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目的应用RNA干扰技术,构建小鼠水通道蛋白1(AQP1)基因重组慢病毒载体并鉴定。方法对小鼠AQP1 mRNA分析,设计合成的单链引物经退火形成双链寡核苷酸序列,连接入经Age I和EcoR I双酶切线性化的pLKO.1-TRC慢病毒质粒载体中,菌液PCR鉴定并经测序验证。测序正确后与慢病毒包装辅助质粒共转染293T细胞,收集病毒上清经高速离心浓缩后感染小鼠小胶质细胞BV-2,Western blot法分析筛选有效shRNA序列。结果成功退火合成3对发夹shRNA序列并将其克隆到pLKO.1-TRC载体中,构建重组质粒pLKO-AQP1-SH1、2、3,经菌液PCR鉴定和测序验证载体构建成功。Real-time PCR检测发现重组质粒pLKO-AQP1-SH2感染BV-2细胞后AQP1 mRNA表达最低,Western blot结果进一步验证结果的准确性。结论成功构建出AQP1基因shRNA慢病毒载体。
Objective To construct and identify recombinant lentiviral vector of aquaporin 1 (AQP1) gene of mouse by RNAi technique. Methods After analyzing the AQP1 mRNA in mice, a single-stranded primer was designed and synthesized to anneal to form a double-stranded oligonucleotide. The double-stranded oligonucleotide was ligated into pLKO.1-TRC lentiviral plasmid vector, Bacterial PCR identification and verification by sequencing. 293T cells were co-transfected with lentiviral packaging helper plasmids after sequencing. The virus supernatants were collected and concentrated in high speed centrifugation to infect mouse microglia BV-2. Western blot analysis was performed to screen for effective shRNA sequences. RESULTS: Three pairs of hairpin shRNAs were successfully annealed and cloned into pLKO.1-TRC vector. The recombinant plasmid pLKO-AQP1-SH1,2,3 was successfully constructed and verified by bacterial PCR. Real-time PCR showed that the expression of AQP1 mRNA was the lowest in BV-2 cells infected by recombinant plasmid pLKO-AQP1-SH2, and the accuracy of the results was further verified by Western blot. Conclusion The AQP1 shRNA lentiviral vector was successfully constructed.