番木瓜环斑病毒外壳蛋白基因原核表达蛋白的抗血清制备及其检测应用  

Preparation and Application of the Antiserum Specific to the Coat Protein Expressed Prokatically of Papaya ring spot virus

作　　者：赵芹[1] 李华平[2] 谢大森[1] 何晓明[1] 张曙光[2] 罗少波[1] 

机构地区：[1]广东省农业科学院蔬菜研究所,广州510640 [2]华南农业大学植物病毒研究室,广州510642

出　　处：《园艺学报》2012年第8期1457-1464,共8页Acta Horticulturae Sinica

基　　金：广东省自然科学博士启动基金项目(10451064001006063);广东省农业科学院院长基金项目(1279009475432)

摘　　要：以受番木瓜环斑病毒(Papaya ring spot virus,PRSV)侵染的番木瓜(Carica papaya)叶片为供试材料,采用RT-PCR方法克隆其外壳蛋白基因cp,并将其连接到原核表达载体pET-28b(+)上,酶切鉴定及克隆测序确定开放阅读框的正确性,将获得的重组质粒转化大肠杆菌表达宿主菌。通过摸索转化的表达宿主菌种类、IPTG浓度及诱导时间,获得高效表达PRSVcp的条件。SDS-PAGE分析结果表明,CP融合蛋白分子量为36.8kD。以Ni2+-NTA亲和层析柱纯化的融合蛋白为抗原,免疫注射新西兰大白兔制备得到高效价抗体,间接ELISA测定效价为1︰16384。Western blot检测结果表明,制备的抗血清可与诱导表达的融合蛋白发生特异性反应。通过ID-ELISA检测田间样品证实了制备的抗血清与PRSV侵染病叶发生了良好的特异性反应。本试验为PRSV的快速检测以及PRSV编码蛋白的功能研究奠定了基础。Coat protein gene （cp） was amplified by RT-PCR from papaya leaves infected by Papaya ring spot virus and cloned to prokaryotic expression vector pET28b （＋） . After identification by enzyme digestion and sequencing, the recombinant clone was transformed to Escherichia coli for protein expression. The fusion protein was highly expressed by groping the expressing host bacteria, concentration of IPTG and inducing time. SDS-PAGE indicated that the molecular weight of fusion protein highly expressed was 36.8 kD. The purified protein by Ni2＋-NTA affinity chromatography was used as antigen to immunize the healthy rabbit for antiserum preparation. Indirect-ELISA （ID-ELISA） assay showed the optimal titer of the antiserum was 1 ： 16 384. Western blot analysis confirmed that the antiserum reacted specially with CP protein of PRSV. ID-ELISA testing of a number of field samples demonstrated the sensitivity and specificity of the antiserum. The present study PRSV detection and the research protein function of PRSV.

关 键 词：番木瓜 番木瓜环斑病毒 外壳蛋白基因(cp) 原核表达 抗血清制备 

分 类 号：S667.9[农业科学—果树学]