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.
出处
《园艺学报》
CAS
CSCD
北大核心
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)
关键词
甜瓜
瓜类褪绿黄化病毒
P22
原核表达
抗血清
melon
Cucurbit chloroticyellows virus
P22
prokaryotic expression
antiserum
