【作者】 杨艳；

【导师】 段昌柱；

【作者基本信息】 重庆医科大学 ， 细胞生物学， 2011， 硕士

【摘要】 目的: NIRF蛋白是泛素化E3连接酶,通过构建各个表达质粒,包括真核表达质粒pFLAG-NIRF,原核表达质粒pGST-NIRF、pGST-HBc,并在HEK293细胞或E.coil BL21(DE3)中表达,纯化出GST-NIRF、GST-HBc蛋白;通过GST pull down证明NIRF与HBc在细胞外存在蛋白-蛋白相互作用;建立泛素化体系,探讨HB(cHBV core protein)能否被NIRF蛋白泛素化。方法:(1)根据NIRF基因序列设计引物,NIRF基因经PCR扩增后连接到pMD19T上,经酶切连接后构建pGST-NIRF原核表达质粒;pGST-NIRF转化E.coil BL21(DE3),优化表达条件使GST-NIRF蛋白在上清中大量完整表达,并探索一条高纯度蛋白纯化方法用以获得GST-NIRF蛋白;用脂质体转染法将pCDNA3-HBc转染至HEK293细胞,检测其表达;纯化后的GST-NIRF蛋白与转染pCDNA3-HBc的细胞上清共同孵育,经GST pull down证明2者之间是否存在相互结合。(2)根据HBc基因序列设计引物,HBc基因经PCR扩增后连接到T载体pTArget上,经酶切连接后构建pGST-HBc原核表达质粒;pGST-HBc转化E.coil BL2(1DE3)经优化表达条件后纯化出GST-HBc蛋白;设计NIRF基因引物,基因经扩增后插入到p3*FLAG-CMV-10中构建pFLAG-NIRF质粒;脂质体转染法将pFLAG-NIRF转染至HEK293细胞,检测其表达;纯化后GST-HBc蛋白与转染pFLAG-NIRF的细胞上清共同孵育,经GST pull down证明2者之间是否存在相互结合。(3)纯化后的GST-NIRF、GST-HBc蛋白用以构建细胞外泛素化体系,分别作为泛素化E3连接酶及泛素化底物。泛素化反应完成后,产物通过SDS-PAGE分离,蛋白免疫印迹检测是否有泛素化条带产生,从而确定NIRF是否能泛素化HBc。结果:(1)成功构建pGST-NIRF原核表达质粒并在E.coil BL21(DE3)顺利表达,优化表达条件得到GST-NIRF在细胞上清中的大量完整表达,探索出一条GST-NIRF蛋白的纯化方式获得较高纯度的GST-NIRF全长蛋白,为蛋白相互作用鉴定及泛素化反应奠定基础;通过GST pull down证明NIRF与HBc能够在细胞外相互结合。(2)成功构建pGST-HBc原核表达质粒并在E.coil BL21(DE3)顺利表达,优化表达条件得到GST-HBc较高浓度表达,纯化出高浓度的GST-HBc蛋白;成功构建pFLAG-NIRF真核表达质粒,并在HED293细胞中顺利表达;通过GST pull down证明HBc与NIRF能够在细胞外相互结合。(3)体外泛素化结果提示,HBc可能被NIRF泛素化。HBc抗体检测效果不理想,无法准确判定还需进一步实验证明。结论: GST pull down证明NIRF蛋白及HBc能够在细胞外相互结合;泛素化结果表明,NIRF蛋白有可能泛素化HBc蛋白,证据不充足还有待进一步完善,另此结果只能为NIRF对HBc的泛素化作用提供体外证据。更多还原

【Abstract】 Objective: NIRF is an ubiquitination E3 ligase which can bind to E2-Ub recombinant and substrate, fascinating polyubiquity transfer from E2 to substrate. Construct various expression plasmids, including eukaryotic expression plasmid pFLAG-NIRF and prokaryotic expression pGST-NIRF and pGST-HBc, and then to express target proteins and to purify GST-NIRF and GST-HBc by GST 4B gel. Protein-protein interaction between NIRF and HBc was indentified by GST pull down. Develop an ubiquitination system to explore whether NIRF can ubiquitylize HBc in vitro by ubiquitination analysis.Methods:(1) Primers were designed according to the sequences of NIRF, and NIRF genes were amplified by PCR and then inserted into prokaryotic expression plasmid pGEX-4T-1 to construct pGST-NIRF. pGST-NIRF was transformed into E.coil BL21 (DE3) to express GST-NIRF proteins and culture conditions were optimized to produce high concentration and full-length target proteins. Purification process was optimized to obtain high-purity GST-NIRF proteins. Eukaryotic plasmid pcDNA3-HBc was transfected into HEK293 cells to produce HBc proteins. Purified GST-NIRF and cells lysate with HBc in which were incubated together to allow the NIRF capture HBc and then products were purified by GST 4B gel and subjected to SDS-PAGE analysis.(2) Primers were designed according to the sequences of HBc, and HBc genes were amplified by PCR and then inserted into prokaryotic expression plasmid pGEX-4T-1 to construct pGST-HBc. pGST-HBc was transformed into E.coil BL21 (DE3) to express GST-HBc proteins and culture conditions were optimized to produce high concentration and full-length target proteins. NIRF genes was amplified by the methods above mentioned and then inserted into eukaryotic plasmid p3*FLAG-CMV-10 to get pFLAG-NIRF. pFLAG-NIRF was transfected into HEK293 cells to produce FLAG-NIRF fusion proteins. GST-pull down was processed through the methods above to prove the protein-protein interaction between HBc and NIRF.(3) Purified GST-NIRF and GST-HBc were added into ubiqutination system including E1, E2 (Ubc5b), ubiquity and so on. Products via ubiquitination reactivates were separated by SDS-PAGE and then subjected to western blot to identify whether NIRF can ubiquity HBc in vitro.Results: Construct eukaryotic plasmid pFLAG-NIRF successfully, two prokaryotic plasmids pGST-NIRF and pGST-HBc too. The eukaryotic plasmid was well expressed in HEK293T cells, so did the two prokaryotic plasmids in BL21 (DE3). GST-NIRF and GST-HBc proteins were purified by GST 4B gel. Combination between NIRF and HBc was confirmed through GST pull down, whether GST-NIRF pulls down HBc or GST-HBc pulls down NIRF. We have a clue that NIRF can ubiquity HBc in vitro.Conclusion: Protein-protein interaction between NIRF and HBc was confirmed by GST pull down analysis. Ubiquitination experiment showed that NIRF may ubiquinate HBc in vitro, which is not confirmed because of the lack of proofs.更多还原