增强型绿色荧光蛋白真核表达载体的构建和表达

CONSTRUCTION AND EXPRESSION OF EUKARYOTIC EXPRESSION VECTOR pcDNA3.1(+)-EGFP

目的 构建增强型绿色荧光蛋白(Enhanced green fluorescent protein, EGFP)编码基因的真核表达载体 pcDNA3.1(+) GFP,并观察其在Hep 2细胞中的表达情况。 方法 据已知的EGFP基因序列,设计合成 1 对引物,并引入Hind Ⅲ和EcoR Ⅴ酶切位点。应用PCR技术,从含有 EGFP的 pAdTrack CMV中扩增 EGFP编码基因。通过 TA连接将其克隆入pGEM T easy载体,经PCR及限制性内切酶鉴定后,插入真核表达质粒pcDNA3.1(+)中,转化Esche richia coli DH5α感受态细胞,于Amp+ LB平板上筛选阳性克隆。重组子经 Hind Ⅲ和EcoRⅤ双酶切、PCR鉴定,将该载体转染人喉癌细胞 Hep 2 后 48 h观察 EGFP表达情况。 结果 成功构建了含 EGFP 编码基因的真核表达载体pcDNA3.1(+) GFP,并成功转染Hep 2细胞,在倒置荧光显微镜下呈现绿色光。 结论 获得可产生绿色荧光的 EG FP,能方便地用作报告基因和筛选标记。

Objective To construct the eukaryotic expression vector pcDNA3.1(+) EGFP and to detect transient expression of enhanced green fluorescent protein(EGFP) in laryngocarcinoma cells Hep 2. Methods According to the open reading frame of known EGFP gene, a pair of primers were designed and synthesized. A specific fragment of EGFP gene was obtained by PCR amplification from pAdTrack CMV containing EGFP gene.The PCR products were subcloned into pGEM T easy vector. Following digestion by HindⅢ/EcoRⅤ, the fragments of recombinants were cloned into the expression vector pcDNA3.1(+), then transformed into Escherichia coli DH5α, a recombinant pcDNA3.1(+) EGFP was constructed and confirmed by PCR and HindⅢ/EcoRⅤdigestion. It's transient expression was observed in 48 hours after it was transfected into laryngocarcinoma cells Hep 2. Results The eukaryotic expression vector pcDNA3.1(+) GFP was successfully constructed and GFP showing green fluorescence could be observed in laryngocarcinoma cells Hep 2 under fluorescent microscopy. Conclusion GFP was successfully expressed in laryngocarcinoma cells Hep 2. It was a good reporter and selection marker molecule in mammalian cells.