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利用原核系统表达病毒编码的结构蛋白为抗原制备多克隆抗体是提高检测灵敏度和降低检测成本的重要途径。根据已报道的苹果茎沟病毒Apple stem grooving virus(ASGV)外壳蛋白(coatprotein,CP)基因的核苷酸序列设计合成引物,利用RT-PCR方法克隆了ASGV的cp基因(命名为ASGV cp BJ),该基因由714个核苷酸组成,与GenBank中已报道的ASGV的cp基因核苷酸相似性为86%~96%。将ASGV的cp基因克隆到原核表达载体pET-28a上,转化大肠杆菌BL21(DE3),筛选得到阳性克隆pET-ASGVcp。SDS-PAGE电泳分析发现,经IPTG诱导,ASGVcp在大肠杆菌中得到高效表达,获得的融合蛋白的分子量约为33kD。利用Ni珠吸附法纯化原核表达的目的蛋白,并以此蛋白为抗原制备了抗血清,ACP-ELISA检测结果显示,此抗血清效价为1∶4 096。Western blot分析结果显示,该抗血清具有高度特异性,能够用于ASGV的快速检测。
The use of prokaryotic expression of the virus-encoded structural proteins as antigens to prepare polyclonal antibodies is an important way to improve the detection sensitivity and reduce the cost of detection. The ASGV cp gene (named as ASGV cp BJ) was cloned by RT-PCR from the reported nucleotide sequence of the coat protein of apple stem grooving virus (ASGV) This gene consists of 714 nucleotides and has nucleotide similarity of 86% -96% with that of ASGV reported in GenBank. The cp gene of ASGV was cloned into the prokaryotic expression vector pET-28a and transformed into E. coli BL21 (DE3) to obtain the positive clone pET-ASGVcp. SDS-PAGE electrophoresis analysis showed that induced by IPTG ASGVcp highly expressed in E. coli, the molecular weight of the fusion protein obtained was about 33kD. The target protein expressed in prokaryotic cells was purified by Ni beads adsorption method, and the antiserum was prepared using this protein as antigen. The result of ACP-ELISA showed that the antiserum titer was 1:4 096. Western blot analysis showed that the antiserum was highly specific and could be used for the rapid detection of ASGV.