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以被瓜类褪绿黄化病毒(Cucurbit chlorotic yellows virus,CCYV)侵染的甜瓜叶片为供试材料,采用RT-PCR方法克隆其P22蛋白基因,并将其连接到原核表达载体pGex-4T-3上,PCR验证及克隆测序确定开放阅读框的正确性。将重组载体pGexp22转化大肠杆菌BL21菌株,诱导表达,SDS-PAGE分析表明,经IPTG诱导,p22基因在大肠杆菌BL21中得到了高效表达。以表达的蛋白作为抗原,免疫家兔,制备了CCYV P22的特异性抗血清。ACP-ELISA检测结果表明,血清效价高达1.28×105。Western blot检测甜瓜叶片,结果表明抗血清能够特异性地检测CCYV侵染的甜瓜叶片中的CCYV P22蛋白。
Melon leaves infected by Cucurbit chlorotic yellows virus (CCYV) were used as experimental materials. The P22 protein gene was cloned by RT-PCR and ligated into prokaryotic expression vector pGex-4T- 3, PCR validation and cloning sequencing to determine the correctness of the open reading frame. The recombinant vector pGexp22 was transformed into E. coli strain BL21 to induce expression. SDS-PAGE analysis showed that the p22 gene was highly expressed in E. coli BL21 induced by IPTG. The expressed protein was used as antigen to immunize rabbits and the specific antiserum of CCYV P22 was prepared. ACP-ELISA test results showed that the serum titer up to 1.28 × 105. Western blot analysis of melon leaves showed that the antiserum specifically detected CCYV P22 protein in CCYV-infected melon leaves.