GL1-EGFP融合蛋白的原核表达及对神经胶质瘤细胞的靶向作用分析

Prokaryotic expression of GL1-EGFP fusion protein and its targeting of glioma cells

关键词:  GL1-EGFP  融合蛋白；神经胶质瘤  靶向作用

Key words:  GL1-EGFP  fusion protein  glioma  targeting

摘要:

[目的]构建、纯化神经胶质瘤靶向肽GL1和EGFP融合蛋白表达载体（GL1-EGFP），并且分析其靶向作用。[方法]通过PCR扩增目的基因GL1-EGFP，并连接到pET-22b(+)载体上构建原核表达质粒，重组体转到感受态大肠杆菌BL21（DE3）中进行蛋白诱导表达，重组蛋白利用Ni2+-树脂柱亲和层析纯化，利用SDS-PAGE和Western blotting鉴定表达纯化蛋白，激光共聚焦显微镜检测GL1-EGFP对神经胶质瘤细胞的靶向作用。[结果]构建了GL1-EGFP原核表达载体，在IPTG诱导下获得了高效表达。Western blotting分析证明纯化蛋白为目的蛋白GL1-EGFP。体外细胞实验表明GL1-EGFP融合蛋白对神经胶质瘤细胞U87△EGFR具有靶向作用。[结论]GL1-EGFP靶向融合蛋白对恶性神经胶质瘤细胞有明显的靶向定位作用。

Abstract:

[Objective] To construct expression vector of glioma targeting fusion protein (GL1-EGFP), purify and analyze its targeting function. [Methods] Full-length GL1-EGFP DNA was amplified by polymerase chain reaction (PCR) and ligated with pET-22b(+) expression vector to construct prokaryotic expression plasmid. The recombinant was transformed into competent E.coli BL21(DE3) for protein expression with IPTG induction. Recombinant protein was purified with Ni2+-resin affinity chromatography and identified by SDS-PAGE and western blot analysis. Targeting of GL1-EGFP to glioma cells was observed with confocal microscope. [Results] The prokaryotic expression vector for GL1-EGFP was constructed and fusion protein was expressed at a high level with IPTG induction. Western blotting analysis indicated that purified protein was GL1-EGFP. GL1-EGFP could target to glioma cell U87△EGFR in vitro. [Conclusion] Targeting fusion protein GL1-EGFP has the function targeting glioma.