摘要
目的:构建重组质粒PGCsi-Pyk2shRNA,并检测所引起的Pyk2mRNA和蛋白在Lovo结肠癌细胞系中的表达.方法:设计合成3对Pky2基因shRNA序列,形成双链后将其依次连入带有U6启动子并含有潮霉素B的pGCsi空载体,构建成能产生Pyk2短发卡RNA的质粒;采用双酶切和测序分析鉴定插入基因的序列;脂质体介导重组质粒pGCsi-Pyk2shRNA稳定转染Lovo细胞系并通过潮霉素B筛选,获得比较单一的转染细胞;分别采用RT-PCR、Westernblot等检测转染前后Pyk2的水平表达.结果:酶切鉴定和测序分析表明重组表达质粒pGCsi-Pyk2shRNA构建无误;绿色荧光照相及PCR表明质粒转染成功;RT-PCR和Westernblot均表明,与转染空质粒PGCsi组和转染仅含免疫荧光基因组细胞相比,转染重组表达质粒pGCsi-Pyk2shRNA细胞中Pyk2mRNA及蛋白表达水平均明显降低.结论:成功构建重组质粒pGCsi-Pyk2shRNA,并证明他能降低Pyk2在Lovo细胞株系中的表达,为进一步研究Pyk2如何调控Hic-5/ARA55,Paxillion等下游靶基因的表达和参与结肠癌表观遗传学发生机制奠定了基础.
AIM： To construct the recombinant small hairpin RNA （shRNA） plasmids targeting the prolinerich tyrosine kinase 2 （Pyk2） gene （pGCsi-Pyk2 shRNA） and detect their expression in Lovo cells. METHODS： Three pairs of Pyk2 shRNA sequences were designed and ligated to the pGCsi vector that contains U6 promoter and hygromycin B to obtain shRNA expression plasmids targeting the Pyk2. The recombinant pGCsi- Pyk2 shRNA plasmids were introduced into Lovo cells by liposome-mediated transfectionand selected with hygromycin B. The expression of Pyk2 mRNA and protein was detected by reverse transcription-polymerase chain reaction （RT-PCR） and Western blot, respectively. RESULTS： Restriction enzyme digestion and sequence analysis showed that recombinant pGCsi-Pyk2 shRNA plasmids were successfully constructed. The expression levels of Pyk2 mRNA and protein in Lovo cells transfected with pGCsi-Pyk2 shRNA plasmids were signifi-cantly lower than those in Lovo cells transfected with empty or negative plamsids. CONCLUSION： Recombinant pGCsi-Pyk2 shRNA plasmids are successfully constructed. Their transfection can silence the expression of Pyk2 gene in Lovo cells. The pGCsi-Pyk2 shRNA plasmids obtained lay a foundation for further study of the role of the Pyk2 gene in the pathogenesis of colorectal cancer.
出处
《世界华人消化杂志》
CAS
北大核心
2010年第9期877-882,共6页
World Chinese Journal of Digestology
基金
国家自然科学基金资助项目
No.30572105~~
关键词
PYK2
结肠癌
RNA干扰
小发夹RNA
pGCsi载体
Proline-rich tyrosine kinase 2
Colon cancer
RNA Interference
Small hairpin RNA
pGCsi vector
