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目的构建人视黄酸诱导蛋白16(retinoic acid induced 16,RAI16)与增强型绿色荧光蛋白(enhanced greenfluorescent protein,EGFP)的融合基因真核表达载体pEGFP-C1-RAI16,使RAI16-EGFP融合蛋白在人肝癌细胞株HepG2中得到表达。方法采用PCR技术,从含有目的基因的质粒克隆模板中钓取并扩增出RAI16全长编码基因,构建RAI16与EGFP的融合基因真核表达载体pEGFP-C1-RAI16,用脂质体转染技术将pEGFP-C1-RAI16导入HepG2,激光共聚焦分析RAI16亚细胞定位及Western blot检测RAI16-EGFP融合蛋白的表达。结果经转化细菌、抽提质粒、酶切鉴定和DNA序列分析证实,RAI16基因已正确插入pEGFP-C1中EGFP基因的下游,获得融合基因表达载体pEGFP-C1-RAI16。将pEGFP-C1-RAI16导入HepG2细胞24 h后,激光共聚焦分析显示RAI16-EGFP融合蛋白主要表达于细胞质内。Western blot结果显示,转染pEGFP-C1-RAI16 24、48 h和72 h后,RAI16基因在蛋白水平的表达逐渐增高,而AFP的表达逐渐下调。结论成功构建了真核表达载体pEGFP-C1-RAI16,并在HepG2细胞中进行了表达。
Objective To construct eukaryotic expression vector pEGFP-C1-RAI16, which is a fusion gene of human retinoic acid induced 16 (RAI16) and enhanced green fluorescent protein (EGFP), so that RAI16-EGFP fusion protein Human hepatoma cell line HepG2 was expressed. Methods The full length coding gene of RAI16 was amplified by PCR from the plasmid cloning template containing the gene of interest. The eukaryotic expression vector pEGFP-C1-RAI16 of fusion gene of RAI16 and EGFP was constructed. The recombinant plasmid pEGFP-C1-RAI16 was transfected into HepG2 cells. The expression of RAI16-EGFP fusion protein was detected by confocal laser scanning microscopy and Western blot. Results The transformed bacteria, plasmid extraction, restriction enzyme digestion and DNA sequence analysis confirmed that the RAI16 gene was correctly inserted into the downstream of the EGFP gene in pEGFP-C1 and the fusion gene expression vector pEGFP-C1-RAI16 was obtained. After transfecting pEGFP-C1-RAI16 into HepG2 cells for 24 h, confocal laser scanning confocal microscope showed that RAI16-EGFP fusion protein was mainly expressed in cytoplasm. Western blot results showed that after transfection of pEGFP-C1-RAI16 for 24,48 h and 72 h, the expression of RAI16 protein gradually increased while the expression of AFP decreased gradually. Conclusion The eukaryotic expression vector pEGFP-C1-RAI16 was successfully constructed and expressed in HepG2 cells.