HBx-EGFP-TLM融合蛋白表达载体的构建、蛋白表达纯化及活性检测

Expression, purification, and bio-activity analysis of fusion protein HBx-EGFP-TLM

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。

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.