HBc蛋白与NIRF蛋白细胞外相互作用鉴定

Identification of protein-protein interaction between HBc and NIRF in vitro

构建原核表达质粒pGST-HBc,真核表达质粒pFLAG-NIRF,表达并纯化GST-HBc蛋白,表达FLAG-NIRF蛋白,利用GST pull down技术鉴定HBc与NIRF蛋白的相互作用。利用PCR技术分别扩增HBc编码框和NIRF基因全长,并将其分别插入到原核表达载体pGEX-4T-1和真核表达载体p3*FLAG-CMV-10中,构建出pGST-HBc及pFLAG-NIRF。pFLAG-NIRF转染HEK293,培养48 h,收获前10 h加入MG132抑制蛋白酶体,收获细胞,经NP40裂解后离心收集上清pGST-HBc转化BL21(DE3)细菌,优化培养条件使GST-HBc在上清中大量表达,然后用GST4Bgel亲合纯化该蛋白,结合有GST-HBc蛋白与含FLAG-NIRF的细胞上清孵育后,离心收集GST 4Bgel,经洗脱液洗脱后蛋白电泳,western blotting分析。构建了pGST-HBc及pFLAG-NIRF,在上清中成功表达GST-HBc并纯化该蛋白,在细胞中成功表达FLAG-NIRF,GST pull down结果显示两者存在体外相互结合。HBc与NIRF能够在细胞外发生相互结合。

To identify the protein-protein interaction in vitro between HBc and NIRF,prokaryotic expressing plasmid pGST-HBc was constructed and protein GST-HBc was purified,and so as eukaryotic expressing plasmid pFLAG-NIRF.HBc and NIRF gene was amplified by PCR and then inserted into pGEX-4T-1 and p3＊FLAG-CMV-10 respectively.pFLAG-NIRF was transfected into HEK293 cells for 48 h and 10 h before collected MG132 added into cells to inhibit proteasomes.Cells were lysed by NP40 and then centrifuged to collect supernatant.pGST-HBc was transformed into BL21（DE3）.Culturing conditions were optimized to maximize protein expressing level in supernatant.The recombination proteins were purified by GST-Sepharose 4B gel and then incubated with cells supernatant including FLAG-NIRF.After incubation,gels were collected and proteins were eluted by elution buffer.Samples were separated by SDS-PAGE and then analyzed by western blot.Result showed that pGST-HBc and pFLAG-NIRF were constructed and expressed in bacteria supernatant or in cells successfully.GST-HBc was purified by GST-Sepharose 4B gel and then was proved to interact with NIRF by GST pull down.