Eukaryotic Expression Of Sheep C-Myc And EGFP Fusion Protein And Its Regulation On The Cell Proliferation-related Genes Expression

c-Myc is a transcription factor of the helix-loop-helix-leucine zipper(HLH-LZ)family and plays a critical role in cell proliferation.The aims of this study were to construct the eukaryotic expression vector of sheep c-Myc and EGFP fusion gene and to explore the regulatory effects of c-Myc on the cell proliferation-related genes expression.The coding sequence of sheep c-Myc gene followed by the flexible linker peptide(G4S)3 sequence was synthesized,the eukaryotic expression vector pc-Myc-EGFP of the c-Myc-(G4S)3-EGFP fusion gene was constructed and its structure and physicochemical characteristics were analyzed by bioinformatics.After transfection,the effects of c-Myc on the cell proliferation-related genes expression were detected by real-time PCR in sheep embryonic fibroblasts.The corresponding expression regulation mechanism was analyzed by the promoter analysis software.Results showed that:(1)The results of restriction enzyme digestion and sequencing indicated that the vector was successfully constructed.(2)The bioinformatics analysis showed that the sheep c-Myc protein was mainly located in the nucleus and contained the helix-loop-helix(HLH)domain.The molecular weight of sheep c-Myc protein was 48 474.78 u,the isoelectric point was 5.47 and the grand average of hydropathicity was-0.78.The amino acid sequence of sheep c-Myc protein is closest to that of goats and cattle in the evolutionary distance.The fusion protein c-Myc-(G4S)3-EGFP was highly hydrophilic and contained 31 phosphorylation sites.Because(G4S)3 had a high degree of flexibility which was 9.848 5,the target proteins c-Myc and EGFP on both sides maintained their respective spatial conformation and did not interfere with each other.(3)The further cellular assay results indicated that the recombinant plasmid pc-Myc-EGFP was expressed successfully in sheep embryonic fibroblasts.The overexpression of c-Myc promoted the proliferation of sheep embryonic fibroblasts,and increased the expression levels of cyclin D2,Cdk4,Cdk6,E2F1,cyclin E1,cyclin A2 and cdc25 A m RNA up to 5.20 times(P<0.01),3.10 times(P<0.01),6.54 times(P<0.01),1.83 times(P<0.05),6.52 times(P<0.01),23.37 times(P<0.01)and 8.22 times(P<0.01),respectively.(4)The bioinformatics prediction indicated that there were binding elements of c-Myc and E2F1 on the 5’ regulatory region of the above genes.In conclusion,the fusion expression vector of sheep c-Myc and EGFP was successfully constructed.c-Myc up-regulated the expression of the cell proliferation-related genes,which might be through directly acting on E-box elements of the 5’ regulatory region along these target genes,or through the indirect regulatory role of other transcription factors such as E2F1.