瓜类褪绿黄化病毒p22基因在大肠杆菌中的表达及抗血清的制备  

Expression of p22 Gene of Cucurbit chlorotic yellows virus in Escherichia coli and Preparation of Antiserum

作　　者：施艳[1] 王振跃[1] 袁媛[1] 刘珊珊[2] 孙虎[3] 古勤生[2] 

机构地区：[1]河南农业大学植物保护学院,郑州450002 [2]中国农业科学院郑州果树所,郑州450009 [3]河南省农业科学院植物保护所,郑州450002

出　　处：《园艺学报》2013年第4期762-766,共5页Acta Horticulturae Sinica

基　　金：现代农业产业技术体系建设专项(CARS-26-13);NSFC–河南人才培养联合基金项目(U1204319)

摘　　要：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。p22 gene was amplified by RT-PCR from CCYV infected melon leaves and cloned into the prokaryotic expression vector pGex-4T-3. After verification by PCR and sequencing, the recombinant plasmid was transformed to Escherichia coli strain BL21 for protein expression. The SDS-PAGE analyses showed that 48 kD specific fusion protein was highly expressed after induction by IPTG. The expressed protein was purified from SDS-PAGE and the antiserum against the protein was raised in rabbit. The titer of antiserum was 1.28 × 10^5 estimated by ACP-ELISA. Western blot analysis confirmed that the antiserum reacted specifically with P22 protein of CCYV.

关 键 词：甜瓜 瓜类褪绿黄化病毒 P22 原核表达 抗血清 

分 类 号：S652[农业科学—果树学]