REV囊膜蛋白的原核表达、抗体制备及鉴定  

Prokaryotic Expression,Antibody Preparation and Identification of REV Envelope Protein

作　　者：朱丽霖 王后坤 陈雪阳 高可丽 方春 梁雄燕[1] 杨玉莹[1] ZHU Lilin;WANG Houkun;CHEN Xueyang;GAO Keli;FANG Chun;LIANG Xiongyan;YANG Yuying(College of Animal Science,Yangtze University,Jingzhou 434025,China)

机构地区：[1]长江大学动物科学学院,湖北荆州434025

出　　处：《江西农业大学学报》2021年第5期1159-1166,共8页Acta Agriculturae Universitatis Jiangxiensis

基　　金：国家自然科学基金面上项目(31972646)。

摘　　要：【目的】禽网状内皮增生症病毒(reticuloendothelial virus,REV)囊膜蛋白与细胞表面受体结合启动病毒感染,并在REV的复制、致病过程中具有重要作用。为探索REV囊膜蛋白相关生物学特性,研究获得了高效表达的禽网状内皮增生症病毒囊膜蛋白,制备多克隆抗体。【方法】利用原核表达载体pET-28a和pGEX-6p-1表达REV囊膜蛋白gp90,并通过Ni2+亲和层析柱对融合蛋白REV-His-gp90进行纯化,纯化的蛋白免疫Balb/c小鼠,制备了多克隆抗体;纯化的REV-GST-gp90蛋白用于检测多克隆抗体效价。【结果】pET-28a-REV-gp90和pGEX-6p-1-REV-gp90表达质粒构建成功,通过SDS-PAGE验证蛋白能在大肠杆菌BL21菌株中表达;Western-blot检测结果显示制备的多克隆抗体具有良好的反应性和特异性;采用间接ELISA法检测抗血清效价为1∶64000;IFA结果显示,制备的多克隆血清能与REV特异性反应。【结论】研究制备的多克隆抗体效价高、特异性良好,为进一步开展REV囊膜蛋白的生物学特性研究及其特异性抗体应用奠定基础。[Objective]Reticuloendothelial virus(REV)envelope protein binds to cell surface receptors to initiate viral infection,and plays an important role in the replication and pathogenic process of REV.In order to explore the biological characteristics of the REV envelope protein,this study obtained the highly expressed avian reticuloendotheliosis virus envelope protein and prepared polyclonal antibody.[Method]The envelope protein gp90 was expressed by the prokaryotic expression vector pET-28a and pGEX-6p-1 respectively.The fusion protein REV-His-gp90 was purified by the Ni2+affinity chromatography column.This purified protein was used to immunize Balb/c mice and prepared polyclonal antibody.Purified REV-GST-gp90 was used to detect titer of the polyclonal antibody.[Result]The pET-28a-REV-gp90 and pGEX-6p-1-REV-gp90 expression plasmids were successfully constructed,and the SDS-PAGE verified that the protein could be expressed in E.coli BL21 strain.The results of Western-blot analysis showed that the prepared polyclonal antibody had good reactivity and specificity.The antibody titer by indirect ELISA method was 1∶64000;the IFA result showed that the prepared polyclonal serum could be specific to the REV wild strain reaction.[Conclusion]The above results comprehensively indicate that the polyclonal antibody prepared in this study has high titer and good specificity and this study has laid a foundation for the further study on the biological characteristics of REV capsule membrane proteins and the application of specific antibodies.

关 键 词：禽网状内皮增生症病毒 原核表达 囊膜蛋白 多克隆抗体 

分 类 号：S852.657[农业科学—基础兽医学]