鸡、鸭、鹅MC5R真核表达载体及突变型的构建及信号通路初探

Construction and Signaling of the Eukaryotic Expression Vectors and Mutants of Gallus gallus, Anas platyrhynchos and Anser cygnoides domesticus Melanocortin

试验旨在构建鸡、鸭、鹅黑素皮质素受体5(Melanocortin Receptor-5,MC5R)及其突变体的真核表达载体,并比较野生型与突变体MC5R在人源胚胎肾细胞(HEK293T)中的表达及功能的差异。从三种家禽血液中分别提取全基因组DNA,以其为模板,用聚合酶链式反应(PCR)扩增目的基因编码区。用同源重组方法将扩增的目的基因片段定向插入表达载体pcDNA3.1(+)中。单点突变构建pcDNA3.1(+)-MC5Rs突变型。用脂质体法将三种禽类野生型和突变型重组质粒pcDNA3.1(+)-MC5Rs分别瞬时转染入HEK293T细胞中,用双萤光素酶报告基因法检测胞内第二信使cAMP水平的变化来反应受体的表达和激活情况。结果表明:成功构建了鸡、鸭、鹅MC5R真核表达载体与各3个突变型(D119A、F254A和H257A)。在HEK293T细胞中表达时,与野生型相比,F254A的基础活性大幅升高,D119A与H257A的基础活性变化不大;D119A与H257A对激动剂α-MSH的反应活性完全丧失,F254A对于激动剂α-MSH的反应活性降低,为进一步研究体外鸡、鸭、鹅MC5R功能奠定基础。

This study was to construct eukaryotic expression vector plasmids of Gallus gallus,Anas platyrhynchos and Anser cygnoides domesticus MC5Rs,then test the differences in expression and function of wild-type and mutant MC5Rs in HEK293 T cell.Whole genome DNA was extracted from the fresh blood of Gallus gallus,Anas platyrhynchos and Anser cygnoides domesticus.The coding region of target gene was amplified by polymerase chain reaction(PCR),using genomic DNA as template.The amplified target gene fragment was inserted into the expression vector pcDNA3.1(+)by homologous recombination kit.Single point mutations were used to construct pcDNA3.1(+)-MC5Rs mutants.Three poultrys wild-types and mutants recombinant plasmid pcDNA3.1(+)-MC5Rs were transfected into HEK293T cell by liposomal method,and the production of the in-cell second messenger cAMP was detected by luciferase repoter gene method to reflect the expression and activation of the receptor.The results showed that MC5R eukaryotic expression vectors of Gallus gallus,Anas platyrhynchos and Anser cygnoides domesticus and their mutants(D119A,F254 A,H257A)were successfully constructed in this study.When expressed in HEK293T cell,the basic activity of D119 A and H257 A did not change significantly,while that of F254A significantly increased.D119A and H257A completely lost their sensitivity to agonistα-MSH and the basic activity of F254A was decreased to agonistα-MSH.These results laid foundation for further study on Gallus gallus,Anas platyrhynchos and Anser cygnoides domesticus MC5R gene function in vitro.