HIF1α基因RNAi慢病毒载体构建与鉴定.docVIP

HIF1α基因RNAi慢病毒载体构建与鉴定 　　【摘要】 目的 构建HIF-1α基因RNA干扰慢病毒载体。方法 选定HIF-1α基因RNAi靶序列,合成靶序列的Oligo DNA,退火形成双链DNA,与经XhoⅠ和HpaⅠ双酶切后的慢病毒载体pLentilox3.7(pLL3.7)连接,产生pLL3.7 HIF-1α慢病毒载体,转化大肠杆菌DH5α菌株,抽取质粒后,用限制性内切酶XbaⅠ和NotⅠ进行酶切鉴定,将阳性克隆送金斯特公司进行测序鉴定;用PHR、pVSVG和pLL3.7慢病毒载体共转染包装细胞293T细胞(磷酸钙转染法),包装产生慢病毒, 将收集的病毒上清经高速离心浓缩后,按一定比例稀释,然后感染293T细胞,用流式细胞仪检测293T细胞GFP蛋白的表达水平,从而测定病毒滴度。结果 酶切和测序证实,构建出HIF-1αshRNA的慢病毒载体pLL3.7 HIF-1α;包装慢病毒,测得离心的浓缩病毒悬液的滴度为2×10?8 TU/ml。结论 成功构建HIF-1α基因RNAi慢病毒载体。 　　【关键词】 HIF-1α,RNAi,慢病毒 　　 　　Construction and Identification of HIF-1αRNAi Lentiviral Vector 　　 　　SHEN Shang-hang, WANG Zhan-xiang, CHEN Yu-ying,et al. 　　Department of Neurosurgery, First Hospital of Xiamen Affiliated to the Fujian Medical University, Xiamen 361003,China 　　 　　【Abstract】 Objective To construct a lentiviral vector of RNA interfered(RNAi)HIF-1αgene. Methods To confirm the targeting sequence of HIF-1α gene that can be effectively silenced by RNA inference. The cDNA containing both sense and antisense Oligo DNA fragments of the targeting sequence was designed, and cloned into the pLentilox3.7(pLL3.7) vector which was digested by XhoI and HpaI. The obtained lentiviral 　　 　　基金项目:厦门市卫生局资助项目(项目编号:3502; 　　厦门市科技局资助项目(项目编号:3502 　　作者单位:361003 厦门,福建医科大学附属厦门第一医院 　　 　　 　　 vector containing HIF-1α shRNA was confirmed by digestion and sequencing. Using lentiviral vector PHR、pVSVG and pLL3.7 HIF-1α to cotransfect 293T cells, then the collected lentivirus. The titer of virus was tested according to the expression level of GFP. Results Digestion and DNA sequencing demonstrated that the constructed lentivirus vector pLL3.7 HIF-1α which can produce HIF-1α shRNA. The titer of concentrated virus was 2×108 TU/ml. Conclusion The lentivirus RNAi vector targeting HIF-1α is constructed successfully. 　　【Key words】 HIF-1α;RNAi;Lentivirus 　　 　　 　　缺氧诱导因子-1α(Hypoxia-inducible factor-1α,HIF-1α)是目前所发现的正常细胞以及肿瘤细胞适应缺氧的最关键的转录因子[1],其基因的功能研究具有非常重要的意义。很多肿瘤存在HIF-lα的高表达,并与肿瘤的侵袭转移,耐药等相关[2]。已有研究表明 HIF-1α参与了 VEG