泡桐丛枝植原体抗原膜蛋白抗血清的制备及应用

Preparation and application of antiserum against antigenic membrane protein of Paulownia witches′ broom phytoplasma

根据报道的泡桐丛枝植原体(Paulownia witches'-broom phytoplasma,PaWB)抗原膜蛋白(antigenic membrane pro-tein,AMP)基因的核苷酸序列设计引物,提取发病泡桐总DNA,经PCR扩增并成功克隆泡桐丛枝植原体amp基因。序列分析表明,amp基因由696个核苷酸组成,编码231个氨基酸残基,与GenBank中登录的2个泡桐丛枝植原体的膜蛋白核苷酸序列同源性为100%。将amp基因91-604 nt部分序列(命名为ampd)克隆到原核表达载体pGEX-4T-3,诱导后,经SDS聚丙烯酰胺凝胶电泳分析,表明融合蛋白在大肠杆菌BL21(DE3)中得到了高效表达。以纯化的带GST标签的AMPD融合蛋白为抗原免疫德国大白兔,制备了PaWB-AMPD抗血清。利用该血清,通过Western印迹、点印迹、ELISA、间接免疫荧光和免疫捕获PCR试验均能在发病泡桐组织中特异检测到泡桐丛枝植原体。

Antigenic membrane proteins（AMPs） encoded by phytoplasma play important roles in recognition between pathogen and host.To study the function of AMP of Paulownia witches′broom（PaWB） phytoplasma and detect the pahogen,specific primers were designed and used for amplification of the amp gene.The sequencing results showed that the open reading frame（ORF） of amp gene consisted of 696 nt and encoded 231 amino acid residues,which were 100% identical to two other sequenced amp genes of PaWB phytoplasma.For antibody preparation,the partial sequence of amp from nt 91 to 604 was cloned into the expression vector pGEX-4T-3.After induction by IPTG,the total protein was separated by SDS-PAGE,which showed that the fusion protein was overexpressed in E.coli.The antiserum against the fusion protein was raised in rabbit and applied specifically for detection of PaWB phytoplasma in Western blot,dot blot,ELISA,indirect immunofluorescence and immunocapture PCR assays.