摘要
目的构建PRL-3基因特异性shRNA慢病毒干扰载体,感染结肠癌SW480细胞,研究PRL-3基因在结肠癌细胞株中的表达抑制情况。方法设计并合成3对特异性针对PRL-3基因的shRNA序列,构建到慢病毒载体中,测序鉴定载体构建情况,转染293T细胞,荧光显微镜下观察转染效率。收集到的病毒上清感染结肠癌SW480细胞,RT-PCR检测各组细胞中PRL-3 mRNA表达水平。结果经测序鉴定成功构建3对PRL-3基因的RNAi重组慢病毒载体,包装、浓缩后其滴度为5×108 TU·mL-1,3对PRL-3 shRNA病毒载体感染结肠癌SW480细胞株后,Real-time PCR显示,各组PRL-3基因敲减效率分别达到89.6%、83.2%和88.8%,PRL-3 shRNA1重组慢病毒载体干扰效率最佳。结论本研究成功构建了靶向PRL-3基因的shRNA慢病毒载体,PRL-3 shRNA1可抑制SW480细胞中PRL-3基因表达水平。
Objective To construct PRL-3 gene-specific shRNA lentivirus interference vector and infect SW480 colon cancer cells, and study the expression inhibition of PRL-3 gene in colon cancer cell lines. Methods Three interfering sequences shRNAs targeting PRL-3 were synthesized and constructed the lentiviral vector, and the recombinant was identificated by DNA sequencing analysis, then the lentiviral interference vectors of PRL-3 shRNAs were transfected and packaged into 293T cells. The transfection efficiency was detected under fluorescence microscope. The lentiviral particles were collected to infect human colon cancer SW480cells. The expression of PRL-3 mRNA was detected by real-time quantitative polymerase chain reaction (RT-PCR). Results DNA sequencing identified successful construction of the three PRL-3 shRNA lentivirus vectors. The recombinant lentivirus gained from 293T cells had a titer 5×10^8TU·mL^-1.three PRL-3 shRNA lentivirus vectors infect SW480 cells, real-time PCR demonstrated that the interference efficiency of PRL-3 mRNA were 89.6%, 83.2%, 88.8%, respectively. PRL-3 shRNA1 showed the highest interference efficiency. Conclusion In this study, shRNA lentiviral vector targeting PRL-3 gene was successfully constructed, and PRL-3 shRNA1 is shown to inhibit the expression level of PRL-3 gene in SW480 cells.
作者
王燕
乔立娇
陈耀平
WANG Yan;Qiao Lijiao;CHEN Yaoping(Dept. of Medical Oncology,the General Hospital of Ningxia Medical University,Yinchuan 750004,China;Dept. of Reproductive Medicine Center,Ningxia Medical University,Yinchuan 750004,China)
出处
《宁夏医科大学学报》
2019年第1期28-32,共5页
Journal of Ningxia Medical University
基金
宁夏自然科学基金(NZ13147)
宁夏医科大学校级课题(XM2012009)
