p62增加HBsAg^+ HepG2细胞DRibbles中HBsAg含量的研究

Over-expression of p62 increases the level of HBsAg in the DRibbles extracted from HepG2 cells expressing HBsAg

目的:探讨过表达p62能否提高自噬小体中泛素化蛋白的水平。方法:构建pIRES2-EGFP-HBsAg重组质粒和pIRES2-EGFP-Ub-HBsAg重组质粒;脂质体法分HBsAg、Ub-HBsAg、Ub-HBsAg+p62组3组转染HepG2细胞,在相同细胞数量和转染效率下,经万珂、雷帕霉素和氯化铵处理转染细胞并提取DRibbles,ELISA法检测其中HBsAg含量。结果:万珂、雷帕霉素和氯化铵处理后,Ub-HBsAg+p62组HepG2细胞DRibbles中HBsAg显著增高,而且明显高于药物处理的其他两组。结论:当蛋白酶体被抑制时,过表达p62蛋白促进泛素化蛋白(HBsAg)进入自噬小体,阻断自噬降解途径提取的DRibbles能够募集到更多的泛素化蛋白(HBsAg)。

Objective：To approach whether over-expressed p62 could aggregate more ubiquitinated proteins in DRibbles.Methods：Recombinant pIRES2-EGFP vector containing the HBsAg or UbHBsAg gene was constructed and transfected into HepG2 cell line respectively and pIRES2-EGFP-UbHBsAg and pIRES2-EGFP-p62 were cotransfected into HepG2 cell line.Then the transfected cells were cultured in the medium including velcade,rapamycin and ammonium chloride.After 16 hours,we extracted and lysed DRibbles from the medium and detected HBsAg by ELISA.Results：HBsAg in DRibbles extracted from HepG2 that was co-transfected with pIRES2-EGFPUbHBsAg and pIRES2-EGFP-p62 vectors rapidly increased when the cells were cultured with the drugs,and was much higher than the other groups which had been under the same treating.Conclusion：Over-expression of p62 aggregates more ubiquitinated proteins（HBsAg） in the DRibbles extracted from HepG2 cells as the proteasome pathway is blocked by the drugs.