ARRB1融合蛋白表达载体ARRB1-EGFP的构建及其在神经胶质瘤细胞中的表达

Construction of ARRB1-EGFP fusion protein and its expression in glioma cells

目的构建重组表达载体ARRB1-EGFP,在细胞中表达与鉴定,为进一步研究其功能奠定基础。方法 RT-PCR获得编码人ARRB1的全基因序列,克隆到真核表达载体pEGFPN1,构建重组表达载体ARRB1-EGFP,并转化于E.coli DH5α中筛选阳性重组子,通过限制性内切酶酶切电泳鉴定和DNA序列测定正确后,转染入HEK293细胞中表达,表达产物经Western blot检测蛋白的特异性,应用免疫荧光方法比较融合蛋白与内源性ARRB1在SNB19细胞的定位。结果成功构建了ARRB1融合蛋白的真核表达载体,并经Western blot鉴定正确。免疫荧光提示融合蛋白与内源性ARRB1定位相似。结论 ARRB1-EGFP融合蛋白可以安全、高效地转导入HEK293以及SNB19细胞中。

Objective To construct the recombinant expression vector ARRB1-EGFP, identify its expression in cells, and lay foundations for further functional study. Methods The full gene sequence encoding human ARRB 1 was obtained by RTopCR and subcloned into the eukaryotic expression vector pEGFPN1. Recombinant expression vector, ARRB 1-EGFP, was transformed into E.coli DH5 a and screened by restriction enzyme digest, gel electrophoresis and DNA sequence. Then the expression vectors were transfected into HEK293 cells, and its expression products were detected by Western Blot. The subcellular localization between endogenous ARRB 1 and ARRB 1-GFP in the SNB 19 cells was detected and compared by using immunofluorescence staining. Results ARRB 1-EGFP fusion protein eukaryotic expression vector was successfully constructed, and identified by Western Blot. Localization of the fusion protein, ARRB 1-GFP, was similar with that of the endogenous ARRB 1. Conclusion ARRB 1-EGFP fusion protein can be safely and efficiently transfected into HEK293 cells and SNB 19.