犬黑皮质素受体-4突变体D298N真核表达载体的构建及其在MDCK细胞中的表达

Construction of Canine Melanocortin Receptor-4 Mutant D298N Eukaryotic Expression Vector and Its Expressed in MDCK Cells

[目的]引进犬MC4R突变点,构建突变体D298N表达载体并在MDCK细胞中获得表达。[方法]设计带突变点D298N引物,以犬MC4R基因组DNA为模板,采用普通PCR和Overlap-PCR扩增编码区,将产物克隆、酶切、测序鉴定。将测序正确的基因连接到pcDNA3.1-myc-His/A载体上,将重组体pcDNA3.1-myc-His/A-cMC4R-D298N酶切、鉴定,将测序正确的重组体转染到MDCK细胞中,继续培养3 d,提取细胞总RNA,并采用RT-PCR方法检测基因表达。提取细胞总蛋白,Western Blot鉴定蛋白表达。[结果]构建带D298N突变点的真核表达载体,提取质粒后有2处碱基不同,分别是777位碱基T→C,892位碱基G→A(引入的突变位点)。转染到MDCK后,RT-PCR和Western Blot均检测到重组体在基因水平和蛋白水平的表达。[结论]成功构建犬的重组体真核表达载体并在MDCK细胞中表达。

[Objective]The canine MC4R mutation site was introduced,the mutant D298N expression vector was constructed and expressed in MDCK cells.[Method]We designed D298N primers with mutation site.Using canine MC4R genomic DNA as template,we amplified the coding region of mutation MC4R by normal PCR and overlap PCR.The product was cloned,digested by enzymes and sequenced.The correctly sequenced gene was connected to pcDNA3.1-myc-His/A vector.The recombinant pcDNA3.1-myc-His/A-D298N was digested by enzymes and identified;The correctly sequenced recombinant was transfected into MDCK cells and cultured for 3 days.The total RNA was extracted and the gene was detected by RT-PCR;The total protein was extracted and the protein was identified by Western blot.[Result]The eukaryotic expression vector with D298N mutation was constructed.We extracted the plasmid.There were two different bases in the recombinant expression plasmid,which were 777 base T→C,892 base G→A(here is the introduced mutation site).After MDCK cells transfecting,the expression of recombinant at gene level and protein level was detected by RT-PCR and Western blot.[Conclusion]The recombinant eukaryotic expression vector of canine was successfully constructed and expressed in MDCK cells.