摘要
为了获得猪源干扰素α(PoIFN-α)的单克隆抗体,本研究首先将PoIFN-α基因连接到pcDNA3.1真核表达载体从而构建rPoIFN-α质粒,通过真核表达系统(ExpiCHOTM)表达蛋白,并用亲和层析技术纯化后对重组蛋白进行鉴定;以重组蛋白为抗原免疫BALB/c小鼠后进行细胞融合,用建立的间接ELISA方法筛选阳性杂交瘤细胞,亚克隆后通过制备腹水得到单克隆抗体,纯化制备的抗体并鉴定。结果显示,成功构建了rPoIFN-α质粒,通过ExpiCHOTM表达系统成功表达出正确大小的可溶性蛋白,分子质量为20 ku;通过间接ELISA筛选和5次亚克隆,得到1株能稳定分泌抗IFN-α蛋白的单克隆细胞株(3c6),亚型鉴定为IgG1亚型、IgGκ轻链;Western-blot结果显示,抗体可以特异识别IFN-α,抗体纯化后效价检测可达1∶256000。上述结果表明,本试验制备的rPoIFN-α蛋白纯度高,单克隆抗体特异性强,可以在以后的研究中用于建立不同的IFN分子的检测方法,为研究猪病毒感染和免疫过程中不同细胞因子的应答情况提供便利。
In order to obtain monoclonal antibodies against porcine interferon-α(rPoIFN-α),the open reading frame of PoIFN-αwas cloned into pcDNA3.1 eukaryotic expression vector to obtain rPoIFN-αplasmid.The recombined proteins were expressed in eukaryotic expression system(ExpiCHOTM)and purified by affinity chromatography,and then validated it.Mice were immunized with the recombinant proteins and cell fusion was performed.Indirect ELISA method was established to screen positive hybridoma cells.After subcloning,monoclonal antibodies were obtained by preparing ascites,and prepared antibodies were purified and tested.Result:the rPoIFN-αplasmid was successfully constructed and the correct size of soluble protein with molecular weight of 20 ku was successfully expressed by ExpiCHOTM expression system.A monoclonal cell line(3c6)stably secretes anti-IFN-αantibody was obtained by indirect ELISA screening and subcloning for 5 rounds.The antibody was identified as IgG1 subtype and IgGκlight chain.Western-blot showed the antibody could specifically detect IFN-α.After purification,titer of the antibody could be as high as 1∶256000.The rPoIFN-αprotein prepared in this study has high purity,and the monoclonal antibody is highly specific,detection methods of IFN molecules can be established in future research to provide some experimental convenience for studying the secretion of different cytokines in the process of swine virus infection and immunity.
作者
孙波涛
张涛清
孙冰洁
王常英
李亚军
杨锐
秦晓东
冷青文
郑海学
SUN Bo-tao;ZHANG Tao-qing;SUN Bing-jie;WANG Chang-ying;LI Ya-jun;YANG Rui;QIN Xiao-dong;LENG Qing-wen;ZHENG Hai-xue(State Key Laboratory for Animal Disease Control and Prevention,Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Lanzhou 730046,China;School of Animal Science and Technology,Shihezi University,Shihezi 832001,China)
出处
《中国兽医科学》
CAS
CSCD
北大核心
2023年第6期684-690,共7页
Chinese Veterinary Science
基金
国家自然青年科学基金项目(C1804)
科技重大专项-国家重点实验室重组项目(22ZD6NA001)
国家生猪产业技术体系项目(CARS-35)
“十四五”广东省揭榜挂帅项目(2022SDZG02)。
关键词
干扰素
蛋白表达纯化
单克隆抗体
interferon
protein expression and purification
monoclonal antibody
