hsa-microRNA-96-5p down慢病毒表达载体的构建与鉴定

Construction and Identification of Hsa-micorRNA-96-5p Down Lentivirus Expression Vector

目的 构建hsa-microRNA-96-5p down慢病毒表达载体并对其进行鉴定.方法 根据hsa-miR-9-5p基因序列设计合成hsa-miR-96-5p-inhibition-a和hsa-miR-96-5p-inhibition-b,将合成后成对的引物干粉溶解于退火缓冲液中引物退火.将慢病毒GV280载体酶切产物通过T4DNA连接酶将双酶切线性化的载体和退火双链DNA连接.经菌落筛选,菌落PCR及测序鉴定,荧光法及药筛法测定病毒效价.结果 菌落PCR和测序验证成功构建了hsa-miR-96-5p down慢病毒载体,经荧光法及药筛法测定病毒效价为3×108TU/ml.结论 成功构建了hsa-miR-96-5p down慢病毒表达载体,为后续深入研究miR-9-5p在肺癌细胞中的生物学行为及机制提供了前期实验基础.

Objective To construct and identify a hsa-micorRNA-96 down lentivirus expression vector.Methods According to the gene sequence of hsa-microRNA-96,we designed and synthesized two pairs of single stranded oligonucleotides hsa-microRAN-96-5p-inhibition-a and hsa-miR-96-5 p-inhibition-b.The powder of primers was then dissolved in the annealing buffer and to be annealed.The double enzyme digested GV280 lentivirus vectors were linked to the annealing double-stranded DNA via T4 DNA ligase.The constructed recombinant vectors were identified by single colony PCR and DNA sequencing after the colony isolation.The concentration of lentivirus was detected by the fluorescence and drug screening method.Results The single colony PCR and DNA sequencing confirmed that the purpose anti-hsa-microRNA-96 oligonucleotide was correctly inserted into the lentivirus vectors.The concentration of lentivirus was 3 × 108 TU/ml determined by the fluorescence and drug screening method.Conclusion It could be concluded that hsa-microRNA-96 down lentivirus expression vector was successfully constructed and identified,providing an experimental basis for further study on the biological behavior of microRNA-96 and its mechanism in lung cancer cells.