RECK基因GFP融合蛋白真核表达载体的构建及鉴定

The construction and verification of RECK gene expressive plasmid in eukaryocyte

目的使用真核表达载体pCDNA3.1,构建含有GFP蛋白标签的RECK真核表达载体,研究RECK基因在口腔鳞状细胞癌转移中的作用。方法使用人胚肝cDNA文库,通过聚合酶链反应扩增出带有XhoⅠ及BamHⅠ核酸内切酶位点的目的 RECK基因序列后连接入真核载体pCDNA3.1质粒中,在RECK基因序列上游通过聚合酶链反应扩增构建GFP蛋白标签。经过DNA琼脂糖凝胶电泳检测后,将GFP蛋白标签连接入真核表达载体pCDNA-RECK中。经过测序分析比对后,转化入DH5a感受态细胞后,提取质粒。使用Lipofectamine2000脂质体方法转染RECK基因入舌癌细胞系SCC-25细胞中。经过RNA提取、反转录后,通过RT-PCR及Western-blot技术鉴定RECK基因体外表达质粒转染效果及RECK蛋白表达情况。结果通过序列比对,证实了体外表达质粒与目标序列一致。将pCDNA-GFP-RECK质粒转染入真核细胞SCC-25细胞后,通过RT-PCR及Western-blot分析,验证RECK基因真核体外表达质粒能有效表达RECK基因及蛋白。结论通过分子生物学基因克隆的方法成功地在体外构建了RECK基因真核表达质粒,为研究RECK基因在口腔鳞状细胞癌转移中的机制奠定了实验基础。

Objective To construct the RECK gene expressive plasmid with GFP protein tag in eukaryocyte. The plasrnid will show light on the RECK functions in the head and neck carcinoma. Methods The pCD- NA3.1 plasmid was used to construct the RECK gene expression plasrnid. The RECK gene was cloned from human liver cDNA library. The sequence of the gene was constructed with XhoI and BamH I sites, meanwhile with the GFP marker. After sequencing, the RECK plasrnid was transfect into SCC-25 eukaryo- cyte. The RT-PCR and Western-blot methods were carried out to test the expression of the RECK gene in the SCC-25 cells. Results By sequencing the plasmid of the RECK the gene was ligated into the pCDNA 3.1 plasmid successfully. After analyzing the expression of the RECK gene in SCC-25 cells, we got the plasmid which can effectively express the RECK gene and protein. Conclusion We constructed the RECK gene expressive plasmid in vitro. This will provide the gene function and gene therapy in head and neck carcinoma. Through biology method. good fundamental for study the gene function and gene therapy in head and neck carcinoma.