利用免疫共沉淀技术研究RSV CP、SP和NSvc4三个蛋白的互作情况

为了检测水稻条纹病毒(Rice stripe virus, RSV)CP、SP和NSvc4蛋白自身和两两之间的互作关系，首先通过PCR扩增获得了分别融合有HA或c-Myc标签的RSV CP、SP和NSvc4基因，并将其插入到植物表达载体pEGAD或pKYLX71:35S2中。重组质粒转化农杆菌EHA105后，将含有不同重组质粒的农杆菌菌液混合后注射烟草(Nicotiana benthamiana)叶片。提取烟草中瞬时表达的蛋白，以HA抗体或c-Myc抗体进行免疫共沉淀实验，最后免疫混合物通过c-Myc抗体或HA抗体进行Western blot检测，确定蛋白的互作情况。结果表明，RSV CP蛋白自身能够发生互作，而CP与SP，CP与NSvc4，SP与SP，SP与NSvc4，及NSvc4与NSvc4蛋白之间均未有检测到互作现象。

Studies on the Interactions between RSV CP、SP and NSvc4 Proteins by Using Co-immunoprecipitation Technology

In order to detect the interaction between Rice stripe virus (RSV) CP, SP and NSvc4, the HA-tagged or c-Myc-tagged CP, SP and NSvc4 of RSV were amplified by PCR, and then inserted into plant expression vector pEGAD or pKYLX71:35S2. The recombinant plasmids were transformed into Agrobacterium tumefaciens EHA105, and the EHA105 containing different recombinant was injected into Nicotiana benthamiana. The total proteins were isolated from Agrobacterium-infiltrated N. benthamiana leaves, and co-immunoprecipitated with HA or c-Myc antibody. The immunocomplexes were detected by Western blot with c-Myc or HA antibody to identify the interaction between these proteins. The results showed that RSV CP can interact with itself, but no interaction between other proteins was detected.