江建新,吴渊,高珊,孙诚谊
JIANG Jianxin1,WU Yuan1,GAO Shan2,SUN Chengyi1(1.Department of Hepatobiliary Surgery

• 1:贵阳医学院附院肝胆外科
• 2:贵阳医学院附院消化内科

摘要(Abstract)：

目的:构建hsa-microRNA-138慢病毒表达载体。方法:PCR扩增pri-miR-138-2前体序列,克隆至plenti-GFP慢病毒表达载体,双酶切及测序鉴定正确后进行慢病毒包装与滴度检测;构建成功后感染人胰腺癌细胞PANC-1,48 h后Real-time Q-PCR检测miR-138的表达。结果:酶切、测序鉴定证明插入序列正确,测定病毒滴度为1×109TU/ml,病毒感染48 h后的PANC-1胰腺癌细胞观察可见绿色荧光,Real-time Q-PCR显示被感染细胞的miR-138表达量较未感染细胞显著增高。结论:建立了高效稳定表达hsa-miR-138的慢病毒转染系统。
Objective: To construct hsa-microRNA-138 lentiviral vector.Methods: Pre-miR-138-2 amplified by PCR was cloned into plenti-GFP vector,and was identified by restriction endonuclease digestion and nucleotide sequencing,and then the lentivirus was packaged and virus titer was detected.After successfully constructed,the vector was transfected into human pancreatic cancer cell PANC-1.After 48h,the expression of hsa-miR-138 was detected by Real-time Q-PCR.Results: Restriction enzyme digestion and DNA sequencing demonstrated that the inserted sequences were correct.The titer of virus was 1×109 TU/mL.After lentiviral infection,green fluorescence was detectable under fluorescence inverted microscope in PANC-1 cells,and real-time Q-PCR showed that expression amount of hsa-miR-138 in transfected cells was more than that in non-transfected cells.Conclusions: The lentiviral vector expressing hsa-miR-138 efficiently and stably is successfully constructed.

关键词(KeyWords)： RNA,小干扰;慢病毒属;载体;波形蛋白;胰腺肿瘤
RNA,small interfering;lentivirus;vector;vimentin;pancreatic neoplasms

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金资助(81160311);; 贵州省科技厅贵阳医学院社发联合基金资助[黔科合(2010)3171];; 贵阳市科技局社会发展与民生科技计划资助[筑科合(2011103)22号]

作者(Author): 江建新,吴渊,高珊,孙诚谊
JIANG Jianxin1,WU Yuan1,GAO Shan2,SUN Chengyi1(1.Department of Hepatobiliary Surgery

DOI: 10.19367/j.cnki.1000-2707.2012.05.004

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