| 张 熠 杨 靖 吕运成 钟 警 文格波.hsa-miR-20a 低表达慢病毒载体的构建及其表达鉴定[J].,2015,15(5):804-807 |
| hsa-miR-20a 低表达慢病毒载体的构建及其表达鉴定 |
| Construction of a Recombinant Lentiviral Vector to Inhibit hsa-miR-20aExpression and Validation of its Transduction Efficiency |
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| DOI： |
| 中文关键词: Hsa-miR-20a 慢病毒载体 HL-60 |
| 英文关键词: Hsa-miR-20a Lentivirus HL-60 |
| 基金项目:国家自然科学基金项目（ 81 100560） |
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| 中文摘要: |
| 目的： 构建 hsa-miR-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-miR-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-miR-20a 表达载体， 通过与包装
质粒共转染 293T 细胞， 获得携带 hsa-miR-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， qRT-PCR 检测 HL-60 细胞 hsa-miR-20a
的表达变化。 结果： 成功构建 LV-hsa-miR-20a 低表达慢病毒载体，其病毒滴度为 (8E+8)TU/mL。 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-miR-20a 表达水平。 结论： 成功构建了 hsa-miR-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。 |
| 英文摘要: |
| 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. |
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