苹果茎沟病毒外壳蛋白基因的克隆、原核表达及抗血清制备

Cloning,prokaryotic expression of the coat protein gene of Apple stem grooving virus and antiserum preparation

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。

Production of polyclonal antibody using prokaryotic expressed recombinant viral structure proteins as antigen is an efficient method for virus detection at low cost.In this study,the coat protein（ASGV cp BJ） gene of Apple stem grooving virus（ASGV） was amplified by RT-PCR using total nuclei acid extracted from apple fruit skin.The amplified fragment was cloned and confirmed by sequence analysis,which consisted of 714 nucleotides.To express recombinant ASGV CP in Escherichia coli,the cloned ASGV cp gene was inserted into expression vector pET-28a.The resulted recombinant plasmid pET-ASGV cp was transformed into E.coli BL21（DE3）.After induction by IPTG,the recombinant ASGV cp was expressed as a 33kD inclusion body protein at high level in SDS-PAGE analysis.Through Ni beads affinity purification,the expressed protein was used as antigen to produce antiserum in rabbit.By antigen coating plate-ELISA（ACP-ELISA）,the antiserum＇s titer was determined to be 1∶4096.Western blot results showed that the antiserum was specific to ASGV and could be used for detection.