重组P16蛋白在大肠杆菌中的表达纯化及其功能研究

【摘 要】： 目的 在原核细胞中表达P16蛋白并纯化，研究外源性P16蛋白对肿瘤细胞周期的影响。方法 构建外源性P16融合蛋白的原核表达载体pQE31-p16。IPTG诱导大肠杆菌BL21菌株表达P16融合蛋白，通过亲和层析柱纯化目的蛋白，经Western-blot鉴定后, 将P16蛋白转导入人肺腺癌细胞系A549中, 利用免疫细胞化学方法对蛋白进行细胞内定位, 同时绘制细胞生长曲线，并用流式细胞仪分析细胞周期的变化。结果 所构建P16融合蛋白的原核表达载体pQE31-p16，可在大肠杆菌中稳定表达，通过纯化获得P16融合蛋白。利用lipofectamine2000转染试剂可将P16蛋白转入A549细胞中，生长曲线与流式细胞术结果显示P16蛋白可抑制肿瘤细胞增殖。结论 原核细胞表达的P16蛋白经纯化，转导进入A549细胞，可影响其生长周期，抑制A549细胞的增殖。

Study on the Function of Expression and Purification of Recombinant P16 Protein in E. coli

Objective To express and purify P16 protein in prokaryotic cells and research the effect of recombinant exogenous P16 fusion protein on the cell cycle.Methods The prokaryotic expression vector pQE31-P16 for exogenous P16 fusion protein was constructed by inducing E. coli BL21 to express P16 fusion protein by IPTG. After purified by affinity chromatography, the protein was analyzed by Western blot, then the purified P16 protein was transducted into human lung adenocarcinoma cells A549. Intra-cellular location of the protein was demonstrated by fluorescence immunocytochemistry, and the growth curve for the cells was drawn to analyze the change of the cell cycle by flow cytometer(FCM). Results The prokaryotic expression vector pQE31-P16 for exogenous p16 fusion protein could be stably expressed in E. coli. After the fusion protein was purified by affinity chromatography the protein was transducted into A549 cells using lipofectamineTM 2000. The results of growth curve and flow cytometry demonstrated that P16 protein could inhibit the growth of the cancer cells. Conclusion The purified P16 protein expressed by E. coli can inhibit the growth of the human lung adenocarcinoma of lung cells A549 after tranducted into the cells, and it can influence the cell cycles of the cancer cells.