奶牛ARPC3多克隆抗体制备及ARPC3在乳腺组织中的表达  

Preparation of Bovine(Bos taurus)ARPC3 Polyclonal Antibody and Expression of ARPC3 in Mammary Tissue

作　　者：白文凯 刘佩雯 陈艳 王安 方博 杨庭基 张全伟 王琪[1,2] 赵兴绪[1,2] 董伟韬 张勇[1,2] BAI Wen-Kai;LIU Pei-Wen;CHEN Yan;WANG An;FANG Bo;YANG Ting-Ji;ZHANG Quan-Wei;WANG Qi;ZHAO Xing-Xu;DONG Wei-Tao;ZHANG Yong(College of Veterinary Medicine,Gansu Agricultural University,Lanzhou 730070,China;Gansu Key Laboratory of Animal Generational Physiology and Reproductive Regulation,Lanzhou 730070,China;College of Life Science and Technology,Gansu Agricultural University,Lanzhou 730070,China)

机构地区：[1]甘肃农业大学动物医学院,兰州730070 [2]甘肃省动物生殖生理及繁殖调控重点实验室,兰州730070 [3]甘肃农业大学生命科学技术学院,兰州730070

出　　处：《农业生物技术学报》2025年第4期933-944,共12页Journal of Agricultural Biotechnology

基　　金：甘肃省教育厅青年博士支持项目(2024QB-064);甘肃省科技厅青年科技基金(24JRRA65);国家自然科学基金(U21A20262)。

摘　　要：肌动蛋白相关蛋白2/3复合体亚基3(actin associated protein 2/3 complex subunit 3,ARPC3)是肌动蛋白骨架组织的重要组成部分,对细菌的侵袭、炎症发展和细胞凋亡具有重要作用。为了研究ARPC3蛋白对奶牛(Bos taurus)乳房炎的作用,本研究利用在线软件对奶牛ARPC3进行生物学特性分析,构建pQE-80L-ARPC3重组质粒,通过原核表达系统诱导表达蛋白,并对异丙基-β-D-硫代半乳糖(isopropylbeta-D-thiogalactopyranoside,IPTG)浓度和诱导时间进行优化。将表达的蛋白经亲和层析法纯化,通过SDS-PAGE和质谱鉴定后免疫新西兰大白兔(Oryctolagus cuniculus)以制备多克隆抗体,采用ELISA和Western blot分别鉴定多克隆抗体的效价和特异性。结果显示,成功构建了重组质粒pQE-80L-ARPC3,最佳诱导表达条件为IPTG浓度1 mmol/L诱导8 h,经IPTG诱导后获得了以包涵体形式表达的重组蛋白(大小约为20.5 kD)。ARPC3多克隆抗体血清效价为1∶128000。天然蛋白Western blot鉴定表明,ARPC3多克隆抗体血清能特异性识别目的蛋白和乳腺组织中的ARPC3。免疫组织化学(immunohistochemical,IHC)结果表明,ARPC3蛋白的表达与相关凋亡蛋白的表达呈正相关,所制备多克隆抗体能很好地应用于奶牛乳房炎的检测。本研究成功制备出特异性良好的奶牛ARPC3多克隆抗体,可以用于奶牛乳房炎快速检测新技术的建立及应用。Actin associated protein 2/3 complex subunit 3(ARPC3)is an important component of actin skeleton tissue,which plays an important role in bacterial invasion,inflammation development and apoptosis.To investigate the role of ARPC3 protein in bovine mastitis,in this study,rabbit polyclonal antibody against bovine(Bos taurus)ARPC3 was prepared and its activity was identified.The biological characteristics of bovine ARPC3(NM 001034271.2)were analyzed by online software.pQE-80L-ARPC3 recombinant plasmid was constructed.The prokaryotic expression system was used to express ARPC3.The concentration of isopropyl-beta-D-thiogalactopyranoside(IPTG)and induction time were optimized.The expressed protein was purified by affinity chromatography,identified by SDS-PAGE and mass spectrometry,and then immunized New Zealand white rabbits(Oryctolagus cuniculus)to prepare polyclonal antibody.The titer of polyclonal antibody was detected by ELISA,and the specificity of polyclonal antibody was identified by Western blot.The results showed that the recombinant plasmid pQE-80L-ARPC3 was successfully constructed,and the recombinant protein(about 20.5 kD)was expressed in the form of inclusion bodies after IPTG induction.The optimal expression conditions were as follows:IPTG concentration was 1 mmol/L for 8h.The titer of ARPC3 polyclonal antibody was 1∶128000.Western blot identification of natural protein showed that ARPC3 polyclonal antiserum could specifically recognize ARPC3 in target protein and mammary tissue.Immunohistochemical(IHC)results showed that the polyclonal antibody could be effectively applied for the detection of bovine mastitis,and the expression of ARPC3 protein was positively correlated with the expression of related apoptotic proteins.In this study,the polyclonal antibody of ARPC3 with good specificity was successfully prepared,which can be used to the establishment and application of a new technology for rapid detection of bovine mastitis.

关 键 词：奶牛 肌动蛋白相关蛋白2/3复合体亚基3(ARPC3) 原核表达 抗体制备 乳房炎 

分 类 号：S852.43[农业科学—基础兽医学]