小鼠SOCS1基因沉默重组慢病毒载体的构建及其在DC2.4细胞中的表达

Construction of a recombinant lentiviral vector with a silenced SOCS1 gene and identification its expression in DC2.4 cells

目的构建携带小鼠SOCS1基因沉默重组慢病毒载体,鉴定其在小鼠树突状细胞系(DC2.4)中SOCS1基因的表达并筛选稳定沉默表达SOCS1基因的小鼠树突状细胞系。方法根据小鼠SOCS1基因筛选相应靶序列,与表达载体pYr-Lvsh连接后转化感受态DH5α细胞,挑取细胞克隆进行酶切及测序鉴定,鉴定正确的载体转染293FT细胞,进行慢病毒包装及滴度测定。将包装的慢病毒PLV-musSOCS1-shRNA转染DC2.4细胞系,通过荧光显微镜、流式细胞仪及real-time PCR检测DC2.4细胞中SOCS1基因的表达,通过台盼蓝染色检测转染后细胞活性。结果表达载体pYr-Lvsh测序显示引物完全连接到载体上。将测序所得序列与引物比对,PLV-musSOCS1-sh构建成功。扩增后检测慢病毒滴度约为4×109 TU/ml。慢病毒转染24h后荧光显微镜下可见少量表达绿色荧光的DC2.4细胞,且随着时间的延长,转染成功的细胞逐渐增多,细胞内荧光强度渐次增强,48h后表达量明显增多,72h后更加明显。经流式细胞仪检测,转染48h及72h细胞绿色荧光表达率均达100%。对照组的细胞活性为(92.27±0.80)%,转染组转染后48h的细胞活性为(88.40±0.92)%,差异有统计学意义(t=5.499,P<0.01);转染组转染后72h的细胞活性为(44.97±3.70)%,与对照组及48h转染组比较差异有统计学意义(t值分别为21.637和19.733,P<0.01)。real-time PCR检测,转染组SOCS1基因相对表达量较空病毒载体对照组下调约75.3%(t=-10.179,P<0.01)。结论构建的慢病毒PLV-musSOCS1-shRNA在DC2.4细胞中成功沉默SOCS1基因并稳定表达,成功筛选出低表达SOCS1基因的小鼠树突状细胞系,为通过SOCS1基因沉默调控DC免疫状态抗真菌感染免疫等相关研究奠定了基础。

Objectives To construct a recombinant lentiviral vector containing a silenced mouse suppressor of cytokine signaling 1（SOCS1）gene and to identify its expression in a mouse dendritic cell line（DC 2.4）in order to generate a mouse dendritic cell line with a stably silenced SOCS1 gene. Methods The sequence of mouse SOCS1 mRNA was identified and ligated into a pYr-Lvsh expression vector.DH5αcells were transformed with the ligated product.Selected clones were identified using enzyme digestion and sequencing.Once the expression plasmid was verified as correct,it was transfected into 293 FT cells.Lentivirus particles were packaged,and the viral titer was determined using a serial dilution assay.After the lentivirus（PLV-musSOCS1-shRNA）was transfected into DC2.4 cells,the level of SOCS1 expression in DC2.4 cells was evaluated using fluorescence microscopy,FCM,and a real-time polymerase chain reaction.The activity of transfected cells was detected using trypan blue staining. Results Sequencing of the expression vector（pYr-Lvsh）indicated that the primer was fully ligated to the carrier.A comparison of the sequence and primer indicated that the vector（PLV-musSOCS1-sh）was successfully constructed.The lentivirus titer after amplification was 4×109 TU/ml.Twenty-four h after transfection,few successfully transfected DC2.4 cells emitted green fluorescence according to fluorescence microscopy.The successfully transfected DC2.4 cells increased and the intensity of green fluorescence gradually in-creased over time.The fluorescence intensity increased markedly after 48 hand was even more evident after 72 h.Cells emitted green fluorescence at a rate of 100%according to FCM.The activity of control cells was 92.27±0.80% while the activity of cells 48 hafter transfection was 88.40±0.92%.Levels of cell activity differed significantly（t=5.499,P0.01）.The activity of cells 72 hafter transfection decreased significantly compared to that of the control cells and cells 48 h after transfection.Levels of cell activity differed significantly（the t values were 21.637 and 19.733,respectively,P0.01）.The level of expression of the SOCS1 gene in transfected cells（about 75.3%）was lower than that in the empty viral vector control group（t=-10.179,P0.01）according to real-time PCR. Conclusion The SOCS1 gene can be successfully silenced and stably expressed in DC2.4 cells infected with a lentivirus（PLV-musSOCS1-shRNA）.A mouse dendritic cell line can be generated with a lower level of SOCS1 gene expression,and this may lay the foundation for further study of dendritic cells with a silenced SOCS1 gene and the immune response to a fungal infection.