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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。
Primers were designed according to the nucleotide sequence of the reported antigenic membrane pro-tein (AMP) gene of Paulownia witches’-broom phytoplasma (PaWB), and the total DNA of P. paulowniae was extracted. Successfully cloned the Paulownia tomentosa amphibian amp gene. Sequence analysis showed that the amp gene consisted of 696 nucleotides and encoded 231 amino acid residues, which shared 100% homology with the membrane protein sequences of two S. pampus plants recorded in GenBank. The 91-604 nt amp gene was cloned into the prokaryotic expression vector pGEX-4T-3, and was analyzed by SDS polyacrylamide gel electrophoresis. The results showed that the fusion protein was obtained in E. coli BL21 (DE3) Efficient expression. PaWB-AMPD antiserum was prepared by immunizing German white rabbits with purified GST-tagged AMPD fusion protein. Using this serum, P. paeoniflorus was detected specifically in S. paulownia tissues by Western blot, dot blot, ELISA, indirect immunofluorescence and immunocapture PCR.