泡桐丛枝植原体pPaWBNy-1-ORF5编码蛋白的原核表达和抗体制备

Prokaryotic expression of pPaWBNy-1-ORF5 encoding protein of paulownia witches'-broom phytoplasma and preparation of the polyclonal antiserum

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 hydrophilic polypeptide-coding region of ORF5 was amplified by PCR using the clone containing a 3.0 kb fragment of pPaWBNy-1 as DNA template.The amplified DNA fragment was inserted into the prokaryotic expression vector pET28a（＋）.The recombinant plasmid pET28a-ORF5-5 was transformed into the E.coli Rosseta（DE3） strain.During the mid-exponential growth （OD600 =0.52）,cultures was added IPTG to induce the expression of His-ORF5 protein for 5 hours.The fusion protein was then performed with SDS-PAGE.Western blot analysis was carded out with anti-His tag polyclonal antibody.The results indicated that the 15 kDa His-ORF5 fusion protein was expressed in E.coli Rosseta（DE3） strain.The fusion protein was purified and injected into a German white rabbit to raise antiserum.The titer of the antiserum was 1∶8 100 tested by indirect ELISA.One 17 kDa specific protein band was detected by Western blot analysis in PaWB-infected vector stink bug Halyomorpha haly,while no ORF5 protein was detected in PaWB-infected Paulownia spp.and non-infected H.haly.Therefore,it is presumed that ORF5 protein is involved in the transmission of insect vector.