洋葱黄矮病毒检测试剂盒的研制

根据已报道的洋葱黄矮病毒(OYDV)序列设计引物,扩增洋葱黄矮病毒(OYDV)六安分离物外壳蛋白(CP)基因.序列同源性分析结果表明,与目前已报道的OYDV不同分离物CP基因核苷酸序列同源性为84.0%～95.8%,相应推测的氨基酸序列同源性为86.8%～97.6%.将CP基因插入原核表达载体pSBET后在大肠杆菌BL21(DE3)Plys S中诱导表达.通过12%SDS-PAGE和5%～20%梯度SDS-PAGE两次制备电泳纯化诱导产物,免疫家兔获得抗CP血清,Western blot分析对CP具有高度特异性.硫酸铵沉淀初步IgG,然后用Protein A-Red Sepharose亲和层析进一步纯化IgG,IgG与甘油1: 1混合获得效价1: 4 800的一抗.将一抗、羊抗兔二抗和4-硝基苯基磷酸二钠组成OYDV检测试剂盒,可用于田间OYDV病样检测.

Prokaryotic Expression of OYDV CP Gene and Preparation of the Antibody Against CP

Specific primer was designed to amplify onion yellow dwarf virus(OYDV)Liuan isolate CP genes.It shared 84.0%～95.8%nucleotide acids identities and 86.8%～97.6%amino acids identities with the sequences of CP genes isolates reported in the world.The CP gene was then inserted into pSBET and expressed in Escherichia coli BL21(DE3)plys S strain.The object protein was purified by 12%SDS-PAGE firstly and subsequently 5%～20% gradient SDS-PAGE.The antiserum against the CP was raised in rabbit and its specificity was confirmed by Western blot analysis.IgG against OYDV-CP was purified first by ammonium sulfate sedimentation and then by Protein A-Red Sepharose affinity chromatography.The purified IgG and glycerol were mixed by the ratio of 1: 1 and IgG mixture with 1: 4 800 value was obtained.Test kit consists of IgG against OYDV-CP,IgG raised in sheep(conjugated with alkaline phosphatase)against rabbit IgG and p-Nitrophenyl Phosphate.The test kit prepared in this study could be suitable for OYDV detection.