马MAVS MAb的制备及其应用于EIAV感染机制的初步研究  

Preparation of MAVS MAb and its application in EIAV infection mechanism research

作　　者：孙克会 王欣慧 陈克伟 杨光璞 王晓钧[2] 杜承[2] 郝力力[1] SUN Ke-hui;WANG Xin-hui;CHEN Ke-wei;YANG Guang-pu;WANG Xiao-jun;DU Cheng;HAO Li-li(College of Animal and Veterinary Sciences,Southwest Minzu University,Chengdu 610041,China;Chinese Academy of Agricultural Sciences,Harbin Veterinary Research Institute,Harbin 150069,China)

机构地区：[1]西南民族大学畜牧兽医学院,四川成都610041 [2]中国农业科学院哈尔滨兽医研究所,黑龙江哈尔滨150069

出　　处：《中国预防兽医学报》2022年第12期1317-1323,共7页Chinese Journal of Preventive Veterinary Medicine

基　　金：西南民族大学优秀学生培养工程项目(2021NYYXS83);国家自然科学基金(31772720)。

摘　　要：线粒体抗病毒信号蛋白(MAVS)是机体先天免疫系统中维甲酸诱导基因I(RIG-I)信号通路唯一的接头蛋白,该蛋白对I型干扰素(IFN-I)的激活以及促炎因子的表达至关重要。因人源MAVS抗体不能识别马MAVS(eqMAVS)蛋白,限制了对马属动物病原体调控RIG-I信号通路的研究。为了制备eqMAVS单克隆抗体(MAb),本研究利用PCR方法克隆缺失跨膜域的eqMAVS(eqMAVSΔTM)并构建重组蛋白表达质粒pET32a-eqMAVSΔTM-his、pGEX-6p-1-eqMAVSΔTM-GST,转化大肠杆菌Rosetta(DE3)并经IPTG诱导后,分别利用His和GST亲和层析柱纯化,结果获得eqMAVSΔTM-his蛋白和eqMAVSΔTM-GST蛋白。以eqMAVSΔTM-his蛋白为免疫原免疫6周龄BALB/c小鼠,经常规免疫程序后进行杂交瘤细胞融合,并以eqMAVSΔTM-GST为检测蛋白以筛选杂交瘤细胞。结果显示共获得两株阳性杂交瘤细胞(4E9、3C4)。选用4E9制备腹水并将MAb纯化后,利用SDS-PAGE鉴定纯化的MAb,结果显示,制备的MAb包含重链、轻链,表明MAb纯化成功。对MAb进行亚类鉴定,结果显示其重链均为IgM型,轻链均为kappa型。间接ELISA检测结果显示1 mg/mL MAb的效价可达2×105。将其应用于western blot检测马单核巨噬细胞eMDM和马胎皮肤细胞NBL-6内源性的MAVS和在HEK293T细胞中过表达的eqMAVS;另外,将MAb应用于western blot检测不同剂量EIAV感染NBL-6后内源性eqMAVS蛋白的表达变化。结果显示,该MAb能够特异性识别NBL-6细胞和eMDM细胞内源性表达的eq MAVS蛋白,以及能够特异性识别HEK293T细胞中过表达的eqMAVS蛋白。EIAV感染试验结果显示,随着EIAV感染剂量的增加,内源性eqMAVS蛋白的表达量递减。上述结果表明,本研究制备了eqMAVS的MAb,并利用该MAb首次证明EIAV对eqMAVS的表达具有负调控作用,该MAb为研究EIAV对抗马源RIG-I信号通路的作用机制提供了重要的研究工具。The mitochondrial antiviral signaling(MAVS) protein is the only adaptor protein of the retinoic acid-induced gene I(RIG-I) signal pathway in the innate immune system, which is essential for the activation of type I interferon(IFN-I) as well as the expression of proinflammatory factors. Human MAVS antibodies do not recognize equine MAVS(eq MAVS) proteins, limiting studies on the regulation of RIG-I signaling pathways induced by equine pathogens. In order to prepare eq MAVS monoclonal antibody(MAb), in this study, eq MAVS lacking the transmembrane domain(eq MAVSΔTM) was cloned by PCR and the recombinant protein expression plasmids p ET32a-eq MAVSΔTM-his and p GEX-6p-1-eq MAVSΔTM-GST were constructed,transformed into E. coli Rosetta(DE3) and induced by IPTG, then purified by using His and GST affinity chromatography columns,resulting in 7mg of eq MAVS ΔTM-his protein and 6mg of eq MAVSΔTM-GST protein. Six-week-old BALB/c mouse were immunized with eq MAVSΔTM-his protein as immune antigen, hybridoma cell fusion was performed after routine immunization procedures, and eq MAVSΔTM-GST protein was used as detection protein for screening hybridoma cell, and a total of two positive hybridoma cells(4E9, 3C4) were obtained. 4E9 was selected for the preparation of ascites and the antibody purification. The purified MAb was identified by SDS-PAGE. The results showed that the prepared MAb contained heavy chain and light chain,indicating that the MAb was successfully purified. The subtype of MAb 4E9 was identified as Ig M type for the heavy chain and kappa type for the light chain. The titer for 1mg/m L of MAb is up to 2×105in the indirect ELISA test. The endogenous MAVSs in both the equine monocyte-derived macrophages e MDM and equine fetal skin cell NBL-6, and eq MAVS recombinant protein overexpressed in HEK293T cells were all tested with western blot by using MAb 4E9;in addition, MAb 4E9 was also applied to detect endogenous eq MAVS protein expressed in NBL-6 cells infected with EIAV by western blot. The results showed

关 键 词：单克隆抗体 线粒体抗病毒信号蛋白 原核表达 蛋白纯化 

分 类 号：S852.65[农业科学—基础兽医学]