【摘要】 目的 利用基因工程原理构建人胰岛素样生长因子Ⅰ(IGF-Ⅰ)全长基因的真核表达载体pIRES2-EGFP-IGF-Ⅰ。方法 应用PCR方法从人肝细胞cDNA文库中提取人IGF-Ⅰ全长cDNA序列,克隆人T载体pMD18中,经测序证实序列正确后,进行双酶切并定向插入真核表达载体pIRES2-EGFP中,利用双酶切、电泳和PCR证实插入片段的正确性。结果经测序证实,目的基因人IGF-Ⅰ的全长基因序列被正确扩增;构建的真核表达载体经双酶切和PCR鉴定证实,hIGF-Ⅰ被定向插入到真核表达载体pIRES2-EGFP的多克隆位点中。结论 利用基因工程原理可以正确地构建人IGF-Ⅰ全长基因的真核表达载体pIRES2-EGFP-IGF-Ⅰ。更多还原

【Abstract】 Objective To construct the eukaryotic expression recombinant plasmid pIRES2- EGFP-IGF-Ⅰ by genetic engineering. Methods The full length cDNA sequence of human insulin-like growth factor Ⅰ(IGF-Ⅰ) was isolated from the human hepatocyte cDNA library by polymerase chain reaction and cloned into the T vector pMD18. After being verified by DNA sequencing, the target gene was cut off by EcoR Ⅰ and Xho Ⅰ and then inserted into the eukaryotic expression vector pIRES2-EGFP digested by the same two enzymes. The recombinant plasmid was verified by double digestion with EcoR Ⅰ and Xho Ⅰ, and PCR analysis. Results The full length cDNA sequence of human IGF-I was isolated from the human hepatocyte cDNA library and cloned into the multiple cloning site of the vector pIRES2-EGFP successfully. Conclusion The eukaryotic expression recombinant plasmid pIRES2-EGFP-IGF-Ⅰ, which contains the full length sequence of the human IGF-Ⅰ gene, could be constructed successfully.更多还原