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目的构建大鼠PAX2慢病毒表达载体,以期在大鼠肾脏中高效、稳定表达。方法设计引物引入AgeⅠ酶切位点,使用PCR方法从质粒pcDNA3.1-PAX2中扩增小鼠PAX2基因的编码区序列,对所扩增出的目的片段回收纯化。用In-Fusion技术将AgeⅠ内切酶消化后目的片段交换连接入AgeI酶切的pGC-FU载体,构建PAX2慢病毒表达载体pGC-FU-PAX2。酶切验证并测序正确后,将质粒pGC-FU-PAX2与慢病毒辅助包装载体共转染293T细胞,Western-blot验证PAX2在转染293T细胞中表达。结果通过PCR扩增获得了PAX2基因,将PAX2克隆到慢病毒转移质粒pGC-FU中,并在293T细胞中包装产生慢病毒颗粒。结论成功构建了PAX2慢病毒表达载体,为进一步从分子水平探讨PAX2功能奠定了基础。
Objective To construct rat PAX2 lentivirus expression vector with a view to efficiently and stably express in rat kidney. Methods The primers designed to introduce Age Ⅰ restriction sites were used to amplify the coding region of mouse PAX2 gene from plasmid pcDNA3.1-PAX2 by PCR. The target fragment was recovered and purified. In-Fusion technology was used to digest the gene fragment after AgeⅠ endonuclease into the pGC-FU vector of AgeI digestion to construct the PAX2 lentivirus expression vector pGC-FU-PAX2. 293T cells were co-transfected with plasmid pGC-FU-PAX2 and lentivirus helper vector after digestion and sequencing. The expression of PAX2 in transfected 293T cells was confirmed by Western-blot. Results The PAX2 gene was obtained by PCR amplification, PAX2 was cloned into lentiviral transfer plasmid pGC-FU and packaged in 293T cells to produce lentivirus particles. Conclusion PAX2 lentiviral vector was successfully constructed, which laid the foundation for the further exploration of PAX2 function at the molecular level.