人RASSF1A基因慢病毒表达载体的构建及鉴定  

Construction and identification of lentiviral vector for RASSF1A gene of human

作　　者：孔丽君[1] 胡金霞[1] 于媛[1] 庞敏[1] 

机构地区：[1]滨州医学院山东省高校肿瘤分子生物学重点实验室,生物化学与分子生物学教研室,烟台264003

出　　处：《滨州医学院学报》2013年第3期161-165,168,共6页Journal of Binzhou Medical University

基　　金：烟台市科学技术发展计划项目(2008162)

摘　　要：目的应用pLV.Des3d.P/neo构建人RASSF1A基因的慢病毒表达载体并对其进行鉴定。方法采用Oligo核酸软件对Genbank上所发表的RASSF1A序列进行分析,设计一对RASSF1A基因上下游引物,利用Gateway克隆方法,分别在其两端添加attB重组位点,运用PCR扩增RASSF1A和IRES/eGFP,将PCR产物进行BP反应然后转化到Stbl3感受态菌,通过卡那霉素抗性筛选、PCR鉴定,选取鉴定正确的pDown-RASSF1A和pTail-IRES/eGFP克隆测序。将pDown-RASSF1A和pTail-IRES/eGFP与pLV.EX3d.P/neo混合进行LR反应,构建慢病毒载体pLV.EX3d.P/neo-EF1A>RASSF1A>IRES/eGFP,再次进行PCR、测序鉴定。结果成功地构建了hRASSF1A基因的重组慢病毒表达载体pLV.EX3d.P/neo-EF1A>RASSF1A>IRES/eGFP。结论基因序列克隆正确,为进一步探讨RASSF1A在肿瘤基因治疗中的作用奠定了基础。Objective To construct the lentiviral vector encoding RASSF1A gene of human with clone vector pLV. Des3d. P/neo and identify its titer. Methods Oligo nucleic acid software was used to design a couple of primers according to RASSF1A gene se- quence of human which is publicated on Genbank. The attB recombination site was added to the two ends of the couple of primers, and RASSF1A and IRES/eGFP were amplified by PCR. The PCR product after BP reaction was transferred to Stb13, and the correct pDown-RASSF1A and pTail- IRES/eGFP were selected and sequenced. The mixture of pDown - RASSF1 A, pTail- IRES/eGFP and pLV. EX3d. P/neo was used to do LR reaction. The lentiviral vector pLV. EX3d. P/neo-EFIA 〉 RASSF1A 〉 IRES/eGFP was amplified and equeneed again. Results The lentiviral vector pLV. EX3d. P/neo-EF1A 〉 RASSF1A 〉 IRES/eGFP was constructed successfully. Conclusion The results of PCR and sequencing showed that the lentiviral vector encoding RASSF1A gene of human was constructed successfully, and it establishs the basis on further functional assay in cancer gene therapy.

关 键 词：RASSF1A基因 人 目的载体pLV Des3d P NEO 慢病毒 

分 类 号：R34[医药卫生—基础医学]