猪外周血淋巴细胞干扰素诱导跨膜蛋白的拓扑学分析及其在猪繁殖与呼吸综合征病毒感染后转录水平的变化  

opological analysis of IFN-induced transmembrane proteins in porcine peripheral blood lymphocytes and changes of transcription level after infection with porcine reproductive and respiratory syndrome virus

作　　者：蒙小刚 宋扬 鲁会军 霍晓伟[1] 吕香玉 刘锴[1] 温树波 MENG Xiaogang;SONG Yang;LU Huijun;HUO Xiaowei;LV Xiangyu;LIU Kai;WEN Shubo(College of Animal Science and Technology,Inner Mongolia Minzu University,Tongliao 028000,Inner Mongolia Autonomous Region,China;不详)

机构地区：[1]内蒙古民族大学动物科技学院,内蒙古通辽028000 [2]内蒙古自治区布鲁氏菌病防控工程技术研究中心,内蒙古通辽028000 [3]内蒙古民族大学人畜共患病创新研究院,内蒙古通辽028000 [4]军事科学院军事医学研究院军事兽医研究所,吉林长春130012

出　　处：《中国生物制品学杂志》2023年第5期551-558,共8页Chinese Journal of Biologicals

基　　金：国家重点研发计划(2018YDF0500803,2018YDF0500104);内蒙古自治区布鲁氏菌病防治工程技术研究中心开放课题(MDK2019082);内蒙古民族大学博士启动基金(BS583,BS584)。

摘　　要：目的对猪外周血淋巴细胞(peripheral blood lymphocytes,PBMCs)干扰素诱导跨膜蛋白(IFN-induced transmembrane,IFITM)进行拓扑学分析,并检测PBMCs在体外感染猪繁殖与呼吸综合征病毒(porcine reproductive and respiratory syndrome virus,PRRSV)后IFITM基因mRNA转录水平的变化。方法无菌采集PRRSV、猪圆环病毒2(porcine circovirus 2,PCV2)和乙型脑炎病毒(Japanese encephalitis virus,JEV)阴性的仔猪抗凝血,分离PBMCs,PCR扩增猪IFITM CDS序列,测序并进行拓扑学分析。PBMCs体外感染PRRSV,分别于感染后12、24、36和48 h收集细胞样品,RT-PCR法检测PBMCs中PRRSV感染情况,qRT-PCR法检测IFITM1、IFITM2和IFITM3基因mRNA转录水平的变化。结果成功分离到猪PBMCs,并克隆获得了PBMCs源IFITM CDS全长序列,猪IFITM蛋白可能存在两种拓扑学结构。PBMCs接种PRRSV后24 h可产生明显的细胞病变。PRRSV可在PBMCs中复制。PRRSV感染早期可显著上调PBMCs中IFITM1、IFITM2和IFITM3基因mRNA的转录水平,且均在感染后第12 h达高峰,随后逐渐下降;IFITM1基因mRNA的转录水平在病毒感染后第36 h有所回升,然后又迅速回落。结论PRRSV体外感染PBMCs可显著上调猪IFITM基因mRNA的转录水平,表明IFITM参与PBMCs抗病毒免疫反应。本研究为揭示机体抗PRRSV天然免疫反应提供了参考。Objective To analyze the topology of IFN-induced transmembrane(IFITM)protein in porcine peripheral blood lymphocytes(PBMCs)and detect the change of IFITM mRNA transcription in PBMCs after porcine reproductive and respiratory syndrome virus(PRRSV)infection in vitro.Methods PRRSV,porcine circovirus 2(PCV2)and Japanese encephalitis virus(JEV)negative anticoagulant blood of piglets were collected aseptically and isolated for PBMCs.Porcine IFITM CDS sequence was amplified by PCR,sequenced and analyzed for topology.PBMCs were infected with PRRSV in vitro.Cell samples were collected at 12,24,36 and 48 h after infection,detected for PRRSV infection by RT-PCR,and detected for mRNA transcription level changes of IFITM1,IFITM2 and IFITM3 by RT-PCR.Results The porcine PBMCs were successfully isolated and the full-length sequence of IFITM CDS derived from PBMCs was cloned.The porcine IFITM protein might have two topological structures.PBMCs inoculated with PRRSV for 24 h produced obvious cytopathic effect.PRRSV was replicated in PBMCs.The transcription levels of IFITM1,IFITM2 and IFITM3 mRNA in PBMCs were significantly up-regulated at the early stage of PRRSV infection,and reached the peak at 12h after infection,and then gradually decreased;The transcription level of IFITM1 mRNA increased at 36 h after virus infection and then declined rapidly.Conclusion PRRSV infection in vitro significantly up-regulated the transcription level of IFITM mRNA in PBMCs,indicating that IFITM was involved in the antiviral immune response of PBMCs.This study provided a reference for revealing the natural immune response against PRRSV in vivo.

关 键 词：猪繁殖与呼吸综合征 外周血淋巴细胞 干扰素刺激基因 干扰素诱导跨膜蛋白 

分 类 号：S855.3[农业科学—临床兽医学]