p21WAF1与p27KIP1真核双表达载体的构建鉴定及在胃癌细胞中的表达

目的 构建p21WAF1与p27KIP1真核双表达载体,体外转染胃癌7901细胞,观察其在胃癌细胞中的表达及相关特性.方法 提取人全血总RNA,利用逆转录-聚合酶链反应(RT-PCR)扩增p21WAF1和p27WAF1基因并将其分别克隆到真核双表达载体pIRES中构建重组质粒pIPES-p27KIP1-p21WAF1,经酶切和测序鉴定重组质粒.脂质体介导重组质粒plRES-p27KIP1.p21WAF1"转染胃癌7901细胞,收集并提取转染后12、24、48、72 h和5 d各细胞的总RNA,逆转录成cDNA,荧光定量聚合酶链反应(FQ-PCR)检测各细胞中p21WAF1和p27KIP1在RNA水平的表达.带有绿色荧光蛋白的pIRES-EGFP作为对照.结果 RT-PCR扩增分别获得约500和600 bp大小的产物,重组质粒pIRES-p27KIP1.p21WAF1经酶切和测序鉴定证实为p21WAF1和p27KIP1基因.转染胃癌7901细胞48 h时转染率为68%.与空质粒对照组比较,重组质粒转染胃癌7901细胞12、24、48、72 h和5 d后FQ-PCR证实p27KIP1的Ct值(cycle threshold,Ct)分别减少8.2、10、10、7.4和6.7,p21WAF1的Ct值分别减少7.4、8.2、8.2、6.3和5.70其中24 h和48 h的Ct值减少最大.结论 真核双表达载体pIRES-p27KIP1.p21WAF1构建成功并能在胃癌7901细胞内高效表达.

Construction and identification of recombinant eukaryotic double expression vector pIRES-p27KIP1-p21WAF1 and its expression in human gastric carcinoma cells SGC-7901

Objective To construct and identify recombinant eukaryotie double expression vector pIRES-p27KIP1-p21WAF1 and observe the expression of the recombinant plRES-p27KIP1-p21WAF1 in human gastric carcinoma cells SGC-7901. Methods Total RNA was isolated from healthy human blood. The full-length open reading frames of human p21WAF1 and p27KIP1 gene were amplified by RT-PCR and cloned into eukaryotic double expression plasmid pIRES. After identified by enzyme digestion and DNA sequencing, the recombinant plasmid was transfected into human gastric carcinoma cells SGC-7901 with liposomes. The fluorescence quantitative polymerase chain reaction (FQ-PCR) was used to detect the mRNA expression. The pIRES-EGFP plasmid with green fluorescence protein served as control. Results The recombinant plasmid was constructed successfully,which was confirmed by enzyme digestion and DNA sequencing. The transfection rate of human gastric carcinoma cells was 68% 48 h after transfection. Compared with blank vector group,12,24,48,72 h and 5 days after transfection with plRES-p27KIP1-p21WAF1 ,the Ct value of p27KIP1 was reduced by 8.2,10,10,7.4 and 6.7, and that of p21WAF1 was reduced by 7.4,8.2,8.2,6.3and 5.7 respectively. Conclusion The recombinant pIRES-p27KIP1-p21WAF1 was constructed successfully and expressed effectively in human gastric carcinoma cells.