细胞因子信号转导抑制因子1 RNA干扰慢病毒载体的构建与鉴定*☆

背景：以往关于细胞因子信号转导抑制因子1的研究多用脂质体或其他载体转染的技术，但存在效率低和安全性差等问题。 目的：构建细胞因子信号转导抑制因子1 RNA干扰慢病毒载体并进行鉴定。 方法：实验设计3组针对细胞因子信号转导抑制因子1的短发夹RNA序列，应用基因重组技术插入pPll3.7载体，测序正确的重组病毒质粒与包装质粒通过共转染293T细胞，培养48 h后，收集细胞培养上清液，感染A549细胞，western blot检测目的蛋白细胞因子信号转导抑制因子1在靶细胞中的表达。 结果与结论：实验通过对重组载体进行测序分析证实短发夹RNA插入慢病毒载体，慢病毒载体上清成功转染A549细胞后western blot检测结果显示该载体抑制了细胞因子信号转导抑制因子1蛋白在A549细胞有表达。说明实验成功构建了细胞因子信号转导抑制因子1 RNA干扰慢病毒载体。 关键词：细胞因子信号转导抑制因子1；慢病毒载体；RNA干扰；A549细胞；组织构建 doi:10.3969/j.issn.1673-8225.2012.07.030

Construction and identification of a lentiviral vector for RNA interference of suppressors of cytokine signaling 1

BACKGROUND: The previous studies mainly used liposome transfection or other vectors for research of suppressors of cytokine signaling 1 (SOCS1). However, those transfections have the disadvantages of low efficiency and less security. OBJECTIVE: To construct a lentiviral vector for RNA interference of SOCS1 gene and identify it.  METHODS: Three pairs of complementary short hairpin RNA (shRNA) oligonucleotides targeting the SOCS1 gene were designed, and then were inserted into pPll3.7 vectors by gene recombination technology. The recombinant plasmids with correct sequence were cotransfected with packaging plasmids into 293T cells. The cell culture supernatant was obtained after 48 hours in order to transfect A549 cells. The expression of SOCS1 in target cells was detected by western-blot. RESULTS AND CONCLUSION: The shRNA had been confirmed to insert into pPll3.7 vectors by sequencing of the recombinant plasmids. The western-blot detection showed that the expression of SOCS1 in A549 cells was inhibited after the cells were successfully transfected by the lentiviral vector supernatant. A lentiviral vector for RNA interference of SOCS1 gene was successfully constructed in this study.