瓜类褪绿黄化病毒外壳蛋白基因在大肠杆菌中的表达及抗血清的制备

 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2.5 μg/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。

Expression of coat protein gene of Cucurbit chlorotic yellows virus in E.coli and preparation of antiserum

Primers were designed according to the published nucleotide sequence for amplifying the CP gene by RT-PCR. CP gene was cloned into the prokaryotic expression vector pGEX-4T-3 and the recombinant plasmid, named as pGexCP, was transformed into Escherichia coli BL21. SDS-PAGE analyses showed that specific fusion protein was efficiently expressed by IPTG induction. The expressed protein was purified as antigen and the antiserum against the protein was raised in rabbit. The titer of antiserum was 5.12?10⁵ estimated by ACP-ELISA. Western blot confirmed the detection specificity by polyclonal antiserum with diseased leaf. The results suggested that the antiserum could be applied for detection of field samples. This experiment laid the foundation for field detection of CCYV.