猪MC4R基因突变体真核表达载体的构建及功能研究

Construction and Function Confirmation of MC4R Gene Eukaryotic Expression Vector with Different Mutations

为研究MC4R基因不同突变体的功能差异,本研究从猪肌肉组织中克隆得到MC4R基因的编码序列,将克隆片段与pcDNA3.1真核表达载体进行KpnⅠ和EcoRⅠ双酶切,连接形成重组表达载体MC4R1,将载体转染BHK细胞后Western blotting检测蛋白表达。通过点突变法得到MC4R基因在707/892位点的3个突变载体,将突变载体瞬时转染到BHK细胞,采用免疫荧光对细胞进行标记,通过激光共聚焦显微镜和流式细胞检测荧光表达,结果发现,克隆的MC4R1基因序列长度大约1 000bp,在707/892位点序列为G/G,将构建的真核表达载体MC4R1转染BHK细胞,经过Western blotting检测发现MC4R1蛋白正常表达;通过点突变法得到在707/892位点突变的突变载体MC4R2(G/A)、MC4R3(A/G)、MC4R4(A/A),经过激光共聚焦观察荧光表达后发现,突变受体在细胞膜上均有正常表达,流式检测发现4个突变受体表达的荧光平均强度并没有显著差异(P>0.05)。通过构建MC4R基因突变载体后发现,707/892位点突变对于MC4R受体在细胞膜的表达没有显著影响,其具体机制作用还需深入研究。

To investigate the function difference of the different Melanocortin-4receptor（MC4R）gene mutants,the complete coding sequence（CDS）of MC4 Rgene were cloned from pig muscle tissues.The CDS sequence was linked with pcDNA3.1vector by digested with restriction endonuclease of Kpn Ⅰand EcoR Ⅰand the recombinant eukaryotic expression vector MC4R1 was constructed.After recombinant MC4R1 plasmid transfected into BHK cells,the protein expression of MC4R1 was confirmed by Western blotting.Simultaneously,3site-directed MC4 RcDNA mutant（707/892site）vectors were generated.The protein expression of MC4 Rmutations vector was ob-served in transiently transfected BHK cell by Laser Scanning Confocal Microscopy and Flow Cytometry ananlysis.The results indicated that the CDS sequence of MC4R1 gene was about 1 000 bp and the sequence of 707/892 site was G/G.The protein expression of MC4R1 was confirmed in BHK cells using Western blotting.Three site-directed mutant eukaryotic vector MC4R2,MC4R3,MC4R4 were constructed and the sequence of 707/892 site was G/A,A/G,A/A,respectively.Confocal microscopy showed that every mutant of MC4 Rreceptor were expressed well on the cell surface by the Immunofluorescence.There were no significant difference of the fluorescence intensity between mutants of MC4Rreceptors（P0.05）by FCM.The construction and functional research of MC4 Rvector suggest that the mutants of 707/892 site has no significant impact on the expression of MC4 Rreceptor on cell surface.This will provide technological platform for further research in the role of MC4 Rgene.