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

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.