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  • 林士波1,程张军1,2*,石 欣1, 沈 锋2.连环蛋白p120 shRNA重组慢病毒载体构建及鉴定[J].第二军医大学学报,2010,31(5):553-557    [点击复制]
  • LIN Shi-bo1, CHENG Zhang-jun1,2*, SHI Xin1, SHEN Feng2.Construction and identification of recombinant lentiviral vector expressing shRNA of p120 catenin gene[J].Acad J Sec Mil Med Univ,2010,31(5):553-557   [点击复制]
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连环蛋白p120 shRNA重组慢病毒载体构建及鉴定
林士波1,程张军1,2*,石欣1,沈锋2
0
(1.东南大学附属中大医院普通外科,南京 210009;2.第二军医大学东方肝胆外科医院,上海 200438)
摘要:
目的 构建连环蛋白p120特异性shRNA重组慢病毒载体,感染胰腺癌PANC-1细胞,鉴定干扰效果。方法 针对p120ctn mRNA设计合成3对shRNA序列,连接入酶切线性化的慢病毒载体pGCSIL-GFP,PCR鉴定及测序正确后与构建成功的含p120ctn基因的载体pGC-FU-p120ctn-3FLAG共转染293T细胞,蛋白质印迹分析筛选有效shRNA序列,包装慢病毒,测定病毒滴度。重组慢病毒感染胰腺癌PANC-1细胞,实时定量PCR及蛋白质印迹分析鉴定干扰效果。结果 PCR及测序证实成功构建出p120ctn-shRNA-LV慢病毒载体,病毒滴度达到3×109TU/ml。重组慢病毒感染PANC-1细胞后,实时定量PCR提示PANC-1细胞p120ctn mRNA表达下降82.6%(P<0.05),蛋白质印迹分析提示p120ctn蛋白表达较转染前显著下降。结论 成功构建出p120ctn基因shRNA慢病毒载体,为进一步研究p120ctn在胰腺癌浸润及转移中的作用奠定了基础。
关键词:  连环蛋白p120  RNA干扰  慢病毒  胰腺肿瘤
DOI:10.3724/SP.J.1008.2010.0553
投稿时间:2009-10-19修订日期:2010-04-10
基金项目:国家自然科学基金面上项目(30700801).
Construction and identification of recombinant lentiviral vector expressing shRNA of p120 catenin gene
LIN Shi-bo1, CHENG Zhang-jun1,2*, SHI Xin1, SHEN Feng2
(1. Department of General Surgery, Zhongda Hospital, Southeast University, Nanjing 210009, Jiangsu, China;2. Eastern Hepatobiliary Hospital, Second Military Medical University, Shanghai 200438, China)
Abstract:
Objective To construct the recombinant lentiviral vector expressing specific shRNA of p120 catenin gene, and to identify the RNA interference efficiency by infecting PANC-1 cells. Methods Three pairs of shRNA targeting p120ctn mRNA were designed, synthesized and ligated into enzyme-digested and linearized pGCSIL-GFP lentiviral vectors. The recombinant lentiviral vectors were co-transfected with the pGC-FU-p120ctn-3FLAG containing p120ctn gene into 293T cells after identification by PCR and sequencing analysis. Western blotting analysis was applied to select the most effective shRNA. Recombined lentivirus was packaged and the concentration of the virus titer was measured. Real-time PCR and Western blotting analysis were used to identify the interference efficiency after infection of the PANC-1 cells by recombined lentivirus.Results PCR and DNA sequencing analysis confirmed that p120ctn-shRNA-LV was successfully constructed and the concentration of the virus titer was 3×109TU/ml. Real-time PCR showed that expression of p120ctn mRNA was decreased by 82.6% in PANC-1 cells infected with the recombined lentivirus (P<0.05). Western blotting analysis showed that the expression of p120ctn protein was also greatly deceased after infection. Conclusion We have successfully constructed lentiviral vector expressing shRNA of p120 catenin gene, and this lays a foundation for studying the role of p120ctn in invasion and metastasis of pancreatic carcinoma.
Key words:  p120 catenin  RNA interference  lentivirus  pancreatic neoplasms