中文摘要：

目的：构建真核表达载体pEGFP-N1-ND CyclinB1,并观察其在人胃癌细胞SGC-7901中的表达及对SGC-7901活性的影响。方法：通过RT-PCR法扩增不可降解CyclinB1（NDCyclinB1）,将其插入到pEGFP-N1载体的XhoⅠ和BamHⅠ酶切位点,构建真核表达载体pEGFP-N1-ND CyclinB1,并以PCR、双酶切、测序鉴定。通过脂质体法转染胃癌细胞SGC-7901,进行荧光检测、Western blotting分析和流式细胞仪分析。结果：PCR、酶切及测序证明重组质粒pEGFP-N1-ND CyclinB1构建成功。转染胃癌细胞SGC-7901 24 h后,荧光显微镜下观察到绿色荧光。West-ern blotting检测到CyclinB1的表达明显增加。流式细胞仪检测重组质粒细胞组凋亡指数高于对照组,差异有显著性。结论：成功构建pEGFP-N1-ND CyclinB1荧光真核表达载体,并可在SGC-7901细胞中有效表达。

英文摘要：

Aim：To construct an eukaryotic expression vector pEGFP-N1 Non-degrable CyclinB1,and to observe its expression in human gastric cancer cell lines SGC-7901 and effects on SGC-7901 cells. Methods： ND-CyclinB1was amplified by reverse transcription polymerase chain reaction（RT-PCR）.The eukaryotic expression vector pEGFP-N1-ND CyclinB1 was constructed by introducing ND-CyclinB1 DNA fragment into the sites of Xho I and BamH I in pEGFP-N1 vector.The plasmid was transfected into SGC-7901 cells by using lipofectamine.The expressed EGFP was observed by fluorescent microscope.Then the CyclinB1 was analyzed by Western blotting,and the apoptosis index was detected under a FACScan flow cytometry.Results： Identification of pEGFP-N1-ND CyclinB1 by enzyme digestion and PCR showed that the recombinant vector was correctly constructed.The EGFP was observed 24 hours after transfection,the expression of CyclinB1 was increased,and the apoptosis index was found to be increased too.Conclusion：The eukaryotic expression plasmid pEGFP-N1-ND CyclinB1has been successfully constructed and it can be expressed in SGC-7901 cells.