p21^WAF1与p27^KIP1真核双表达载体的构建鉴定及在胃癌细胞中的表达

Construction and identification of recombinant eukaryotic double expression vector pIRES-p27^KIP1- p21^WAF1 and its expression in human gastric carcinoma cells SGC-7901

目的构建p21^WAF1与p27^KIP1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p21^WAF1和p27^KIP1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pIRES-p27^KIP1-p21^WAP1，经酶切和测序鉴定重组质粒。脂质体介导重组质粒pIRES-p27^KIP1-p21^WAP1转染胃癌7901细胞，收集并提取转染后12、24、48、72h和5d各细胞的总RNA，逆转录成cDNA，荧光定量聚合酶链反应（FQ-PER）检测各细胞中p21^WAF1和p27^KIP1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600bp大小的产物，重组质粒pIRES-p27^KIP1-p21^WAF1经酶切和测序鉴定证实为p21^WAF1和p27^KIP1。基因。转染胃癌7901细胞48h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、72h和5d后FQ-PCR证实p27^KIP1。的Ct值（cycle threshold，Ct）分别减少8．2、10、10、7、4和6．7，p21^WAF1的Ct值分别减少7．4、8．2、8．2、6．3和5．7。其中24h和48h的Ct值减少最大。结论真核双表达载体pIRES-p27^KIP1-p21^WAF1构建成功并能在胃癌7901细胞内高效表达。

Objective To construct and identify recombinant eukaryotic double expression vector pIRES-p27^KIP1-p21^WAF1 and observe the expression of the recombinant pIRES-p27^KIP1-p21^WAF1 in human gastric carcinoma cells SGC-7901. Methods Total RNA was isolated from healthy human blood. The fulllength open reading frames of human p21^WAF1 and p27^KIP1 gene were amplified by RT-PCR and cloned into eukaryofic 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 pIRES-p27^KIP1-p21^WAF1, the Ct value of p27^KIP1 was reduced by 8.2,10,10,7.4 and 6.7,and that of p21^WAF1 was reduced by 7.4,8.2,8.2,6.3 and 5.7 respectively. Conclusion The recombinant pIRES-p27^KIP1-p21^WAF1 was constructed successfully and expressed effectively in human gastric carcinoma cells.