靶向肿瘤细胞PD-L1的shRNA慢病毒干扰载体的构建  

Construction of shRNA Lentiviral Interference Vectors Targeting PD-L1 of Tumor Cell

作　　者：孙杰[1,2,3] 贾裕 贾旭 陈阳 佟莹莹 金微洋 杨光华 Sun Jie;Jia Yu;Jia Xu;Chen Yang;Tong Yingying;Jin Weiyang;Yang Guanghua(International Research Center for Biological Sciences,Ministry of Science and Technology,Shanghai Ocean University,Shanghai 201306;National Aquatic Animal Pathogen Collection Center,Shanghai Ocean University,Shanghai 201306;Aquatic Animal Genetics and Breeding Center,Shanghai Ocean University,Shanghai 201306;Zhejiang Huayi Health Industry Development Co.,Ltd.,Hangzhou 311500,China)

机构地区：[1]上海海洋大学,科技部生物科学国际研究中心,上海201306 [2]上海海洋大学,国家水生动物病原体收集中心,上海201306 [3]上海海洋大学,水产动物遗传育种中心,上海201306 [4]浙江华医健康产业发展有限公司,浙江杭州311500

出　　处：《广东化工》2022年第23期36-40,共5页Guangdong Chemical Industry

基　　金：本研究由上海市科研计划项目(19411951100)资助。

摘　　要：目的:利用RNAi技术构建靶向抑制肿瘤细胞PD-L1表达的shRNA慢病毒干扰载体,用于解除肿瘤细胞的免疫逃逸机制。方法:利用Designer3.0(Genepharma)软件以PD-L1为靶点设计4种shRNA基因序列,将shRNA序列插入到慢病毒载体中,连接产物转化后在选择性培养基上筛选阳性菌落并提取重组质粒,进行双酶切及测序鉴定,荧光显微镜观察慢病毒转染效果,westernblot对目的基因PD-L1四个靶点筛选获得PD-L1蛋白表达并计算出重组慢病毒的干扰效率。结果:限制性核酸内切酶Xba I和Nhe I双切鉴定结果皆为1000 bp大小的阳性重组载体;测序结果与shRNA模版一致;荧光显微镜观察结果显示慢病毒转染效果较好;western blot结果显示SH1、SH4组PD-L1表达无抑制,SH2、SH3组PD-L1表达有抑制;SH1组干扰效率19.75%,SH2组干扰效率32.10%,SH3组干扰效率40.74%,SH4组干扰效率7.41%。讨论:SH3组重组慢病毒对PD-L1表达有抑制作用且干扰效率最高,成功构建了一款靶向肿瘤细胞PD-L1的shRNA慢病毒干扰载体,为利用RNAi技术干扰PD-L1靶点的肿瘤免疫治疗奠定了实验基础。Purpose: ShRNA lentiviral interference vectors targeting PD-L1 expression of tumor cells were constructed using RNAi technology to remove the immune escape mechanism of tumor cells. Method: Using designer 3.0(genepharma) software designed four shRNA gene sequences with PD-L1 as the target. The shRNA sequences were inserted into the lentivirus vector. After the ligation product was transformed, the positive colonies were screened on the selective medium and the recombinant plasmid was extracted for double enzyme digestion and sequencing identification. The lentivirus transfection effect was observed by fluorescence microscope. The four targets of PD-L1 gene were screened by western blot to obtain PD-L1 protein expression and calculate the interference efficiency of the recombinant lentivirus. Result: The restriction endonuclease Xba I and Nhe I double cleavage identification results were positive recombinant vectors with the size of 1000 bp;The sequencing results were consistent with the shRNA template;Fluorescence microscopy showed that lentivirus transfection was effective;Western blot showed that there was no inhibition of PD-L1 expression in sh1 and SH4 groups, but there was inhibition of PD-L1 expression in SH2 and SH3 groups;The interference efficiency of SH1 group is 19.75 %, that of SH2 group is 32.10 %, that of SH3 group is 40.74 %, and that of SH4 group is 7.41 %. Discuss: SH3group recombinant lentivirus inhibited the expression of PD-L1 and had the highest interference efficiency. A shRNA lentivirus interference vector targeting tumor cell PD-L1 was successfully constructed, which laid an experimental foundation for tumor immunotherapy using RNAi technology to interfere with PD-L1 target.

关 键 词：PD-L1基因 RNA干扰 慢病毒载体 

分 类 号：TQ[化学工程]