Hsa-mir-196b慢病毒表达载体的构建及鉴定  

Construction of Lentiviral Vector for the Expression of Hsa-mir-196b

作　　者：向星宇[1] 马文丽[1] 宋艳斌[1] 姜茂竹[1] 王妮莎[1] 郑文岭[1,2] 

机构地区：[1]南方医科大学基因工程研究所,广州510515 [2]华南基因组研究中心,广州510800

出　　处：《热带医学杂志》2010年第5期513-515,F0002,共4页Journal of Tropical Medicine

基　　金：教育部科学技术研究重点项目(No.208100)

摘　　要：目的构建Hsa-mir-196b慢病毒表达载体并对其进行鉴定。方法以正常人外周血DNA为模板,PCR扩增得到目的基因。通过HpaⅠ/XhoⅠ双酶切及其后的连接将其插入Lentilox3.7(pLL-3.7)质粒中。PCR筛选阳性克隆,测序鉴定。用pLL-3.7-mir-196b、pCMV-VSV-G和pCMV-dR8.91三质粒系统共感染HEK-293FT细胞,包装生产慢病毒。将所得病毒悬液梯度稀释后感染HEK293FT细胞,以检测病毒滴度,并用实时定量PCR检测慢病毒感染后Hsa-mir-196b的表达变化。结果 PCR及测序结果证明成功构建了pLL-3.7-mir-196b重组质粒。所得慢病毒上清滴度为(7.2±1.1)×107TU/ml。感染慢病毒的239FT细胞中,Hsa-mir-196b的表达量相对于未感染细胞提高了约24倍。结论成功构建Hsa-mir-196b慢病毒表达载体。Objective To construct a lentiviral vector for the expression of Hsa-mir-196b. Methods Hsamir-196b gene amplified from the peripheral blood DNA of healthy subjects was inserted into the Lentilox 3.7 （pLL-3.7） plasmid by double digestion with HpaⅠ / XhoⅠand subsequent ligation. Plasmids pLL-3.7-mir-196b,pCMV-VSV-G and pCMV-dR8.91 were co-transfected into HEK-293FT cells for packaging of the lentivirus. HEK293FT cells were then transduced with an appropriately diluted lentivirus supernatant for the titration of virus titer. Expression of Hsa-mir-196b was investigated using real-time PCR. Results Results from the PCR and DNA sequencing confirmed that the recombinant plasmid pLL-3.7-mir-196b was successfully constructed. The titer of supernatant was 7.2 ±1.1× 107 TU / ml. The expression level of Has-mir-196b in the lentivirus transduced HEK-293FT cells was increased by 24-fold. Conclusion Hsa-mir-196b expression lentiviral vector was successfully constructed.

关 键 词：Hsa-mir-196b 慢病毒 肿瘤 

分 类 号：R73[医药卫生—肿瘤]