重组牛IFN-ω12生物学特性及其免疫调节机制的研究  

Biological characterization and immunomodulatory mechanism of recombinant bovine interferon-omega12

作　　者：安东 田其真[1] 曹斌[1] 刘静[1] 周宁 吉大庆 卢炜[1] AN Dong;TIAN Qi-zhen;CAO Bin;LIU Jing;ZHOU Ning;JI Da-qing;LU Wei(Jiangsu Agri-animal Husbandry Vocational College,Taizhou 225300,China)

机构地区：[1]江苏农牧科技职业学院,江苏泰州225300

出　　处：《中国预防兽医学报》2023年第7期737-743,共7页Chinese Journal of Preventive Veterinary Medicine

基　　金：江苏农牧科技职业学院大学生创新创业项目(202112806124Y);江苏农牧科技职业学院课题(NSF2023ZR21)。

摘　　要：为表达重组牛干扰素ω12(rBoIFN-ω12),并分析该蛋白的稳定性和免疫调节机制,本研究首先从牛的肝脏基因组中扩增牛IFN-ω12基因,构建克隆载体,从克隆载体中扩增牛IFN-ω12成熟肽编码基因,并将其克隆于p ET-32a载体构建了重组表达载体p ET-32a-Bo IFNω12,经酶切和测序鉴定正确后转化大肠杆菌诱导表达重组蛋白r Bo IFNω12。在不同细胞系中检测纯化后r Bo IFNω12蛋白的生物学活性,结果显示r Bo IFNω12在MDBK和PK-15细胞中对水泡性口炎病毒(VSV)具有抗病毒活性;此外,40μg/m L的r Bo IFNω12在MDBK细胞中相对r Bo IFNαA有较低细胞毒性。经酸碱或高温处理后,r Bo IFNω12仍具有抗病毒活性。通过双荧光素酶试验在牛肺细胞(BL)中检测r Bo IFNω12蛋白对IFN信号通路中关键元件的影响,结果显示与未加r Bo IFN-ω12的对照细胞相比,r Bo IFNω12能够有效激活NF-κB响应元件、ISRE结合元件和Bo IFN-β启动子调控的荧光素酶活性。通过荧光定量PCR和western blot检测r Bo IFNω12作用MDBK细胞后不同时间IFN刺激因子(ISG)的表达情况,结果显示r Bo IFNω12作用MDBK细胞后,细胞中ISG15、ISG56、Mx-1的m RNA转录水平及表达量在3 h~24 h逐渐增多,Mx-1蛋白表达量在6 h~24 h比未处理组增多。本研究表明牛IFN-ω12具有抗病毒和抗细胞增殖活性,细胞毒性较低,可刺激细胞中NF-κB下游ISG中的Mx1、ISG15、ISG56的表达,并能启动细胞中NF-κB、ISRE、Bo IFN-β启动子激活的荧光素酶活性,具有典型I型IFN的特征,可作为有效的抗病毒药物,为牛IFN-ω系统的进一步研究提供思路。A novel bovine IFN-ω12 was cloned and expressed,the protein stability and biological activity were analyzed in this study.Firstly,bovine IFN-ω12 gene was cloned from bovine liver genome and clonal vector was constructed.Bovine IFN-ω12 mature peptide encoding gene was amplified from clonal vector,and the prokaryotic expression vector pET-32aBo IFNω12 was constructed,then prokaryotic expression vector pET-32a-BoIFNω12 was constructed,and the recombinant vector was transformed into E.coli to express the fusion protein r BoIFNω12.The biological activity of the purified protein was detected in different cell lines.Results showed that r BoIFNω12 had antiviral activity against VSV on MDBK cells and PK-15 cells.In addition,r BoIFNω12 at 40μg/m L was less cytotoxic on MDBK cells than r BoIFNαA.After acid-base treatment or high temperature treatment,r BoIFNω12 still showed antiviral activity.The effect of r BoIFNω12 protein on key elements of interferon signaling pathway was detected by dual-luciferase assay on BL cells.The results showed that r Bo IFNω12 could effectively activate the luciferase activity regulated by NF-κB response element,ISRE combining element and Bo IFN-βpromoter.Fluorescence guantitative PCR and western blot assay were used to detect the expression of ISGs in MDBK cells treated with r Bo IFNω12,the results showed that the m RNA transcription and expression level of ISG15,ISG56 and Mx-1 were gradually increasing over time.The expression of Mx-1 was significantly higher than that of the untreated group.This study revealed that Bo IFN-ω12 has antiviral and anti-proliferating activities with low cytotoxicity.It can stimulate the production of downstream proteins Mx1,ISG15,ISG56,and activate the luciferase activity activated by the promoter of NF-κB,ISRE,and Bo IFN-β,has the typical characteristics of type I interferon,which can not only be a potential candidate for a novel,effective therapeutic agent,but also facilitate further research on the role of bovine IFN system.

关 键 词：牛 IFN-ω12 分子特征 抗病毒活性 干扰素刺激基因 

分 类 号：S852.4[农业科学—基础兽医学]