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目的:构建人snail基因真核表达载体并鉴定。方法:使用RT-PCR法获取人snail基因全长c DNA,经Bam H I、Eco R I双酶切、连接,插入pc DNA3.1(+)真核表达载体,转化TOP10感受态细胞,用含氨苄青霉素的LB培养基筛选阳性克隆,提取质粒双酶切电泳及测序鉴定,瞬时转染siha细胞Western-blot从蛋白水平鉴定重组质粒在真核细胞内的表达。结果:pc DNA3.1-snail重组质粒经酶切电泳符合预期片段,测序鉴定插入片段与NCBI Gen Bank文库中人snail序列一致,重组质粒瞬时转染后snail蛋白表达量明显增高。结论:成功构建pc DNA3.1-snail重组质粒载体,为进一步探讨snail基因生物学功能奠定了基础。
Objective: To construct and identify human snail gene eukaryotic expression vector. Methods: The full length c DNA of human snail gene was obtained by RT-PCR. The recombinant plasmid was digested with Bam HI and Eco RI, ligated into pcDNA3.1 (+) eukaryotic expression vector, transformed into TOP10 competent cells, The positive clones were screened by LB medium containing penicillin. The plasmid was digested by restriction enzymes and identified by sequencing. Transient transfection of siha cells by Western-blot was used to identify the expression of recombinant plasmid in eukaryotic cells from the protein level. Results: The pcDNA3.1-snail recombinant plasmid was confirmed by restriction endonuclease digestion and sequencing. The inserted fragment was identified with the human snail sequence in the NCBI Gen Bank library. The expression of snail protein was significantly increased after transient transfection. Conclusion: The successful construction of pcDNA3.1-snail recombinant plasmid vector laid the foundation for further exploring the biological function of snail gene.