hsa-miR-20a低表达慢病毒载体的构建及其表达鉴定

目的:构建hsa-mi R-20a低表达慢病毒载体,检测其在HL-60中表达。方法:采用In-fusion重组交换克隆法设计并合成hsa-mi R-20a前体序列的扩增引物,扩增获得目的片段插入慢病毒GV159中,得到重组的LV-hsa-mi R-20a表达载体,通过与包装质粒共转染293T细胞,获得携带hsa-mi R-20a的重组慢病毒并测定病毒滴度。取对数生长期HL-60细胞根据病毒滴度及细胞MOI值感染慢病毒,感染后24 h、48 h、72 h、96 h镜下观察荧光表达情况,判断感染效率,q RT-PCR检测HL-60细胞hsa-mi R-20a的表达变化。结果:成功构建LV-hsa-mi R-20a低表达慢病毒载体,其病毒滴度为(8E+8)TU/m L。该病毒感染HL-60细胞的效率可高达到80%,并可有效降低HL-60细胞hsa-mi R-20a表达水平。结论:成功构建了hsa-mi R-20a低表达慢病毒载体,包被的慢病毒可以在HL-60细胞中实现低表达效果,为后续功能研究奠定了基础。

Construction of a Recombinant Lentiviral Vector to Inhibit hsa-miR-20a Expression and Validation of its Transduction Efficiency

Objective:To construct a recombinant lentiviral vector to inhibit hsa-miR-20a expression and validation of its transduction efficiency in HL-60.Methods:Based on BD In-FusionTM PCR cloning method, we designed and synthesized hsa-miR-20a gene with its promoter, then cloned it into a lentivirus vector GV1 59. After being conformed, the LV-hsa-miR-20a plasmid was co-transfected with packaging plasmid into 293T cells to obtain hsa-miR-20a lentivirus vector. Recombinant lentivirus was added to logarithmic growth phase HL-60 cells according to virus titer and MOI (multiplicity of infection) value, and its infection efficiency and hsa-miR-20a level were tested by fluorescent observation and real-time quantitative PCR respectively.Results:Hsa-miR-20a lower expressive lentiviral vector was constructed successfully, and virus titer was (8E+8)TU/mL. The infection efficiency of Recombinant lentiviruse in HL-60 cells reached 80%, and the expression level of hsa-miR-20a reduced effectively.Conclusion:The letiviral vector containing hsa-miR-20a with low expression was successfully constructed, which will be useful for researching the function of hsa-miR-20a in the future.