马铃薯卷叶病毒与S病毒重组双CP多克隆抗体的制备  

Preparation of Polyclonal Antibody Against Recombinant Double CP of Potato leafroll virus and Potato virus S

作　　者：吉祥 宋志成 韦晓玲 杨煜 隋炯明 郭宝太 JI Xiang;SONG Zhicheng;WEI Xiaoling;YANG Yu;SUI Jiongming;GUO Baotai(College of Agronomy,Qingdao Agricultural University,Shandong Province Key Laboratory of Rainfed Agriculture Technology,Qingdao 266109,China;Vegetable and Flower Research Institute,Shandong Academy of Agricultural Sciences,Ji′nan 250100,China)

机构地区：[1]青岛农业大学农学院,山东省旱作农业技术重点实验室,山东青岛266109 [2]山东省农业科学院蔬菜花卉研究所,山东济南250100

出　　处：《华北农学报》2022年第4期212-219,共8页Acta Agriculturae Boreali-Sinica

基　　金：山东省现代农业(薯类)产业技术体系项目(SDAIT-16-03)。

摘　　要：为了制备出马铃薯卷叶病毒(PLRV)与S病毒(PVS)重组双CP多克隆抗体并用于PLRV与PVS这2种病毒的间接与DAS-ELISA检测,构建了PLRV与PVS融合双CP基因的原核表达载体pET22b-LRCP/SCP,用超声破碎法代替溶菌酶法裂解重组菌BL21(pET22b-LRCP/SCP)并提取菌体蛋白,用镍离子亲和层析纯化获得了分子质量为51.2 ku的高纯度的重组双CP。用该重组双CP作抗原免疫家兔获得了高效价(1∶128 k)抗血清。纯化的重组双CP多克隆抗体与PLRV或PVS阳性标准物特异性结合,与另外4种马铃薯主要病毒(PVX、PVY、PVA与PVM)无交叉反应。间接ELISA反应中,稀释度为1∶3200的重组双CP多克隆抗体与PLRV或PVS阳性物都呈现阳性反应;在DAS-ELISA反应中,稀释度均为1∶100的重组双CP多克隆抗体及其碱性磷酸酶标记物与PLRV或PVS的阳性标准物也分别呈现阳性反应。结果表明,制备的重组双CP抗体既可用于PLRV与PVS这2种病毒的间接ELISA检测,也可用于DAS-ELISA检测。The purpose was to prepare polyclonal antibody against the recombinant double CP of Potato leafroll virus(PLRV)and Potato virus S(PVS),and apply the polyclonal antibody to indirect ELISA and DAS-ELISA detections of PLRV and PVS.Prokaryotic expression vector pET22b-LRCP/SCP of the fused double CP gene of PLRV and PVS was constructed.After replacement of lysozyme treatment by ultrasonic disruption,inclusion body protein was extracted from the recombinant strain BL21(pET22b-LRCP/SCP),the target protein(recombinant double CP)was purified by nickel ion affinity chromatography and high-purity target protein of 51.2 ku was obtained.The high-purity recombinant double CP was used as antigen to immunize rabbits to prepare an antiserum with a titer of 1∶128 k.Specific reactions were respectively observed between the purified polyclonal antibody(IgG)against the recombinant double CP and the positive standard of PLRV or PVS and no cross-reaction was found between the purified IgG and other four potato major viruses(PVX,PVY,PVA and PVM).The purified IgG against the recombinant CP with the diluted concentration of 1∶3200 still positively reacted with PLRV or PVS in indirect ELISA detection.The purified IgG and the alkaline phosphatase-conjugated IgG both with the diluted contraction of 1∶100 also positively reacted with PLRV or PVS positive standard in DAS-ELISA detection.The results showed that one type of the prepared IgG against the recombinant double CP could detect two viruses of PLRV and PVS by DAS-ELISA or indirect ELISA.

关 键 词：马铃薯卷叶病毒 S病毒 重组双CP 多克隆抗体 间接ELISA DAS-ELISA 

分 类 号：S432.41[农业科学—植物病理学]