小鼠生长激素促分泌素受体真核表达载体的构建及表达鉴定

Construction and identification of eukaryotic expression vector bearing mouse ghrelin receptor

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 mouse ghrelin receptor （GHS-R） eukaryotic expression vector, and observe its expression in transient transfeted COS-7 cell line. Methods The coding DNA sequence of ghrelin receptor gene （ CDS ） was amplified by SOE- PCR technology from mouse genomic DNA and cloned into PCDNA3 vector. The positive clone was confirmed by enzyme digestion and sequencing. The expression vector was transiently transfected into COS-7 cell line. The expression of GHS-R protein was identified by Western blot. Results The CDS of mouse GHS-R was successfully cloned. The construction of pcDNA3-GHS-R was correct and its expression of GHS-R protein was confirmed by Western blot. Conclusions The eukaryotic expression vector pcDNA3-GHS-R was successfully constructed and mouse GHS-R protein was successfully expressed. This study can be a basis for further study on GHS-R gene function in vitro.