摘要
目的：构建介导小鼠B7-1基因RNAi的慢病毒载体,研究其对L929细胞表面B7-1分子表达的沉默效应。方法：从小鼠B7-1基因编码区选择3段RNA干扰靶序列,制备转录双发夹RNA的前体DNA,并克隆至慢病毒穿梭质粒,构建B7-1的RNAi慢病毒穿梭载体,测序鉴定。将重组质粒及辅助质粒共转染293T细胞产生慢病毒,经超速离心获得浓缩慢病毒颗粒。通过测定293T细胞GFP表达水平确定病毒滴度,流式细胞术检测感染效率以及对细胞表面B7-1的干扰效率。病毒感染细胞经嘌呤霉素筛选及克隆培养获得小鼠B7-1基因RNAi慢病毒稳定感染的L929细胞,流式细胞术分析细胞中GFP及B7-1分子表达的状况;将感染细胞与分离的小鼠脾脏T细胞混合培养,分析B7-1稳定沉默细胞刺激T细胞增殖的能力。结果：成功了构建小鼠B7-1基因RNA干扰的重组慢病毒载体;获得了滴度达（3~5）×10~8TU/ml的浓缩重组慢病毒,重组慢病毒可有效感染L929细胞介导GFP表达以及沉默其表面的B7-1。经筛选获得慢病毒稳定感染的L929细胞,该细胞表面B7-1分子表达受到抑制,刺激T细胞增殖的能力显著下降（P〈0.05）。结论：构建了能够高效感染及沉默小鼠B7-1分子的RNAi慢病毒载体,该载体可稳定沉默L929细胞表面B7-1分子表达,抑制B7-1/CD28信号诱导的T细胞增殖效应。
Objective：To construct lentiviral vector specific for mouse B7-1 RNA interference and study lentivirus-mediated B7-1 gene silencing effects in L929 fibroblast cells.Methods：Three candidate sequences for B7-1 RNAi selected from coding sequence of mouse B7-1 transcription were used to design short hairpin RNA（shRNA） templates and then cloned into lentiviral expression plasmid followed with correctness identification of inserted sequence by DNA sequencing.Recombinant lentivirus were prepared by co-transfecting lentiviral expression vector and packaging plasmids into 293 T cells.Then the resulting culture supernatant containing infectious lentiviral particles was pooled and centrifuged via ultra-centrifugation.Infectious titer of the preparations was determined by detecting the expression of GFP in 293 T cells after transfected by lentivirus.Cultured L929 cells were transfected with lentivirus to deter-mine transduction efficiency and silencing efficacy of B7-1 expression by flow cytometry.Transducted L929 cells were then screened using puromycin to generate stable cell clones followed by flow cytometry analysis of GFP and B7-1 expression.A mixed reaction system consisting of stable B7-1 silencing L929 cells and mouse splenic T cells was used to analyze ability of the established cell line to trigger T cells proliferation.Results：Lentiviral expression vector for mouse B7-1 RNAi was correctly constructed with inserted sequences as designed.Recombinant RNAi lentivirus were prepared with titers ranging（3-5） × 108 TU/ml and efficacy to mediate GFP transgene expression and B7-1 silencing.B7-1 expression and the ability to trigger T cells proliferation of stable L929 cells were suppressed significantly（P 0.05）.Conclusion：We generated lentiviral vector specific for mouse B7-1 RNAi with high performance of transduction efficiency as well as B7-1 silencing efficacy and the recombinant RNAi lentivirus can mediated stable B7-1 gene silencing in L929 cells and inhibition of T cells proliferation induced by B7-1/CD28 co-stimulatory signal.
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
2016年第9期1327-1332,共6页
Chinese Journal of Immunology
基金
国家自然科学基金面上项目(81373236)资助
