在哺乳动物细胞中表达hIGF-1

Expression Human Insulin-like Growth Factor-1( IGF-1) Gene in Mammalian Cells

目的:构建hIGF-1真核表达载体,通过293F细胞表达hIGF-1蛋白。方法:合成已知hIGF-1碱基序列,构建pMD18T-hIGF-1基因工程载体,PCR扩增得到hIGF-1基因序列,构建真核表达载体pcDNA3.1-hIGF-1。转化大肠杆菌DH5α菌株,经菌液PCR法、双酶切法鉴定阳性克隆,并送样测序确保阳性克隆的hIGF-1碱基序列正确。通过脂质体法将基因工程载体pcDNA3.1-hIGF-1转染293F细胞,Western-blot检测hIGF-1蛋白的表达。然后再对hIGF-1的核酸序列和蛋白功能结构进行生物信息学分析。结果:琼脂糖凝胶电泳显示在250bp处出现亮色条带,PCR成功扩增出目的条带,双酶切鉴定显示在5 400bp附近获得亮色条带,说明得到pcDNA3.1-hIGF-1基因工程载体,Western-blot鉴定结果表明成功表达hIGF-1蛋白。结论:成功构建真核表达载体pcDNA3.1-hIGF-1,并表达hIGF-1蛋白。

Objective：To construct the hIGF - 1 eukaryotic expression vector and express hIGF - 1 protein in the 293F cells. Method：The hIGF - 1 gene sequence has been synthesized and constructed as pMD18T - hIGF - 1 genetic engineering vector which was used as tern-plate for PCR to amplify the hIGF - 1 gene sequence, and the amplified hIGF - 1 gene sequence was used to construct the eukaryotic ex- pression vector pcDNA3. 1 - hIGF - 1. Transformed the constructed eukaryotic expression vector pcDNA3.1 - hIGF - 1 into E. coli DI-ISot strain, then identified the recombinant plasmid through PCR and double digestion. The recombinant plasmid was sent for sequencing to en- sure the correctness of the hIGF - 1 gene sequence. The recombinant plasmid pcDNA3.1 - hIGF - 1 was transfected into the 293F cells through lipofection method, and the expression of hIGF - 1 in the 293F ceils was verified through Western - blot. Using bioinformatics anal- ysis software to analyzed the nucleic acid sequence and properties of hIGF - 1. Result：The electrophoresis result showed a bright band of about 250bp, the objective band was successfully amplified by PCR, and the restriction digestion analysis showed a bright band of 5 400bp, it illustrated we gained the genetic engineering vector pcDNA3.1 - hIGF - 1. The Western - blot result showed the hlGF - 1 protein was successfully expressed in the 293F ceUs. Conclusion：The eukaryotic expression vector pcDNA3.1 -hlGF- 1 was constructed correctly, and the hIGF - 1 protein has been successfully expressed in the 293F cells.