洋葱黄矮病毒检测试剂盒的研制

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

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袁明，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）PlysS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，Westernblot分析对CP具有高度特异性。硫酸铵沉淀初步IgG，然后用Protein A-RedSepharose亲和层析进一步纯化IgG，IgG与甘油1：1混合获得效价1：4800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。

Specific primer was designed to amplify onion yellow dwarf virus（OYDV）Liuan isolate CP genes. It shared 84. 0% - 95.8% nueleotide 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, lgG 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.