HCBP1的细胞定位及与HCV核心蛋白在体内的相互作用

Cellular localization of HCBP1 and its interaction with HCV core protein in vivo

目的探讨HCV核心蛋白与(HCBP1)HCV核心蛋白的相互作用蛋白在真核细胞内的相互作用。构建增强型绿色荧光蛋白(EGFP)与HCBP1融合蛋白真核表达载体,分析其在COS-7细胞中的表达和亚细胞定位。方法PCR分别扩增含HCV核心及HCBP1的cDNA片段,克隆入pGEMT载体,经测序正确后,分别克隆入哺乳双杂交的表达质粒pM和pVP16中,并转染入COS-7细胞,48h后检测细胞中报告基因CAT的表达水平。将HCBP1的cDNA片段克隆入绿色荧光表达载体pEGFP-C1中,构建HCBP1-EGFP融合蛋白的表达载体,转染COS-7细胞,以Western blot和荧光显微镜分析融合蛋白的表达及其细胞定位。结果CAT-ELISA检测显示pM-核心蛋白与pVP16-HCBP1实验孔吸光度值高于其他阴性对照,但低于阳性对照组。成功构建HCBP1-EGFP融合蛋白的表达载体,Western blot证实融合蛋白在转染细胞中的表达,荧光显微镜示HCBP1-EGFP融合蛋白分布于细胞浆,而转染空载体的细胞内EGFP的分布无明显改变。结论成功构建HCBP1-EGFP融合蛋白表达载体,并在COS-7细胞中得到表达。哺乳双杂交系统证实HCV核心蛋白与新基因HCBP1确有一定的相互作用。

Objective To analyze the interaction of hepatitis C virus （HCV） core protein with HCBP1 and observe the expression and cellular localization ofHCBP1. Methods The eDNA fi＇agments encoding HCV core protein and HCBP1 were amplified by PCR and subsequently cloned into pGEM T vector, respectively. After sequence verification, the two recombined vectors were respectively subcloned into two hybrid plasmids, pM and pVP16, pM-core, pVP16- HCBP1 and the reporter vector pG5CAT were co-transfected into COS-7 cells, and the interaction between HCV core protein and HCBP1 was assayed by detecting CAT gene expression after 48 h. The expression and subcellular localization of the fusion protein in the transfected COS-7 cells were analyzed by Western blotting and fluorescence microscopy, respectively. Results CAT-ELISA showed that the absorbance of the co-transfection group was significantly higher than that of the negative control groups but lower than that of the positive control group. Western blotting confirmed the expression of fusion protein in the transfected COS-7 cells. Fluorescence microscopy showed that the fusion protein was distributed mainly in the cytoplasm, and in contrast, diffuse EGFP expression was detected in COS-7 cells transfected with the empty vector. Conclusion Mammalian two-hybrid assay confirms the capacity of HCBP1 to bind HCV core protein, and the expression vector for HCBP1-EGFP fusion gene has been constructed successfully and expressed in COS-7 cells.