070,China;State Key Laboratory of Veterinary Etiological Biology,College of Veterinary Medicine,Lanzhou University,Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Lanzhou 730000,China;Hebei Key Laboratory of Preventive Veterinary Medicine,College of Animal Science and Technology,Hebei Normal University of Science&Technology,Qinhuangdao 066004,China;Shenzhen Zhenrui Biological Science and Techonology Co.Ltd.,Shenzhen 518000,China)

机构地区：[1]甘肃农业大学动物医学院,兰州730070 [2]中国农业科学院兰州兽医研究所兰州大学动物医学与生物安全学院家畜疫病病原生物学国家重点实验室,兰州730000 [3]河北科技师范学院动物科技学院河北省预防兽医学重点实验室,秦皇岛066004 [4]深圳真瑞生物科技有限公司,深圳518000

出　　处：《畜牧兽医学报》2022年第12期4419-4428,共10页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家自然科学基金(31941002);甘肃省科技重大专项(20ZD7NA006-1);中国农业科学院基本科研业务费(Y2021YJ10)。

摘　　要：旨在初步探究非洲猪瘟病毒(African swine fever virus,ASFV)多基因家族MGF360-13L基因的功能。使用生物信息学软件对MGF360-13L基因进行分析;构建真核表达质粒pVAX1-MGF360-13L并转染至293T细胞进行表达;利用同源重组方法构建基因缺失毒株ASFV-ΔMGF360-13L,以相同的感染复数(MOI=0.01)在猪肺泡巨噬细胞(PAMs)中感染基因缺失毒株和亲本毒株(ASFV CN/GS/2018)后,利用红细胞吸附试验(HAD)计算病毒滴度并绘制体外生长曲线;以MOI=1将ASFV-ΔMGF360-13L和ASFV CN/GS/2018分别感染猪骨髓巨噬细胞(BMDM),利用qPCR和ELISA测定细胞炎性因子IL-1β、IL-6和TNF-α的表达水平。结果表明,MGF360-13L基因在ASFV基因Ⅱ型中相对保守,编码蛋白属于非分泌型、无跨膜区、亲水性好;构建的真核表达质粒pVAX1-MGF360-13L被293T细胞表达;成功获得MGF360-13L单基因缺失毒株ASFV-ΔMGF360-13L,与ASFV CN/GS/2018相比,其体外复制没有显著性差异;在ASFV-ΔMGF360-13L感染BMDM后,IL-1β、IL-6和TNF-α炎性细胞因子在转录和分泌水平都显著高于ASFV CN/GS/2018。本研究成功制备了ASFV的MGF360-13L基因缺失毒株,并证实了MGF360-13L基因作为ASFV复制非必需基因具有抑制炎性因子表达的功能,这为进一步注释ASFV MGF360-13L基因功能提供了线索。The aim of this study was to investigate the function of MGF360-13L gene of African swine fever virus(ASFV)multigene family.The MGF360-13L gene was analyzed by bioinformatics software.The eukaryotic expressing plasmid pVAX1-MGF360-13L was constructed and transfected into 293 T cells for expression.The MGF360-13L gene deleted strain ASFV-ΔMGF360-13L was constructed by homologous recombination.After infecting the gene deleted virus strain and parent virus strain(ASFV CN/GS/2018)in porcine alveolar macrophages(PAMs)with the same multiplicity of infection(MOI=0.01),the virus titer was calculated by erythrocyte adsorption test(HAD)and the in vitro growth curve was drawn;ASFV-ΔMGF360-13L and ASFV CN/GS/2018 were infected with porcine bone marrow macrophages(BMDM)with MOI=1.The expression levels of inflammatory factors IL-1β,IL-6 and TNF-αwere determined by qPCR and ELISA.The results showed that,MGF360-13L gene was relatively conservative in ASFV gene type II.The coding protein belongs to non secretory type,without transmembrane region and with good hydrophilicity;The eukaryotic expression plasmid pVAX1-MGF360-13L was expressed by 293 T cells;The MGF360-13L deleted strain ASFV-ΔMGF360-13L was successfully constructed.There was no significant difference between ASFV-ΔMGF360-13L and ASFV CN/GS/2018 in in vitro replication.After ASFV-ΔMGF360-13L infected BMDM,the levels of transcription and secretion of inflammatory cytokines IL-1β,IL-6 and TNF-αwere significantly higher than those of ASFV CN/GS/2018.In this study,the MGF360-13L gene deleted strain of ASFV were successfully constructed,and it was confirmed as a non-essential gene for ASFV replication and have the function of inhibiting the expression of inflammatory factors.This study provides clues for further annotation of ASFV MGF360-13L gene function.

关 键 词：ASFV MGF360-13L 炎性因子 生长曲线测定 

分 类 号：S852.651[农业科学—基础兽医学]