与HBV X蛋白相互作用的细胞蛋白的筛选及其鉴定

参考GenBank核苷酸序列库中的HBV的核苷酸序列设计合成了一对对应于HBx基因的引物，从原发性肝癌(hepatocellular carcinoma，HCC)患者的血清中提取HBV基因组DNA作为模板，扩增HBx编码区并测定了核苷酸序列．将该编码区克隆到酵母双杂交系统的诱饵蛋白表达载体pGBKT7中，转入酵母细胞AH109，进行表型鉴定．然后通过Mating实验从已制备好用于酵母双杂交系统的肝cDNA表达文库中筛选与HBx相互作用的细胞蛋白，并用体外免疫共沉淀实验进一步验证．研究表明，分离得到的与HBx基因编码的蛋白相互作用的4种新的细胞蛋白，分别是醛缩酶B、C8α亚基、一种丝氨酸蛋白酶Hepsin和一种未知蛋白．

Screening and Identification of HBx-Associated Cellular Proteins

A pair of primers for HBx gene was synthesized according to the sequences of HBV genome in GenBank. The DNA of HBV genome were isolated from a new HBV field strain from a patient with chronic viral infection and used as template for amplification HBx coding sequence. And then the amplified HBx coding sequence was cloned into the Bait vector pGBKT7. Yeast AH109 with the vector expressing HBx protein fused in BD peptide was identified through the phenotype and screened cellular targets that are associated with the viral protein. By screening a pretransformed human liver cDNA library using a Gal 4 yeast two-hybrid system, four cellular proteins were able to be identified as new HBx interacting partners such as Aldolase B, Complement component 8, alpha polypeptide, Hypothetical protein MGC13047 and Hepsin.