绵羊肺炎支原体标准株Y98外膜蛋白MOP30基因真核表达载体的构建及其在烟草中的瞬时表达

以绵羊肺炎支原体（Mycoplasma oumvipneoniae,MO）标准株Y98基因组为模板,设计1对特异引物,PCR扩增得到798bp的P30目的基因（MOP30）,将其定向克隆到pMD19-T Simple载体中进行测序分析。重组质粒进行XbaⅠ/BamHⅠ双酶切并回收目的片段,将目的基因亚克隆入黄色荧光蛋白（yellow fluorescent protein,YFP）表达载体pCAMBIA1300-YFP中构建重组融合表达质粒pCAMBIA1300-MOP30-YFP。双酶切验证后的融合表达质粒转化农杆菌（Agrobacterium）感受态细胞,侵染烟草（tobacco）叶片。通过激光共聚焦成像显微镜（cofocal imagingmicroscope）观察到融合表达的黄色荧光蛋白,Western-blotting试验得到57 000的特异条带,RT-PCR检测到MOP30基因在烟草叶片中转录。MOP30-YFP融合蛋白在烟草中的成功表达,为转基因植物疫苗防治绵羊支原体肺炎奠定了基础。

Construction of eukaryotic expression vector of outer membrane protein P30 from Mycoplasma oumvipneoniae and its transient expression in tobacco

In this study,the gene of the outer membrane protein P30 with the length of 798 bp was amplified from the genome of Mycoplasma oumvipneoniae（MO） Y-98 using a pair of specific primers and was cloned into the pMD19-T Simple vector.After the sequence was confirmed,the MOP30 gene was extracted by digesting the recombinant plasmid,and was subcloned into the pCAMBIA1300-YFP vector to construct eukaryotic expression vector named pCAMBIA1300-MOP30-YFP.Then the constructed plasmid pCAMBIA1300-MOP30-YFP was transformed Agrobacterium competent cells,and infected tobacco leaves.The enhanced yellow fluorescent protein（YFP） was observed under imaging microscope,and the mRNA of MOP30 was detected by RT-PCR.Western-blotting showed a 57 000 protein-specific band.The results indicated that the vector pCAMBIA1300-MOP30-YFP was successfully constructed and expressed in tobacco,laying a foundation of further investigation of transgenic plant vaccines for preventing Mycoplasma pneumonia of sheep.