小鼠Slfn3重组腺病毒载体的构建及其在EPCs中转染效率的测定

Construction of mouse Slfn3 recombinant adenovirus vector and determination of transfection efficiency in EPCs

目的构建携带小鼠Schlafen3(Slfn3)基因的重组过表达质粒,并观察其在内皮祖细胞(EPCs)中的转染效率。方法从GenBank基因数据库获取小鼠Slfn3基因序列,利用VectorNTI软件对其进行引物设计,通过PCR扩增获得目的基因,将所得到的目的基因与穿梭载体pcADV-EF1-mNeonGreen-CMV-MCS-3xFLAG相连接,获得重组腺病毒载体pcADV-EF1-mNeonGreen-CMV-Slfn3-3xFLAG;筛选阳性克隆抽提质粒,通过限制性内切酶EcoRI和BamHI双酶切鉴定,并通过DNA测序验证;采用Admax系统将目的穿梭质粒和腺病毒骨架质粒一同转染HEK293细胞获得重组腺病毒(Ad-pcADV-EF1-mNeonGreen-CMV-Slfn3-3xFLAG),对其进行病毒滴度测定,Western blot验证Slfn3蛋白的表达;体外培养小鼠脾源EPCs 5~7 d,细胞密度达70%后,用Ad-pcADV-EF1-mNeonGreen-CMV-Slfn3-3xFLAG进行转染48 h后,采用荧光显微镜观察并统计绿色荧光细胞数量,计算Ad-pcADV-EF1-mNeonGreen-CMV-Slfn3-3xFLAG转染的效率。结果DNA测序验证Ad-pcADV-EF1-mNeonGreen-CMV-Slfn3-3xFLAG构建成功,病毒滴度为3.16×1010(ifu/mL);腺病毒包装之后的重组腺病毒能够转染HEK293细胞,并在细胞中表达Slfn3-EGFP-3xFLAG融合蛋白,且含有Flag标签的蛋白,大小为57 kDa;重组腺病毒在EPCs中的转染效率为(71.43±2.58)%。结论成功地构建了小鼠Slfn3的过表达重组腺病毒载体,且在EPCs转染效率较高。

Objective To develop and evaluate a recombinant adenoviral vector expressing mouse Schlafen3(Slfn3)in endothelial progenitor cells(EPCs).Methods The mouse Slfn3 gene sequence was retrieved from GenBank and amplified using PCR with primers designed by VectorNTI software.The amplified gene was inserted into the pcADV-EF1-mNeonGreen-CMV-MCS-3xFLAG shuttle vector.Following plasmid extraction from positive clones,construct validation was performed using EcoRI/BamHI restriction digestion and DNA sequencing.The recombinant adenovirus was generated using the Admax system in HEK293 cells.Viral titer was determined and Slfn3 expression was confirmed by Western blot analysis.Mouse spleen-derived EPCs were cultured to 70%confluence and transfected with the recombinant virus.Transfection efficiency was assessed by fluorescence microscopy after 48 hours.Results The recombinant adenoviral vector was successfully constructed as confirmed by DNA sequencing.The viral titer reached(3.16×1010)ifu/mL.The vector effectively transfected HEK293 cells,producing a 57 kDa Slfn3-EGFP-3xFLAG fusion protein.EPC transfection efficiency was(71.43±2.58)%.Conclusion A functional mouse Slfn3 adenoviral expression vector has successfully been generated,and demonstrated high transfection efficiency in EPCs.