含报告基因的凋亡素基因真核表达载体构建研究

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。

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

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...