侵染生姜的黄瓜花叶病毒检测试剂盒的研制  

Preparation of Reagent Kit for Testing CMV Infecting Zingiber offcinale

作　　者：韦传宝[1,2] 邓辉 WEI Chuan-bao;DENG hui(Biological and Pharmaceutical Engineering College, West Anhui University, Luan 237012, China;Antibody Preparation and Preperty Test Reseach Center, West Anhui University, Luan 237012, China)

机构地区：[1]皖西学院生物与制药工程学院,安徽六安237012 [2]皖西学院抗体制备及其质量检测中心,安徽六安237012

出　　处：《中药材》2017年第10期2241-2245,共5页Journal of Chinese Medicinal Materials

基　　金：安徽省教育厅自然科学重点项目(KJ2013A264;KJ2015A454);安徽省自然科学基金(1408085MC43)

摘　　要：目的:研制出可以检测侵染生姜的黄瓜花叶病毒试剂盒。方法:先采用RT-PCR法检测侵染生姜的黄瓜花叶病毒和烟草花叶病毒,选择仅仅含黄瓜花叶病毒的生姜叶片,采用均浆和高速离心的方法提取黄瓜花叶病毒,通过12%SDS-PAGE和5%~20%梯度SDS-PAGE二次制备电泳的方法分离出黄瓜花叶病毒的外壳蛋白CP,将含CP的聚丙烯酰胺凝胶磨细后免疫小鼠,获得抗血清。对抗血清进行质量的检测,特异性检查采用Western blot方法,结合性采用ELISA方法,合格抗血清通过亲和层析获得Ig G,经过浓缩、透析后与甘油混合获得试剂盒。结果:抗血清中的Ig G仅仅与黄瓜花叶病毒的CP结合,抗血清中的Ig G能够结合天然黄瓜花叶病毒颗粒。结论:该试剂盒能够检测生姜叶片中的黄瓜花叶病毒。Objective ： To prepare a reagent kit for testing Cucumber mosaic virus （CMV） infecting Zingiber offcinale. Methods ： CMV and Tobacco mosaic virus（TMV）infecting Zingiber offcinale were tested by RT-PCR. Zingiber offcinale leaves contained CMV was se- lected, and CMV particle was purified by homogenization and centrifugation, CMV CP was purified through preparation electrophoresis including 12% SDS-PAGE and 5%- 20% gradient SDS-PAGE. Polyacrylamide gel contained CMV CP was ground into suspension, mice were immuned with the suspension, and antiserum was obtained. Antiserum quality was tested, the specific examination was detec- ted by Western blot, and the binding examination was detected by ELISA method. IgG was purified through affinity chromatography method, IgG solution was concentrated and dialyzed to a suitable concentration, and the kit was obtained by mixing with glycerol after enrichment and dialysis. Results：The results indicated that IgG in antiserum only combined with CMV CP protein and it could combine with nature CMV particle CP protein. IgG then mixed with glycerol and a reagent kit for testing CMV infecting Zingiber offcinale was obtained. Conclusion：CMV infecting Zingiber offcinale in the field can be tested by reagent kit.

关 键 词：黄瓜花叶病毒 生姜 试剂盒 

分 类 号：R282.2[医药卫生—中药学]