乙肝病毒X与Tab1蛋白相互作用的体内外验证

Identification of Protein-protein Interaction of Hepatitis B Virus X Protein and Tab1 in Vivo and in Vitro

目的：借助实验室前期质谱分析技术和数据分析研究基础,采用免疫共沉淀（Co-IP）和蛋白质沉降实验（GST pull-down）验证HBV X蛋白与Tab1蛋白的相互作用,为进一步研究HBx在HBV慢性感染致癌机制中的作用提供一定的实验依据。方法：成功构建pGEX-2TK-GST-HBx质粒,对GST-HBx融合蛋白进行诱导表达,与GST-beads结合孵育,构建pcDNA3.1/myc-His（-）BTab1,转染293T细胞使其表达,然后GST pull-down体外试验验证二者的相互作用;构建pcDNA3.1/myc-His（-）B-Tab1和pcDNA3.1-3×flag-HBx真核表达质粒,共转染293T和HepG2细胞使其表达,通过Co-IP实验验证抗Myc抗体可以将HBx从细胞裂解液中沉淀下来,证实了它们在两种细胞系中存在相互作用。结果：显示了HBx和Tab1在体内外条件下能够发生相互作用,为进一步明确HBV X蛋白功能及作用机制奠定基础。

With the help of analysis of mass spectrometry and data analysis according to the preliminary laboratory basic research,Co-IP and GST pull-down methods was used to prove the interaction of HBV X protein with Tab1. In order to study the carcinogenic mechanism of HBx protein,Some experimental evidence for further study of the role of HBx in the carcinogenic mechanism of HBV chronic infection were provided. pGEX-2 TKGST-HBx plasmid was successfully constructed,GST-HBx fusion protein was induced and incubated with GSTbeads. pcDNA3. 1/myc-His（-） B-Tab1 plasmid was constructed,then was transfected 293 T cells to express Myc-Tab1. Finally the interaction of GST-HBx and Myc-Tab1 was verified in GST pull-down test. Moreover,the eukaryotic expression plasmids including pcDNA3. 1/myc-His（-） B-Tab1 and pcDNA3. 1-3 × flag-HBx were successfully constructed,then were co-transfected into human embryonic kideny 293 T cells and HepG2 cells to express fusion protein,further Co-IP test was demonstrated that the anti-Myc antibody could precipitate HBx from the cell lysate,and confirmed their intracellular interaction in two human cell lines. In conclusion,all results show that HBx and Tab1 could interact in vitro and in vivo,also established foundation to reveal the function and mechanism of HBV X protein.