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根据已报道的瓜类褪绿黄化病毒(Cucurbit chlorotic yellows virus,CCYV)外壳蛋白(CP)基因的核苷酸序列设计引物,利用RT-PCR方法从感病甜瓜中扩增得到长度为753 bp的CP基因,将其克隆到原核表达载体pGEX-4T-3上,获得重组载体pGexCP,在大肠杆菌BL21菌株中诱导表达,SDS-PAGE分析表明,经IPTG诱导,CP基因在大肠杆菌BL21中得到了高效表达。以表达的蛋白为抗原,免疫家兔,制备了CCYV外壳蛋白的特异性抗血清。ACP-ELISA检测结果表明,当CP蛋白浓度为2.5μg/mL时,血清效价高达5.12×105。Western blot分析表明制备的抗体检测CCYV侵染的甜瓜叶片得到特异性的条带,表明该抗体的特异性较好。ACP-ELISA检测结果表明,制备的抗血清可用于田间甜瓜病样的检测。本试验为CCYV的快速检测提供了重要的研究材料。
According to the reported nucleotide sequence of CP gene of Cucurbit chlorotic yellows virus (CCYV), primers were designed and amplified from susceptible melon by RT-PCR to obtain a length of 753 bp CP gene was cloned into the prokaryotic expression vector pGEX-4T-3 to obtain the recombinant vector pGexCP, which was induced in E. coli BL21 strain. SDS-PAGE analysis showed that the CP gene was induced in E. coli BL21 by IPTG Efficient expression. The expressed protein was used as antigen to immunize rabbits to prepare specific antisera to CCYV coat protein. The result of ACP-ELISA showed that when the protein concentration of CP was 2.5μg / mL, the titer of serum reached 5.12 × 105. Western blot analysis showed that the prepared antibody detected CCYV-infected melon leaves specific bands, indicating that the specificity of the antibody is better. ACP-ELISA test results showed that the prepared antiserum can be used to detect the disease-like melon in the field. This test provides an important research material for the rapid detection of CCYV.