人神经生长调控因子（hNRAGE）基因重组腺病毒载体的构建及其对293细胞周期的影响

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

作者信息

Zhou Li-hua, Xue Bin, Guo Shi-ying, Shi Yi-huan, Wen Chuan-jun, Li Chao-jun

机构信息

Jiangsu Key Laboratory for Molecular and Medical Biotechnology, Nanjing Normal University, Nanjing 210097, China.

出版信息

Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2006 Jan;22(1):18-21.

PMID:16388736

Abstract

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.

摘要

目的

构建人神经生长相关基因（hNRAGE）的重组腺病毒载体，并研究其对293细胞周期的影响。

方法

通过聚合酶链反应（PCR）扩增hNRAGE基因，并亚克隆到穿梭载体pAdTrack-CMV中，构建穿梭质粒pAdTrack-CMV/hNRAGE。测序后，用Pme I酶切使其线性化，并通过电穿孔与腺病毒基因组质粒pAdEasy-1共转化到大肠杆菌BJ5183细胞中，以实现同源重组。用Pac I酶切后，将鉴定的重组质粒DNA通过磷酸钙转染法转染到QBI-293A细胞中以包装腺病毒。利用pAdTrack-CMV中绿色荧光蛋白（GFP）基因的表达，观察Ad-hNRAGE的出现并测定其浓度。通过蛋白质免疫印迹法（Western blot）检测靶基因的表达，并通过噻唑蓝比色法（MTT）和流式细胞术（FCM）检测其对293细胞周期的影响。