绵羊c-Myc与EGFP融合蛋白的真核表达及其对细胞增殖的调控作用

doi:10.11843/j.issn.0366-6964.2020.05.008

Abstract

Abstract: In order to explore the regulatory effects of c-Myc gene on the cell proliferation and the related genes expression, 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 in this experiment. After transfecting into sheep embryonic fibroblasts (SEF), the effects of c-Myc on the cell proliferation and the related genes expression were detected by cell counting and qRT-PCR, respectively, and the corresponding expression regulation mechanism was analyzed by the promoter analysis software. The results of restriction enzyme digestion and sequencing indicated that the vector was successfully constructed. The bioinformatics analysis showed that the sheep c-Myc protein, with a molecular weight of 48 474.78 u, was mainly located in the nucleus and contained a helix-loop-helix (HLH) domain. The fusion protein c-Myc-(G₄S)₃-EGFP was highly hydrophilic and contained 31 phosphorylation sites. (G₄S)₃ had a high degree of flexibility (9.848 5), c-Myc and EGFP on both sides maintained their respective spatial conformation and did not interfere with each other. The further cellular assay results indicated that the recombinant plasmid pc-Myc-EGFP was expressed successfully in SEF. Overexpression of c-Myc promoted the proliferation of SEF and increased the expression level of cyclin D2, Cdk4, Cdk6, cyclin E1, cyclin A2 and cdc25A mRNA up to 5.20, 3.10, 6.54, 6.52, 23.37 and 8.22 times (P<0.01) respectively, while E2F1 mRNA up to 1.83 times (P<0.05). The results of promoter analysis indicated that there were binding elements for c-Myc and E2F1 on the 5'regulatory region of the above genes. In conclusion, sheep c-Myc promoted SEF proliferation by up-regulating the expression of the cell cycle-related genes, which might be through directly acting on E-box elements of the 5'regulatory region of target genes, or through the indirect regulatory role of other transcription factors such as E2F1.

Key words: sheep, c-Myc gene, EGFP, eukaryotic expression, bioinformatics, cell cycle-related genes, regulatory elements

CLC Number:

S826.2

HAN Gaolian, GUO Lirong, LI Mengxin, ZHENG Tengfei, LI Junling, QIN Jian, DU Rong. Eukaryotic Expression of Sheep c-Myc and EGFP Fusion Protein and Its Regulation on the Cell Proliferation[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(5): 965-975.

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Eukaryotic Expression of Sheep c-Myc and EGFP Fusion Protein and Its Regulation on the Cell Proliferation

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