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利用含泡桐丛枝植原体质粒pPaWBNy-1的3.0 kb片段的克隆为模板,PCR扩增pPaWBNy-1-ORF5的亲水性肽段编码区。目的片段连接到原核表达载体pET28a(+),重组质粒pET28a-ORF5-5转化大肠杆菌(Escherichia coli)Rosseta(DE3)感受态细胞,菌液处在对数生长期时(OD600=0.52),添加IPTG诱导5 h后收集菌体,提取蛋白并进行聚丙烯酰胺凝胶电泳。用六聚组氨酸标签抗体进行Western blot检测,发现分子量约为15 kDa的含多聚组氨酸标签的目的蛋白得到表达。切胶回收融合蛋白,免疫德国大白兔以制备抗血清,间接ELISA法测定抗血清的效价约为1∶8 100。用制备的ORF5编码蛋白的抗血清进行Western blot分析,结果在带菌的介体昆虫茶翅蝽(Halyomorpha halys)中检测到大小约为17 kDa的特异蛋白条带,但在健康和感病泡桐(Paulownia sp.)及无菌茶翅蝽中均未检测到,由此推测pPaWBNy-1-ORF5蛋白与介体昆虫传播植原体有关。
The coding region of pPaWBNy-1-ORF5 was amplified by PCR using the 3.0 kb fragment of pPaWBNy-1 containing the plasmid pPaWBNy-1. The target fragment was ligated into the prokaryotic expression vector pET28a (+), and the recombinant plasmid pET28a-ORF5-5 was transformed into Escherichia coli Rosseta (DE3) competent cells. The bacterial suspension was in logarithmic growth phase (OD600 = 0.52) After 5 h IPTG induction, the cells were harvested and the proteins were extracted and subjected to polyacrylamide gel electrophoresis. Western blot analysis using hexahistidine-tagged antibody revealed that the target protein containing a polyhistidine tag with a molecular weight of about 15 kDa was expressed. The gel was recovered and the fusion proteins were recovered. German rabbits were immunized to prepare antiserum. The titer of antiserum was determined by indirect ELISA. Western blot analysis of the prepared antisera encoding the ORF5 protein resulted in the detection of a specific protein band of about 17 kDa in the Halyomorpha halys, but not in healthy and susceptible paulownia Paulownia sp.) And aseptic tea-leaf bugs were not detected, suggesting that the pPaWBNy-1-ORF5 protein is involved in the dissemination of phytoplasmas by mediator insects.