落葵皱缩花叶病毒CP基因的原核表达及多克隆抗血清的制备  

Prokaryotic Expression of Basella rugose mosaic virus CP Gene and Preparation of Its Polyclonal Antiserum

作　　者：李小琴 张鹏远[1,2] 任龙辉 代瑾然[1] 王建光 陈穗云[1] 

机构地区：[1]云南大学生命科学学院,昆明650091 [2]西南林业大学生命科学学院,昆明650224 [3]云南香料烟有限责任公司,云南保山678000

出　　处：《园艺学报》2017年第10期2001-2007,共7页Acta Horticulturae Sinica

基　　金：国家自然科学基金项目(31101416;31660038)

摘　　要：以感病紫茉莉叶片为材料,采用RT-PCR方法克隆落葵皱缩花叶病毒(Basella rugose mosaic virus,Ba RMV)的外壳蛋白(coat protein,CP)基因,并构建p ET30a-Ba RMV-cp原核表达载体。将原核表达载体转化大肠杆菌[Rosetta(DE3)Plys S],成功诱导表达得到大小约为40.0 k D的与预期大小相符的融合蛋白(His-CP)。SDS-PAGE电泳完毕后用0.25 mol·L^(-1)的KCl溶液染色,将目的蛋白切胶纯化后免疫新西兰大白兔,制备抗血清。间接酶联免疫吸附法(ID-ELISA)检测该抗体的效价为1︰16 000,Western-blotting分析表明该抗体具有很强的特异性。对野外感病紫茉莉和实验室内接种发病本氏烟检测结果也表明,所制备的抗体具有良好的特异性,可用于大规模样品检测。The full length cp gene of Basella rugose mosaic virus（Ba RMV）was obtained by RT-PCR in the infected Mirabilis jalapa L.leaves and the prokaryotic experssion vector of Ba RMV cp gene was successfully constructed also.The p ET30 a-Ba RMV-cp vector was transferred into Rosetta（DE3）Plys S.SDS-PAGE results indicated that the His-CP fusion protein was about 40.0 k D in size,which was in accord with the prediction.Target proteins were isolated by cutting the gel slices that contained the 40.0 k D bands which were stained by 0.25 mol · L^（-1） KCl.Purified fusion protein was used to immunize New Zealand white rabbits to produce the antiserum.The polyclonal antiserum was detected with indirect enzyme-linked immunosorbent assay（ID-ELISA）,the titer of the antiserum was1︰16 000,Western-blotting analysis showed that the antiserum has very strong specificity.Detected the field susceptible M.jalapa L.and the laboratory inoculated and infectious Nicotiana benthamiana L.by the antiserum,the test result also showed that the prepared antiserum has good specificity and can be used for large-scale sample testing.

关 键 词：落葵皱缩花叶病毒 紫茉莉 CP基因 多克隆抗血清 ID-ELISA 蛋白质印迹法 

分 类 号：S681.9[农业科学—观赏园艺]