维隆气单胞菌外膜蛋白AII的植物瞬时表达系统的构建

Construction of Transient Expression System Expressing Aeromonas veronii Outer Membrane Protein AII

以维隆气单胞菌HBJY01为模板通过PCR技术扩增其外膜蛋白AII(AvompAII)基因片段,将序列正确的AvompAII基因片段与含有黄色荧光蛋白(yellow fluorescent protein,yfp)基因的表达载体pCAMBIA1300重组,将该重组表达载体转化到农杆菌(Agrobacterium tumefaciens)GV3101中用于转染烟草叶片细胞.通过激光扫描共聚焦成像系统检测方法和RT-PCR的方法来检测融合表达AvompAII基因的黄色荧光蛋白的表达与AvompAII基因在烟草叶片中的转录情况.从维隆气单胞菌HBJY01中克隆出大小为996bp的AvompAII基因序列,并成功构建了AvompAII蛋白的植物瞬时表达系统.通过该方法能方便可靠地构建植物瞬时表达系统,并进一步对维隆气单胞菌所引起的水产疾病的发现和实际生产应用提供了实验基础.

The gene of Aeromonas veronii outer membrane protein AII （AvompAII） was amplified by PCR using A. veronii HBJY01 cells as template and inserted into expression vector pCAMBIA1300 that contained a yellow fluorescent protein gene. The recombinant expression plasmid was transformed into Agrobacteriurn tumefaciens GV3101 competent cells to transfect tobacco leaf cells. The expression of yellow fluorescent protein fused with AvompAII and the transcription of AvompAII gene were detected by confocal laser scanning microscope and RT-PCR, respectively. In this study, the AvompAII gene cloned from A. veronii HBJY01 was 996 bp in length. A plant-based transient expression system that expresses fusion protein of AvompAlI and YFP was successfully constructed. The method can be used to construct plant-based transient expression systems for antigen expression. Meanwhile, the tobacco leaf cells contai- ning AvompAII gene has laid a foundation for further investigation of prevention of limnobios diseases caused by A. veronii with plant vaccine and its practical application.