宿主蛋白SPCS1通过外泌体途径介导BVDV复制的研究
Research on Replication of BVDV Through Exosome Pathway Mediated by Host Protein SPCS1
张成远 1魏玉荣 2陈俊贞 1杨莉 1李泽宇 1李紫仟 1付强 1史慧君1
作者信息
- 1. 新疆农业大学动物医学学院,乌鲁木齐 830052
- 2. 新疆畜牧科学院兽医研究所新疆动物疫病研究重点实验室,乌鲁木齐 830052
- 折叠
摘要
牛病毒性腹泻病(bovine viral diarrhea,BVD)是由牛病毒性腹泻病毒(Bovine viral diarrhea virus,BVDV)引起的在世界范围内发生的牛传染病,为畜牧业带来了巨大的经济损失.前期研究发现敲低牛肾细胞(Madin-Darby bovine kidney,MDBK)中的信号肽酶复合体亚基 1(signal peptidase complex subunit 1,SPCS1)基因的表达有效抑制BVDV复制.为了探究其分子机制,本研究将BVDV感染SPCS1基因敲低(knock-down,KD)细胞后收集细胞上清液外泌体,使用透射电镜、Western blot和纳米颗粒跟踪分析(nanoparticle tracking analysis,NTA)对外泌体进行特征鉴定.使用qPCR检测外泌体中BVDV 5'非翻译区(untranslated region,UTR)RNA水平.将外泌体感染MDBK,通过qPCR检测BVDV RNA水平,利用倒置显微镜观察外泌体致细胞病变(cytopathic effect,CPE)情况,根据Reed-Muench方法测定病毒滴度变化情况.透射电镜观察到外泌体呈现为盘状囊泡,Western blot检测到外泌体标志性蛋白TSG101,纳米粒径追踪分析结果在100 nm左右出现峰值,成功提取外泌体.Western blot检测BVDV感染SPCS1 KD细胞外泌体TSG101蛋白与空载体处理的对照细胞(Scramble细胞)相比表达显著降低(P＜0.001).qPCR检测显示,外泌体中BVDV 5'UTR RNA水平显著降低(P＜0.001).MDBK感染BVDV感染的SPCS1 KD细胞源外泌体后与对照组感染Scramble细胞源外泌体相比BVDV mRNA水平显著降低(P＜0.001),CPE现象推迟并减弱,子代病毒滴度显著下降(P＜0.001).结果表明,SPCS1敲低后影响BVDV通过外泌体途径复制.本研究为BVDV的防控提供新型抗病毒靶点和理论依据.
Abstract
Bovine viral diarrhea(BVD)is an infectious disease of cattle worldwide caused by Bovine viral diarrhea virus(BVDV),which has brought huge economic losses to animal husbandry.The previous study found that knocked down the expression of signal peptidase complex subunit 1(SPCS1)in bovine kidney cells(Madin-Darby Bovine Kidney,MDBK)could inhibit the replication of BVDV effectively.In order to explore its molecular mechanism,this study collected exosomes from the supernatant of SPCS1 knockdown(KD)cells infected with BVDV,and transmission electron microscopy,Western blot and Nanoparticle Tracking Analysis were used to characterize exosomes.qPCR was used to analysis BVDV 5'untranslated region(UTR)RNA levels in the exosomes.The exosomes were infected with MDBK;BVDV 5'UTR RNA levels were detected by qPCR;The cytopathic effect(CPE)was observed by inverted microscope,and the change of virus titer was determined by Reed-Muench method.The exosomes were observed as disk-like vesicles under transmission electron microscopy,Western blot detected exosomes,the volume characteristic protein TSG101 showed a peak value at about 100 nm,indicating exosomes were successfully extracted.Western blot showed that the expression of exosome TSG101 protein in SPCS1 KD cells infected with BVDV was significantly decreased compared with the control cells treated with empty vector(Scramble cells)(P＜0.001).qPCR detection showed that the level of BVDV 5'UTR mRNA in exosomes was significantly decreased(P＜0.001).BVDV 5'UTR RNA levels of SPCS1 KD cell derived exosomes infected with MDBK were significantly lower than those infected with Scramble cell(P＜0.001),the CPE phenomenon was delayed and weakened,and the viral titer of the progeny was significantly decreased(P＜0.001).The results showed that knockdown SPCS1 affected the replication of BVDV through exosome pathway.The research results provide a new antiviral target and theoretical basis for the prevention and control of BVDV.
关键词
信号肽酶复合体亚基1(SPCS1)/牛病毒性腹泻病毒(BVDV)/外泌体Key words
Signal peptidase complex subunit 1(SPCS1)/Bovine viral diarrhea virus(BVDV)/Exosome引用本文复制引用
基金项目
自治区百名博士引进计划()
国家自然科学基金(31902271)
国家自然科学基金(32060042)
自治区国际合作项目(2020E01006)
自治区天山创新团队项目(2020D14005)
新疆动物疫病研究重点实验室开放课题(2023KLA005)
出版年
2024
国家科技期刊平台
NSTL
万方数据