抗Cyclin D1胞内抗体AD5N基因真核表达载体的构建及其对乳腺癌细胞增殖的抑制作用

Construction of expression vector for anti-cyclin D1 intrabody AD5N and its inhibitory effects on cell proliferation of breast cancer

目的:构建细胞核定位的抗Cyclin D1胞内抗体基因的真核表达载体并分析其对乳腺癌细胞增殖的抑制效应。方法:利用PCR技术和分子克隆技术,构建编码细胞核定位的抗Cyclin D1胞内抗体基因(AD5N)的真核表达载体pcDNA3.1-AD5N,利用脂质体介导法转染MCF-7细胞,RT-PCR、间接免疫荧光、Western blot和流式细胞术分析该胞内抗体基因AD5N在MCF-7细胞中的表达、定位及抑瘤效应。结果:经限制性内切酶分析及序列分析发现,成功构建编码细胞核定位的抗Cyclin D1胞内抗体(AD5N)基因的真核表达载体pcDNA3.1-AD5N,基因片段为855 bp,编码285个氨基酸,分子量约为30 kD。RT-PCR、免疫荧光及Western blot实验结果显示,仅在转染胞内抗体基因的MCF-7细胞中存在该胞内抗体的表达,并且主要分布在细胞核区域。与野生型MCF-7细胞和转染空质粒的细胞相比,在MCF-7/pcDNA3.1-AD5N细胞中AD5N的表达可明显抑制MCF-7细胞增殖,显著诱导细胞凋亡(P<0.01),使G0-G1期细胞周期指数增高12.64%,S期指数下降15.22%,凋亡细胞指数上升9.15%。结论:细胞核定位的抗Cyclin D1胞内抗体AD5N基因的表达,可有效抑制乳腺癌细胞的增殖,阻滞细胞周期进展,并诱导细胞凋亡。

Objective：To construct the vector of anti-cyclin D1 intrabody with nuclear localization signal （NLS） and study on anti-tumor effects for MCF-7 celLs.Methods.Using PCR and molecular cloning technique,the vector of anti-cyclin D1 intrabody gene （ADSN） with nuclear localization signal （NLS） was constructed. Then it was subcloned into pcDNA3.1 to construct the recombinant expression vector pcD- NA3.1-ADSN. The intrabody gene was transfected into human breast cancer MCF-7 cells by lipofectamine 2000. The expression, localization and anti-tumor effects of ADSN were analyzed with RT-PCR, immunofluorescence, Western blot and fluorescence activated cell sorting. Results. Analysis of restriction endonuclease and DNA sequencing showed that the ADSN intrabidy gene and expression vector were constructed successfully. The intrabody gene was 855 bp in length, which coded a peptide with 285 amino acids with the molecular weight of 30 kD. ADSN mRNA and proteins were only detected in the MCF-7 cells transfected with intrabody vector and the intrabody was distributed mainly in nuclei. Compared with the control, the expression of ADS N in MCF-7/pcDNA3.1-ADSN cells significantly arrested the cell cycle of MCF-7 cells, with G0- G1 phase cells increasing for 12.64 % and S phase cells decreasing by 15.22% .In addition, the expression of ibtrabidy induced apoptosis of MCF-7 cells. The apoptosis index increaed by 9.15 %. Conclusion： The expression of anti-cyclin D1 intrabody gene with NLS inhibits proliferation,arrests the cell cycle,and induces cell apoptosis of MCF-7 cells.