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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。
Objective To construct RNA interference (RNAi) lentiviral vector containing human dendritic cells (hDCs) signal transduction pathway inhibitor 1 (SOCS1) gene. Methods Based on the human dendritic cell SOCS1 gene (NM-0037), a target sequence was screened and designed to synthesize a complementary single-stranded oligonucleotide containing the target sequence of sense and antisense, which was compared with the lentivirus digested with BamH and Xho The plasmid pRNA-Lenti-enhanced green fluorescent protein (EGFP) (containing the U6 promoter and EGFP) was ligated to produce the lentiviral vector pRNA-Lenti-SOCS1-EGFP. The 293T cells were cotransfected with the lentivirus packaging mixture, Virus, collect virus supernatant, take a series of dilution method to measure lentivirus titer. HDCs were transfected, and the cell transfection was observed by fluorescence microscopy. The expression of SOCS1 in the interference group, negative control group and blank control group was detected by real-time quantitative PCR and Western blot. Results The target sequence was successfully ligated into the vector and confirmed by sequencing analysis that the vector was successfully constructed. Fluorescence real-time quantitative PCR and Western blot showed that the expression of siRNA group mRNA and SOCS1 protein were significantly decreased (P <0.05) compared with the blank control group and the negative control group. Conclusion The constructed pRNA-Lenti-SOCS1-EGFP lentiviral vector can effectively inhibit the expression of SOCS1 in hDCs, which lays the foundation for further study on the anti-tumor immune response effect of DCs.