犬黑皮质素受体4真核表达载体的构建及表达

Construction and Expression of Enkaryotic Expression Vector of Canine Melanocortin Receptor-4

[目的]构建带myc和His标签的犬黑皮质素受体4真核表达载体并在MDCK细胞中进行表达。[方法]以犬MC4R基因组DNA为模板PCR扩增目的基因编码区,将扩增产物进行T-A克隆;酶切、测序鉴定成功后将目的基因亚克隆到真核表达载体pcDNA3.1-myc-His/A。重组体pcDNA3.1-myc-His/A-cMC4R经酶切和测序鉴定;采用FuGENE HD介导转染技术将重组体导入MDCK细胞;转染后继续培养72 h,提取细胞内总RNA,RT-PCR鉴定目的基因的表达;提取细胞总蛋白,Western Blot检测蛋白表达。[结果]构建带标签的pcDNA3.1-myc-His/A-cMC4R重组真核表达载体,测序结果与GenBank公布的序列相似性为99%。RT-PCR和Western Blot检测到目的基因表达。[结论]成功构建犬真核表达载体pcDNA3.1-myc-His/A-MC4R,重组体能在MDCK细胞中表达。

[Objective] The research aimed to construct the eukaryotic expression vector of canine melanocortin receptor-4 with myc and His tags and express in MDCK cells.[Method] With the genomic DNA of canine MC4R as template,the coding region of target gene was amplified by using PCR and T-A clone was made on the amplification products.After successful identification by enzyme digestion and sequencing,the target gene was subcloned into the eukaryotic expression vector pcDNA3.1-myc-His/A vector.The recombinant pcDNA3.1-myc-His/A-Canis MC4R was identified by enzyme digestion and sequencing.The recombinant was introduced into MDCK cells by using FuGENE HD transfecting technique.After 72 h culture,total RNA was extracted from cells and the expression of target gene was identified by using RT-PCR.Total cellular protein was extracted and the protein expression was detected by Western Blot.[Result] The recombinant eukaryotic expression vector pcDNA3.1-myc-His/A-Canis MC4R with tags was constructed.The similarity between the sequenced results and the reported sequence on GenBank website was 99%.The expression of target gene was detected by RT-PCR and Western Blot.[Conclusion] The eukaryotic expression vector pcDNA3.1-myc-His/A-Canis MC4R was successfully constructed and the recombinant could express in MDCK cell.