凋亡素基因序列重组及其真核表达载体的构建  

Study on recombination of sequence of apoptin gene and construction of a eukaryon vector for expression of apoptin

作　　者：朱惠明[1] 杨俊文[1] 王娜[1] 蔡筱彦[1] 邓传珍[1] 

机构地区：[1]暨南大学医学院附属深圳市人民医院消化内科深圳市人民医院临床医学研究中心,518020

出　　处：《现代消化及介入诊疗》2004年第3期143-145,183,共4页Modern Interventional Diagnosis and Treatment in Gastroenterology

摘　　要：目的构建携带Kozak序列的重组凋亡素基因VP3表达载体,为提高凋亡素基因在肿瘤细胞中的表达效率,增强其诱导肿瘤细胞凋亡的生物学活性奠定实验基础。方法通过两次PCR,以pcDNA-VP3质粒为模板,扩增出带Kozak序列(真核核糖体结合位点)的凋亡素基因VP3。将其克隆到真核表达载体pRevTRE的多克隆位点,构建重组凋亡素的真核表达载体pRevTRE-VP3,将该重组表达载体分别以限制性内切酶BamHI和XhoI进行酶切鉴定,进一步将初步鉴定显示插入目的基因的质粒进行测序鉴定。结果测序结果表明,本研究合成的凋亡素基因与Noteborn报告的凋亡素基因序列一致,同源性为100%。在其起始密码子前添加了Kozak序列的凋亡素基因已成功克隆到真核表达载体pRevTRE。结论采用两次PCR法可将提高表达效率的真核核糖体结合位点添加到凋亡素基因序列起始密码子前端,并构建成凋亡素真核表达载体。Objective To increase efficiency in expression of apoptin gene (VP3) in tumor cells and promote its bioactivity to induce apoptosis of tumor cells. Methods The plasmid pcDNA-VP3 was used as a template and the apoptin gene with Kozak sequence, i.e.eukaryn ribosome locus was amplified through twice PCR.The recombined apoptin gene was subcloned into the multiple clone, site of plasmid pRevTRE to abtain a plasmid pRevTRE -VP3. The plasmid pRevTRE -VP3 was cut and identified by means of restriction endonuclease BamHI and XhoI.The sequence of selected plasmid pRevTRE-VP3 was analysed. Results The sequence of recombined apoptin gene was identical with that reported by Noteborn and his colleagues. The apoptin gene in which Kozak sequence was added to the front of ATG code was subcloned into the eukaryn vector pRevTRE. Conclussion The eukaryon ribosome locus which promotes expression efficiency may be added to the front of ATG code of sequence of apoptin gene by twice PCR, and the eukaryon vector for expression of apoptin is constructed.

关 键 词：凋亡素 真核表达载体 基因序列 核糖体 质粒 肿瘤细胞凋亡 测序 携带 结合位点 高表达 

分 类 号：R730[医药卫生—肿瘤] R394[医药卫生—临床医学]