RKIP的克隆、表达及其影响肿瘤细胞迁移性质的初步研究

Cloning,expression and purification of RKIP and preliminary study on its biological properties in tumor metastasis

目的:研究Raf激酶抑制蛋白(RKIP)在细胞外对肿瘤细胞迁移的影响。方法:通过PCR扩增得到RKIP基因片段,使用Bam HⅠ和HindⅢ双酶切并回收,然后插入双酶切过的原核表达载体p ET28a(+),得到p ET28a-RKIP重组质粒。将重组质粒转化表达菌株BL21(DE3),经IPTG诱导表达,表达产物经Ni+金属螯合层析纯化得到His-RKIP重组蛋白。通过伤口愈合实验,观察不同浓度的重组RKIP在细胞外对肿瘤细胞迁移的影响。结果:重组RKIP获得了高效表达和分离纯化,伤口愈合实验表明,在低浓度重组RKIP作用下A549肿瘤细胞迁移速度增加,在高浓度下肿瘤细胞迁移速度降低。结论:重组RKIP在细胞外具有促进和抑制A549肿瘤细胞迁移的双重作用,具体表现为低浓度时促进A549肿瘤细胞迁移,高浓度时抑制A549肿瘤细胞迁移。

Objective：To investigate the Raf kinase inhibitor protein（RKIP）＇s extracellular effect on tumor metastasis.Methods：The RKIP encoding gene fragment was amplified by PCR,digested with BamH Ⅰ and Hind Ⅲ,and inserted into the same digested expression vector pET28a （＋）,thus becoming recombinant plasmid pET28a-RKIP.The expression plasmid pET28a-RKIP was transformed into E.coli BL21 （DE3）,then induced by IPTG.The recombinant protein His-RKIP was purified through Ni ＋ affinity chromatography.Through wound healing experiment,we could understand the extracellular effect of the concentration of RKIP protein on tumor metastasis.Results：Recombinant RKIP was expressed and purified.The wound healing experiment demonstrated that the rate of tumor cell migration at first grew as the concentration grew and then dropped as the concentration continued to grow.Conclusion：The RKIP protein could either promote or inhibit A549 cell migration depending on the concentration,low concentration of RKIP can promote the migration of A549 cells while high concentration inhibits it.