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目的构建小鼠生长激素促分泌素受体(GHS-R)真核表达系统,并观察其在瞬时转染的COS-7细胞系中的表达情况。方法以小鼠基因组为模板,通过拼接PCR技术(SOE-PCR)克隆出GHS-R的编码序列(CDS),插入真核表达载体pcDNA3中,构建重组真核表达质粒pcDNA3-GHS-R,酶切鉴定并测序,瞬时转染COS-7细胞,用Western Blot鉴定该质粒是否能在真核细胞中表达相应的目的蛋白。结果成功扩增了小鼠GHS-R编码序列(CDS),酶切和测序证明pcDNA3-GHS-R构建正确,Western Blot方法证实转染的该质粒能在COS-7细胞中正确表达目的蛋白。结论成功构建了小鼠GHS-R真核表达载体,并正确表达蛋白,为进一步研究GHS-R的功能奠定了基础。
Objective To construct the eukaryotic expression system of growth hormone secretagogue receptor (GHS-R) in mice and observe its expression in transiently transfected COS-7 cell line. Methods The coding sequence of GHS-R (CDS) was cloned by splicing PCR and inserted into the eukaryotic expression vector pcDNA3. The recombinant eukaryotic expression vector pcDNA3-GHS-R was constructed. After identification and sequencing, the COS-7 cells were transiently transfected and identified by Western Blot to confirm whether the plasmid expressed the corresponding target protein in eukaryotic cells. Results The mouse GHS-R coding sequence (CDS) was successfully amplified. The pcDNA3-GHS-R was confirmed by restriction enzyme digestion and sequencing. Western Blot showed that the recombinant plasmid could express the target protein correctly in COS-7 cells. Conclusion The mouse GHS-R eukaryotic expression vector was successfully constructed and the protein was correctly expressed, which laid the foundation for further study on the function of GHS-R.