猪CD1d蛋白多克隆抗体的制备及应用  

Preparation and Application of Polyclonal Antibodies Against Pig CD1d Protein

作　　者：刘传霞 陈欣[1] 王晓[1] 李雪雯 李婷婷 翁长江[1] 郑君[1] LIU ChuanXia;CHEN Xin;WANG Xiao;LI XueWen;LI TingTing;WENG ChangJiang;ZHENG Jun(Division of Fundamental Immunology,National African Swine Fever Para-Reference Laboratory,State Key Laboratory for Animal Disease Control and Prevention/Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Harbin 150069)

机构地区：[1]中国农业科学院哈尔滨兽医研究所/动物疫病预防控制国家重点实验室/国家非洲猪瘟专业实验室/基础免疫创新团队,哈尔滨150069

出　　处：《中国农业科学》2024年第8期1620-1628,共9页Scientia Agricultura Sinica

基　　金：十四五国家重点研发计划(2021YFD1800100);国家自然科学基金(32172874)。

摘　　要：[目的]制备猪源CD1d的多克隆抗体,为探究猪CD1d蛋白在非洲猪瘟病毒(African swine fever virus,ASFV)感染过程中的功能奠定基础。[方法]利用PCR方法扩增了猪CD1d基因,并将其同源重组至pGEX-6p1载体中,构建了原核重组表达质粒pGEX-6p1-CD1d。将重组质粒转化E.coli BL21(DE3)并用IPTG进行诱导表达,表达的GST-CD1d重组蛋白经SDS-PAGE和Western blot(WB)方法鉴定,SDS-PAGE结果显示约50 ku处有一条明显的条带,该蛋白以包涵体形式表达。然后利用谷胱甘肽琼脂糖亲和层析方法进行蛋白纯化,将纯化的GST-CD1d蛋白与等体积的弗氏完全佐剂混合乳化后,将纯化的蛋白免疫新西兰大白兔,采用颈背部多点皮下注射,免疫剂量为200μg/只,首免后第3周和第5周分别进行二免和三免,均采用弗氏不完全佐剂乳化,方法和剂量与首免相同。三免后第7天,通过耳缘静脉采血分离血清。首免后第7周进行第四次免疫,一周后心脏采血。该抗体经Protein G亲和层析纯化后冻存于-80℃冰箱。通过WB和间接免疫荧光(IFA)鉴定瞬时转染表达的外源CD1d蛋白和猪原代巨噬细胞(PAMs)表达的内源CD1d蛋白的表达与细胞定位情况。同样,制备的CD1d抗体可以将外源瞬时转染表达的CD1d通过IP拉下。为了探究CD1d在ASFV感染早期的情况,将ASFV接种于PAMs,制备ASFV感染0、15、30、60 min的样品,以CD1d为一抗,通过WB检测了CD1d蛋白的表达情况。在HEK293T细胞共转pCAGGS-HA-CD1d和pCAGGS-Flag-CD2v质粒,24 h后收取细胞裂解,加入Flag beads过夜结合蛋白,通过WB检测互作情况染,同时,质粒共转染于共聚焦小皿中的HEK293T细胞,用标签抗体对其进行孵育,选择相应的荧光二抗,通过激光共聚焦显微镜观察CD1d与CD2v在细胞中共定位情况。采用Co-IP验证CD1d与ASFV外囊膜蛋白CD2v的相互作用。[结果]原核表达的GST-CD1d蛋白以包涵体形式表达在感受态细胞中,分子质量约为35 ku;实验兔4次免疫CD1【Objective】The aim of this study was to prepare polyclonal antibodies against porcine CD1d protein,so as to lay the foundation for exploring the function of porcine CD1d protein in the process of African swine fever virus(ASFV)infection.【Method】In this study,the pig CD1d gene was amplified using PCR and homologously recombined into the pGEX-6p1 vector,constructing a prokaryotic recombinant expression plasmid pGEX-6p1-CD1d.The recombinant plasmid E.coli BL21(DE3)was transformed and induced for expression using IPTG.The expressed GST CD1d recombinant protein was identified by SDS-PAGE and Western blot(WB)methods.The SDS-PAGE results showed a clear band at approximately 50 ku,which was expressed in the form of an inclusion body.Then,protein purification was performed using glutathione agarose affinity chromatography.The purified GST-CD1d protein was mixed and emulsified with an equal volume of Freund's complete adjuvant.The purified protein was immunized in New Zealand white rabbits and administered subcutaneously at multiple points on the neck and back,with an immune dose of 200μG/piece,and then second and third immunizations were performed at the 3rd and 5th weeks after the first immunization,respectively,using Freund's incomplete adjuvant emulsification,with the same method and dosage as the first immunization.On the 7th day after the third immunization,the blood was collected from the ear vein to isolate the serum.The fourth immunization was conducted at the 7 weeks after the first immunization,and the blood was collected from the heart one week later.The antibody was purified by Protein G affinity chromatography and frozen at-80℃.The expression and cellular localization of endogenous CD1d protein expressed by transient transfection of exogenous and porcine primary macrophages(PAMs)were indentified by using WB and indirect immunofluorescence(IFA).Similarly,the prepared CD1d antibody could pull down CD1d expressed by transient exogenous transfection through IP.In order to investigate the early stage of

关 键 词：CD1d蛋白 原核表达 多克隆抗体 非洲猪瘟病毒 CD2v蛋白 

分 类 号：S852.4[农业科学—基础兽医学]