CXCL12-KDEL融合基因绿色荧光蛋白表达载体的构建与鉴定

Construction and Identification of GFP Expression Vector Combined with CXCL12-KDEL Gene

目的:构建CXCL12-KDEL融合基因绿色荧光蛋白表达载体并鉴定其在人舌癌细胞株Tb3.1(以下简称Tb3.1)中的表达。方法:设计特定引物,以人基因组DNA为模板,PCR扩增目的基因CXCL12-KDEL,经纯化的目的基因连接19T载体,插入pIRES2-EGFP表达载体中,使用Bgl Ⅱ/Sal Ⅰ进行双酶切,酶切初步鉴定后,测序鉴定。测序正确的重组质粒经脂质体(Lipofectamine^(TM)2000)介导体外转染Tb3.1,转染48h及72h在倒置荧光显微镜下观察。利用Western-blot检测重组质粒CXCL12-KDEL表达情况。结果:经酶切及测序鉴定证实目的基因(CXCL12-KDEL)已成功扩增并插入重组质粒;重组质粒转染Tb3.1细胞,倒置荧光显微镜下可见绿色荧光,Western-blot检测转染可见E-tag蛋白表达。结论:成功构建了CXCL12-KDEL融合基因绿色荧光蛋白表达载体,为进一步完成CXCL12-CXCR4生物学轴的阻断实验奠定了基础。

Objective： To construct a green fluorescence protein （GFP） expression vector combined with the CX- CL12-KDEL gene and study its expression in human tongue cells. Methods： The combined genes CXCL12-KDEL were amplified by PCR from human genomic DNA. After purification it was ligated into the 19T vector then inserted into plRES2-EGFP by homologous recombination to construct the CXCL12-KDEL-plRES2-EGFP vector. The constructed plasmids were identified by Bgl II/Sal I restriction digestion and sequence analysis. Recombinant plasmids were transfected into the Tb3.1 cell line by Lipofectamine （TM 2000）. Cells were observed at 48 and 72 hours after transfection using an inverted fluorescence microscope. The expression of CXCL12 was identified by Western blot. Results： The correct constructed plasmids were confirmed by restriction digestion and sequence analysis. The results of cell transfection indicated that the GFP could be observed in Tb3.1 cells. The Tb3.1 cells can express E-tag protein when transfected with the constructed plasmid as shown by Western blot. Conclusion： The GFP expression vector driven by the combined gene CXCL12-KDEL was constructed successfully, resulting in a good foundation block for the CXCL12-CXCR4 axis.