猪传染性胃肠炎病毒ORF7蛋白在ST细胞中定位及其对病毒复制影响的研究  

The subcellular location of transmissible gastroenteritis virus protein ORF7 and its effect on viral replication

作　　者：何雷[1] 董玲娟[2,3] 张彦明 宋嘉兴[1] 郁川 余祖华[1] 张春杰[1] 丁轲[1] 赵战勤[1] HE Lei;DONG Ling-juan;ZHANG Yan-ming;SONG Jia-xing;YU Chuan;YU Zu-hua;ZHANG Chun-jie;DING Ke;ZHAO Zhan-qin(The Key Laboratory of Animal Disease and Public Health,Henan University of Science and Technology,Luoyang 471003,China;College of Veterinary Medicine,Northwest N and F University,YangLing 712100,China;Research and Development Center for Tissue Engineering,The Fourth Military Medical University,Xi'an 710000,China)

机构地区：[1]河南科技大学动物疫病与公共卫生重点实验室,河南洛阳471003 [2]西北农林科技大学动物医学院,陕西杨凌712100 [3]第四军医大学组织工程研发中心,陕西西安710000

出　　处：《中国预防兽医学报》2020年第6期543-548,共6页Chinese Journal of Preventive Veterinary Medicine

基　　金：河南省重点研发与推广专项(科技攻关)项目(192102110078);国家自然科学基金青年基金项目(31302105);河南省高等学校重点科研项目(19B230003)。

摘　　要：为研究猪传染性胃肠炎病毒(TGEV)ORF7蛋白的亚细胞定位及其在TGEV复制中的作用。本研究通过RT-PCR扩增TGEV ORF7基因,将其与真核表达载体pEGFP-C1和pEGFP-N1相连接,构建表达载体pGFP-ORF7(GFP融合在TGEV ORF7的-NH2端)和pORF7-GFP(GFP融合在ORF7的-COOH端)。分别将这两个质粒转染至ST细胞,western blot检测融合蛋白GFP-ORF7和ORF7-GFP的表达。通过生物信息学分析和激光共聚焦确定TGEV ORF7蛋白的亚细胞定位。在此基础上,通过细胞病变观察和荧光定量PCR检测TGEV ORF7蛋白对TGEV复制的影响。结果显示,本研究正确构建了真核表达载体pGFP-ORF7和pORF7-GFP,western blot检测结果表明融合蛋白GFP-ORF7和ORF7-GFP均正确表达,TGEV ORF7蛋白大小约为9 ku;结合生物信息学分析和激光共聚焦共定位结果显示TGEV ORF7特异性的定位于ST细胞的内质网中,其定位和其N端的信号肽序列有关;荧光定量PCR检测结果显示,过表达ORF7蛋白后TGEV mRNA转录水平降低99%,表明ORF7蛋白参与调控TGEV的复制。本研究为TGEV ORF7蛋白功能的研究提供了参考依据。The objective of the present study was to explore the subcellular localization of TGEV ORF7 and its effect on the viral replication.In this study,the TGEV ORF7 gene was amplified by RT-PCR.The eukaryotic expression vectors pGFP-ORF7(GFP fused with the C-terminal of TGEV ORF7)and pORF7-GFP(GFP fused with the N-terminal of ORF7)were constructed by inserting the TGEV ORF7 gene into the pEGFP-C1 and pEGFP-N1 vectors.Then the two obtaining recombinant plasmids were transfected into the ST cells and the expression of the TGEV ORF7 protein was verified by western blot.And the subcellular localization of the ORF7 protein was confirmed by combining the bioinformatics and the laser confocal scanning microscopy methods together.Furthermore,the effect of ORF7 protein on TGEV replication was assessed by observation of cytopathic effect(CPE)and quantitative analysis of real-time PCR.The results showed that the recombinant plasmids pGFP-ORF7 and pORF7-GFP were constructed successfully.Western blot analysis showed that the GFP-ORF7 and ORF7-GFP fusion proteins were expressed and the size of TGEV ORF7 was 9 ku.The analysis of combining the bioinformatics and confocal microscopy results showed that the TGEV ORF7 was located at the endoplasmic reticulum,and the subcellular localization might be induced by the N-terminal signal peptide.Furthermore,the real-time PCR results showed the viral RNAs were decreased by 99%in the cells with ORF7 protein over expression,indicating that TGEV ORF7 protein might regulate the replication of TGEV.These results provide some experimental reference for further revealing the function of ORF7 protein.

关 键 词：猪传染性胃肠炎病毒 ORF7蛋白 亚细胞定位 过表达 病毒复制 

分 类 号：S852.65[农业科学—基础兽医学]