人树突状细胞SOCS1基因RNA干扰慢病毒载体的构建及鉴定

目的构建人树突状细胞（hDCs）信号传导通路抑制因子1（SOCS1）基因RNA干扰（RNAi）慢病毒载体。方法根据人树突状细胞SOCS1基因（NM-0037）,筛选出一个靶序列,设计并合成包含正反义靶序列的互补单链寡核苷酸,与经BamH和Xho酶切后的慢病毒载体质粒pRNA-Lenti-增强型绿色荧光蛋白（EGFP）（含U6启动子和EGFP）连接产生pRNA-Lenti-SOCS1-EGFP慢病毒重组质粒,与慢病毒包装混合物共转染293T细胞,包装产生慢病毒,收集病毒上清,采取系列稀释法测定慢病毒滴度。然后转染hDCs,通过荧光显微镜观察细胞转染情况,利用荧光实时定量PCR和Westernblot检测干扰组、阴性对照组、空白对照组SOCS1的表达情况。结果将目的序列成功连接到载体上,并经测序分析证实载体构建成功。荧光实时定量PCR及Westernblot检测显示慢病毒重组质粒感染hDCs后,与空白对照组及阴性对照组比较,siRNA组mRNA和SOCS1蛋白的表达量显著降低,差异均有统计学意义（P〈0.05）。结论构建的pRNA-Lenti-SOCS1-EGFP慢病毒载体可有效地抑制hDCs的SOCS1的表达,为进一步研究DCs增强抗肿瘤免疫应答效应奠定基础。

Construction and identification of lentiviral-mediated RNA interference vector for human SOCS1 gene in monocyte-derived dendritic cells

Objective To construct a lentiviral-mediated RNA interference （RNAi） vector targeting human suppressors of cytokine signaling 1 （SOCS1） gene of monocyte-derived dendritic cells. Methods The sequence of human SOCS1 mR NA （NM-0037） was identified through literature search and, both sense and antisense oligonucleotides were designed and synthesized. The annealed double-stranded DNA was cloned to pRNA-Lenti-EGFP vector,which contained promoter U6 and EGFP. The recombinant plasmid and lentivector packaging plasmid mix were co-transfected into 293T cells to package lentivirus particles. Culture supernatant was harvested, and the virus titer was determined by serial dilution assay. After transduction of the human monocyte-derived dendritic cells （hDCs） with the constructed l entiviral vectors, real-time polymerase chain reaction and Western blot was performed to evaluate the level of SOCS1 expression. Results The SOCS1 cDNA sequence was successfully cloned to pRNA-Lenti-EGFP. Compared to the control group,mRNA and SOCS1 expression was significantly suppressed in the siRNA group（P〈0.05）. Conclusion The lentiviral-mediated RNAi vector of human SOCS1 gene has been constructed successfully, and this may provide a foundation for the further studies of DCs and anti-tumor immune response.