摘要
根据已报道的小西葫芦黄花叶病毒(ZYMV)序列设计特异引物,扩增ZYMV的全长外壳蛋白(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二次制备电泳纯化诱导产物,免疫小鼠,获得经过Western blot分析为特异的抗CP血清。硫酸铵沉淀法与Protein A-Red Sepharose亲和层析相结合提取IgG,获得效价达1∶4800的一抗,对西瓜和甜瓜田间样品的间接ELISA检测表明,ZYMV在田间普遍发生,研究制备的IgG可用于ZYMV检测。
Specific primer was designed to amplify ZYMV complete coat protein （CP） gene. The CP gene was then inserted into pSBET vector 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 prepared from mice and its specificity was confirmed by Western blot analysis. IgG against ZYMV-CP was purified by ammonium sulfate sedimentation and Protein A-Red Sepharose affinity chromatography. The value of IgG was about 1 ： 4800. Indirect ELISA was used to detect the occurrence of ZYMV in some Chinese watermelon and muskmelon samples. It was indicated that this virus widely spread and commonly existed. The IgG prepared in this study could be suitable for ZYMV detection.
出处
《生物学杂志》
CAS
CSCD
2010年第1期35-38,共4页
Journal of Biology
基金
安徽省教育厅青年项目基金(No.2007iq1184)资助
