靶向PD-1的shRNA慢病毒载体的构建  

Construction of ShRNA Lent iviral Vector Targeting PD-1

作　　者：贾旭 金威洋 贾裕 王华 杨光华 Jia Xu;Jin Weiyang;Jia Yu;Wang Hua;Yang Guanghua(Shanghai Ocean University,Shanghai 200000;Zhejiang Huayi Health Industry Development Co.,Ltd.,Hangzhou 315000;Shanghai Biomedical Technology Co.,Ltd.,Shanghai 200000,China)

机构地区：[1]上海海洋大学,上海200000 [2]浙江华医健康产业发展有限公司,浙江杭州315000 [3]上海比昂生物医药科技有限公司,上海200000

出　　处：《广东化工》2023年第2期50-55,共6页Guangdong Chemical Industry

摘　　要：目的:设计以PD-1基因为靶点的短发夹状RNA(shRNA),构建重组慢病毒载体,鉴定此RNA干扰系列对PD-1基因表达的影响,从设计序列中筛选高(>90%)干扰效率的靶点序列(PD694/791)。方法:设计合成3种PD-1的shRNA干扰序列,与LV3载体连接,进行阳性鉴定,通过免疫印迹法(western)和阳性细胞数量检测筛选高干扰效率靶序列。结果:基于转染目的细胞的PD-1蛋白western检测结果,计算三种shRNA慢病毒载体的干扰效率分别为:PD791干扰效率为78%、PD 238干扰效率为8%、PD 694干扰效率88%。结论:成功构建靶向PD-1的shRNA慢病毒载体,其中干涉片段PD694和PD791对PD-1的干涉抑制效果显著,可以有效抑制原代T细胞PD-1表达,为研究开发PD-1抑制剂治疗肿瘤奠定基础。Objective:A short hairpin RNA(shRNA)targeting PD-1 gene was designed to construct a recombinant lentiviral vector.The effect of this RNA interference series on PD-1 gene expression was identified and the target sequence(PD694/791)with high interference efficiency(>90%)was screened from the designed sequence.Methods:rence efficiency was screened by Western identification and positive cell number detection.Three shRNA interference sequences of PD-1 were designed and synthesized,linked to LV3 vector for positive identification,and high interference efficiency target sequences were screened by western blot and positive cell number detection.Results:Based on the western detection results of PD-1 protein in transfected cells,the interference efficiency of the three shRNA lentiviral vectors was calculated as 78%for PD791,8%for PD 238 and 88%for PD 694,respectively.Conclusion:ShRNA lentiviral vectors targeting PD-1 were successfully constructed.Among them,interference fragments PD694 and PD791 had significant interference inhibition effect on PD-1,which could effectively inhibit PD-1 expression of primary T cells,laying a foundation for the research and development of PD-1 inhibitors for tumor treatment.

关 键 词：慢病毒载体 PD-1 肿瘤治疗 

分 类 号：TQ[化学工程]