【摘要】 乙型肝炎病毒X蛋白(HBx)具有广泛的转录激活作用,但是由于细胞类型和实验条件的差异,外源启动子介导的HBx瞬时表达水平常表现出不均一性,为其功能的研究带来困难。为了解决HBx研究过程中遇到的这些难题,利用PCR扩增获得HBx及含转导肽TLM的EGFP编码序列,经酶切后插入pGEX-4T-1原核表达载体,成功构建重组质粒pGEX-HBx-EGFP-TLM和pGEX-EGFP-TLM。重组质粒转化大肠杆菌BL21(DE3)感受态细胞,融合蛋白经IPTG诱导表达,采用?KTATM Purifier蛋白纯化系统纯化并进行SDS-PAGE和Western blotting分析,确定为目的蛋白。将纯化后的融合蛋白分别与AML12和SMMC-7721细胞共孵育,Western blotting和激光共聚焦显微镜检测证实TLM转导肽能够介导HBx-EGFP和EGFP进入细胞,同时进入细胞的HBx-EGFP-TLM能够发挥转录激活活性,为HBx功能的深入研究奠定了基础。更多还原

【Abstract】 Hepatitis B virus X protein(HBx) has various functions and plays a crucial role in the development of hepatocellular carcinoma(HCC).However,due to different transfection efficiency levels and experimental approaches,it is difficult to correlate the exact functions of HBx to HBV-associated HCC.In this study,we constructed two prokaryotic expression vectors,pGEX-HBx-EGFP-TLM and pGEX-EGFP-TLM,which expressed HBx-EGFP-TLM and EGFP-TLM fusion proteins respectively.Both vectors contained a coding sequence of TLM transduction motif derived from the PreS2-domain of Hepatitis B Virus surface antigens.In addition,EGFP was expressed as a reporter reflecting the transduction efficiency of TLM.The fusion protein HBx-EGFP-TLM or EGFP-TLM purified from Escherichia coli BL21(DE3) by ?KTATM Purifier system was incubated with AML12 and SMMC-7721 cells.Both Western blotting and laser confocal results indicated that the translocation motif TLM could lead HBx-EGFP and EGFP into the cytoplasm.Dual-Luciferase Reporter Assay revealed that the activity of mEZH2 promoter could be up-regulated by the recombinant HBx.In conclusion,we expressed a cell-permeable HBx,which could provide a new method to study the functions of HBx.更多还原