葡萄病毒B外壳蛋白原核表达及抗血清制备

Prokaryotic Expression of Grapevine virus B Coat Protein and Antiserum Preparation

根据已报道的葡萄病毒B(Grapevine virus B,GVB)核苷酸序列设计引物,采用RT-PCR方法从葡萄中扩增获得GVB外壳蛋白基因(cp),大小为594 bp。通过序列测定和分析,选取优势分离物GVB-JFL cp基因克隆到原核表达载体pET-28a,转化大肠杆菌BL21(DE3),IPTG诱导表达并纯化融合蛋白pET-GVB CP。SDS-PAGE电泳结果显示,融合蛋白获得过量表达,分子量约26 kD。以纯化蛋白为抗原免疫新西兰大耳白兔制备抗血清。采用间接ELISA和dot-ELISA对抗血清特异性进行检测,结果表明,抗血清可与感染GVB的西方烟和葡萄样品发生阳性反应,与健康植株及感染同属另一种病毒葡萄病毒A(Grapevine virus A,GVA)的样品不反应,阳性样品检测效价达1︰4 000,16个田间样品摩擦接种至烟草后有9个样品ELISA检测为阳性,表明制备的抗血清可用于GVB的特异性检测。

In this study, the coat protein gene （ cp, 594 bp） of Grapevine virus B （ GVB ） was amplified from grapevine by RT-PCR using primers designed according to previous reported GVB sequences. After sequence determination and analysis, the cp gene of preponderant isolate GVB-JFL was inserted into expression vector pET-28a and constructed recombinant plasmid pET-GVB cp, pET-GVB cp was then transformed into E. coli BL21 （DE3） and induced by IPTG. SDS-PAGE analysis showed that the coat protein （approximately 26 kD） was induced at a high level. The purified coat protein was used as antigen for raising antiserum in rabbits, and the specificity of the antiserum was tested by ELISA and dot-ELISA. The results showed that the antiserum could be successfully used to detect GVB in the infected Nicotiana occidentalis and grapevine, but had no reaction with the healthy plants and Grapevine virus A （GVA, another member ofvitiruses） infected plants, the antibody titer for positive samples could be up to 1 ： 4 000, and 9 of the 16 field samples detected after inoculated in tobacco were positive, these results indicated that the antiserum obtained in the study could be used for efficient and specific detection of GVB.