人CDH22基因RNAi慢病毒载体的构建及稳定干扰CDH22基因的表达  

Construction of a lentiviral vector for RNA interference of human CDH22 gene and its gene silencing effect in SW480 cells

作　　者：周军[1] 李建明[1] 杨发达[1] 柳玉红[1] 丁彦青[1] 

机构地区：[1]南方医科大学南方医院病理科,南方医科大学病理学教研室,教育部广东省共建人类重大疾病转录组学和蛋白组学重点实验室,广东省分子肿瘤病理重点实验室,广东广州510515

出　　处：《南方医科大学学报》2008年第4期589-592,共4页Journal of Southern Medical University

基　　金：国家自然科学基金(30500241,30670968,30700286)~~

摘　　要：目的构建人CDH22基因RNA干扰(RNAi)慢病毒载体,有效沉默大肠癌细胞的CDH22基因表达,为研究CDH22在大肠癌转移中参与的信号转导机制提供研究基础。方法利用在线软件设计人CDH22基因shRNA序列,合成、退火形成双链寡核酸后克隆到pENTRTM/U6载体的黏性末端,测序。得到的阳性重组子再与慢病毒载体进行重组,获得真核表达慢病毒干扰载体。在脂质体的介导下将慢病毒包装辅助质粒和CDH22基因重组慢病毒载体导入293FT细胞包装病毒,测定病毒滴度,感染大肠癌细胞SW480,杀稻瘟菌素筛选获得稳定干扰CDH22基因的细胞亚系。结果成功构建CDH22真核表达慢病毒干扰载体并获得相应的慢病毒,病毒滴度为8×105U/ml。荧光定量PCR结果表明,所获得的细胞克隆11中CDH22 mRNA水平的表达显著降低。结论成功构建出CDH22基因慢病毒RNAi表达载体,并获得CDH22基因稳定干扰的大肠癌细胞亚系,为研究CDH22在大肠癌转移中的作用机制提供了研究基础。Objective To construct a lentiviral expression vector for RNA interference of human CDH22 gene, and assess its gene silencing effect in colorectal cancer cells to provide a basis for investigating the role of CDH22 gene in the signaling pathway involved in human colorectal carcinoma metastasis. Methods Human CDH22 gene short hairpin RNA （shRNA） sequence was designed using a sottware available on-line. After synthesis and annealing, the double-stranded oligonucleotides （dsOligoe） were cloned into the pENTR^TM/U6 plasmid followed by sequence analysis. A positive clone was subcloned into pLentit/BLOCK-iT^TM-DEST vector and transformed into sthl3 competent cells, with also verification by sequencing. The recombinant lentivirus was harvested from 293FT cells contransfected with the positive recombined plasmid and lentiviral packing materials. SW480 cells were infected with the recombinant lentivirus and the cells with stable CDH22 knock-down were screened by blasticidin selection. CDH22 expression in the cells was determined by real-time reverse transcription-polymerase chain reaction. Results A recombinant lentiviral vector expressing shRNAs against CDH22 gene was obtained and confirmed by DNA sequencing. Fifteen clones of SW480 cells infected with the recombinant lentivirus were selected, and clone 11 exhibited substantial knock-down ofCDH22 mRNA expression. Conclusion The lentiviral shRNA expression vector targeting human CDH22 gene capable of stable CDH22 gene knock-down in SW480 cells has been successfully constructed, which provides a basis for further study of the relationship between human colorectal carcinoma and CDH22 gene.

关 键 词：CDH22 RNA干扰 慢病毒 结直肠癌 

分 类 号：R346[医药卫生—基础医学] R735.34