含报告基因的凋亡素基因真核表达载体构建研究  

Study on construction of a eukaryon vector for expression of apoptin gene having reporting gene

作　　者：王福庆[1] 王庆宝[2] 韩国新[2] 

机构地区：[1]滨州市人民医院,山东滨州256600 [2]泰山医学院,山东泰安271016

出　　处：《泰山医学院学报》2010年第8期567-570,共4页Journal of Taishan Medical College

摘　　要：目的构建含报告基因的凋亡素基因真核表达载体。方法对pMD18T-VP3质粒进行EcorI和BamHI双酶切,回收366 bp(含凋亡素基因完整序列)片段。对增强型绿荧光蛋白pEGFP-C2质粒酶切,回收载体大片段。将凋亡素基因通过连接反应,连接到增强型绿荧光蛋白(EGFP)基因C末端的终止密码前,并使两者读框不移位,构建成pEGFP-VP3质粒,并对其进行酶切分析和测序鉴定载体结构。结果通过对构建的pEGFP-VP3质粒酶切、电泳,出现了366 bp的电泳条带,与凋亡素基因片段一致。凋亡素基因真核表达载体测序鉴定结果表明,本研究合成的凋亡素基因与Genbank中报告的凋亡素基因序列一致,同源性为100%。结论将凋亡素基因连接于增强型绿荧光蛋白的C末端,成功构建了凋亡素—增强型绿荧光蛋白表达载体pEGFP-VP3。Objective： To construct a eukaryn vector for apoptin gene（VP3） having reporting gene.Methods： EcorI and BamHI were used to cut pMD18T-VP3 plasmid,and the segment having 366bp（have apoptin gene intact sequence） was reclaimed.EcorI and BamHI were used to cut pEGFP-C2 plasmid,and the big segment was reclaimed as a vector.An apoptin gene was linked to the C terminal of enhanced green fluorescent protein gene to generate pEGFP-VP3 without reading frame shift.The structure of vector was identified by endonuclease analyzing and base sequencing.Results： Through endonuclease and electrophoresis in the constructive pEGFP-VP3,the segment having 366bp was obtaiined,which was consistent with the gene segment of apoptin.The base sequencing result of eukaryn vector for apoptin gene indicated that the constructive gene in the experiment was consistent with the gene in the Genbank,and was 100% homologous.Conclusion： The apoptin gene is linked to EGFP gene and pEGFP-VP3 is generated.

关 键 词：凋亡素基因 增强型绿色荧光蛋白 真核表达载体 构建 

分 类 号：Q78[生物学—分子生物学]