猪MC4R基因突变体真核表达载体的构建及功能研究

doi:10.11843/j.issn.0366-6964.2016.04.006

Abstract

Abstract:

To investigate the function difference of the different Melanocortin-4 receptor (MC4R) gene mutants，the complete coding sequence( CDS) of MC4R gene were cloned from pig muscle tissues.The CDS sequence was linked with pcDNA3.1 vector by digested with restriction endonuclease of Kpn Ⅰ and EcoR Ⅰ and 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，3 site-directed MC4R cDNA mutant (707/892 site) vectors were generated.The protein expression of MC4R mutations vector was observed 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 MC4R receptor were expressed well on the cell surface by the Immunofluorescence.There were no significant difference of the fluorescence intensity between mutants of MC4R receptors（P＞0.05）by FCM.The construction and functional research of MC4R vector suggest that the mutants of 707/892 site has no significant impact on the expression of MC4R receptor on cell surface.This will provide technological platform for further research in the role of MC4R gene.

Key words: pig, MC4R, mutation, expression

CLC Number:

S828
S813.3

HAN Li-qiang，GUO Yu-jie，LU Wei-fei，ZHANG Xin，CHU Bei-bei，WANG Jiang，YANG Guo-yu. Construction and Function Confirmation of MC4R Gene Eukaryotic Expression Vector with Different Mutations[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2016, 47(4): 679-685.

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

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