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

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

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。

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.