人NRAGE基因重组腺病毒载体的构建及其对293细胞细胞周期的影响

目的构建hNRAGE基因的重组腺病毒载体,并研究其对293细胞细胞周期的影响。方法采用PCR方法扩增hNRAGE基因片段,并亚克隆至腺病毒穿梭质粒载体pAdTrack-CMV中,构建穿梭质粒pAdTrack-CMV/hNRAGE。经测序检测后,用PmeI酶切使pAdTrack-CMV/hNRAGE被线性化,然后与腺病毒骨架质粒pAdEasy-1用电穿孔法共转化大肠杆菌BJ5183,进行同源重组。经PacI酶切鉴定后,用磷酸钙法转染QBI-293A细胞,包装成重组体腺病毒Ad-hNRAGE颗粒。利用穿梭质粒pAdTrack-CMV中GFP报告基因的表达,观察重组腺病毒的产生,测定病毒颗粒的浓度。用Western blot检测目的基因的表达。采用MTT法检测293细胞的活力,用流式细胞术分析hNRAGE基因对293细胞周期的影响。结果成功地构建hNRAGE重组腺病毒载体。Western blot检测结果证实,hNRAGE基因可在293细胞中表达。收获病毒后,经测定病毒颗粒的浓度大约为6.5×109个/μL。转染了重组腺病毒Ad-hNRAGE的293细胞与正常细胞相比较,增殖率显著降低,G0-G1和G2-M期的细胞增多,S期的细胞明显减少。结论hNRAGE基因可能有抑制293细胞生长的作用。

Construction of recombinant adenovirus vector of hNRAGE gene and its effect on cell cycle of 293 cells

AIM: To construct the recombinant adenovirus vector of hNRAGE gene and study its effect on the cell cycle of 293 cells. METHODS: hNRAGE gene was amplified by PCR and subcloned into the shuttle vector pAdTrack-CMV to construct a shuttle plasmid pAdTrack-CMV/hNRAGE. After sequencing, it was linearized with Pme I and cotransformed into E.coli BJ5183 cells with adenovirus genomic plasmid pAdEasy-1 by electroporation to achieve homologous recombination. After being digested with Pac I, the DNA of identified recombinant plasmid was transfected into QBI-293A cells by calcium phosphate transfection to package adenovirus. With the use of GFP gene expression in pAdTrack-CMV, the appearance of Ad-hNRAGE was observed and its concentration was measured. The expression of the target gene was detected by Western blot and its effect on cell cycle of 293 cells was examined by MTT colorimetry and FCM. RESULTS: The Ad-hNRAGE was successfully constructed and the expression of hNRAGE gene in 293 cells was proved by Western blot. After harvesting the virus particles, the concentration of Ad-hNRAGE was about 6.5x10(9) Ad/microL. The transfection of Ad-hNRAGE resulted in significantly lower proliferative rate of 293 cells compared with untransfected ones, with cell number of G0-G1 and G2-M phase increasing and that of S phase decreasing. CONCLUSION: hNRAGE gene can inhibit the growth of 293 cells.