PRL-3基因RNA干扰慢病毒载体的构建及在SW480细胞中的表达  

Construction of a lentiviral vector for RNA interference of PRL-3 gene and its stable expression in SW480 cells

作　　者：柳玉红[1] 李建明[1] 周军[1] 丁彦青[1] 

机构地区：[1]南方医科大学南方医院病理科,南方医科大学基础医学院病理学教研室,广东省分子肿瘤病理重点实验室,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,广东广州510515

出　　处：《南方医科大学学报》2008年第4期509-512,共4页Journal of Southern Medical University

基　　金：国家自然科学基金资助项目(30500241、30670968和30700286)~~

摘　　要：目的构建PRL-3基因慢病毒干扰载体及建立PRL-3基因稳定干扰的结直肠癌细胞亚系,为PRL-3基因功能研究奠定基础。方法设计PRL-3基因特异性RNAi靶序列,与pENTRTM/U6线性载体定向连接,构建pENTRTM/U6真核入门表达载体。通过与pENTRTM/U6 entry clone与pLenti6/BLOCK-iTTM-DEST载体进行重组,获得pLenti6/BLOCK-iTTM-DEST真核表达慢病毒干扰载体。利用包装细胞293FT获得重组的慢病毒,感染大肠癌细胞株SW480细胞,杀稻瘟菌素筛选获得稳定干扰PRL-3基因的细胞亚系。结果成功构建PRL-3pLenti6/BLOCK-iTTM-DEST真核表达慢病毒干扰载体并获得相应的慢病毒,病毒悬液的滴度为6×105U/L。RT-PCR、Western blotting结果表明,克隆1PRL-3 mRNA及蛋白显著降低。结论成功构建人PRL-3基因特异性的慢病毒干扰载体,获得PRL-3基因稳定干扰的大肠癌细胞亚系。Objective To construct a lentiviral vector for RNA interference （RNAi） of PRL-3 gene and establish a human colon carcinoma cell line with PRL-3 gene knock-down. Methods The plasmids were constructed expressing two different short hairpin RNAs. （shRNA） targeting PRL-3 gene under control by the U6 promoter by lentiviral vector. An entry clone was generated in the pENTR^TM/U6 vector. After identification with sequencing, a recombinant lentiviral vector was obtained using the pENTR^TM/U6 construct and pLenti6/BLOCK-iT^TM-DEST vector. The recombinant lentivirus was harvested from 293FT cells co-transfected with optimized ViraPowerTM Packaging Mix and the pLenti6/BLOCK-iT^TM-DEST expression construct. SW480 cells were infected with the recombinant lentivirus and the cells with stable PRL-3 gene knock-down were screened by blasticidin selection. PRL-3 expression was detected using real-time reverse transcription-polymerase chain reaction and Western blotting. Results A recombinant lentiviral vector expressing shRNAs targeting PRL-3 gene was successfully established and confirmed by DNA sequencing. The recombinant lentivirus were harvested from 293FT cells with a viral titer of 6×10^5 pfu/L. Twelve clones of SW480 cells infected with the recombinant lentivirus were selected, and the Clone 1 exhibited significant knock-down of PRL-3 protein expression （72.9%）. Conclusion The successful establishment of human colon carcinoma cell line with PRL-3 gene knock-down provide a basis for investigation of the role of PRL-3 gene in the metastasis of human colorectal carcinoma.

关 键 词：RNA干扰 慢病毒 PRL-3 结直肠癌 

分 类 号：R346[医药卫生—基础医学] R735.34