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| Cloning of Chicken (Gallus gallus) CREPT Gene and Its Expression in Chicken DF-1 Cells |
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Abstract This research was conducted to detect the specificity of cell-cycle related and expression-elevated protein in tumor (CREPT) gene expression in different tissues and to clone CREPT gene coding sequence of the Rugao yellow chicken (Gallus domesticus). For primary understanding of biological function of CREPT gene in chicken, mostly involved with regulation of cell cycle, we constructed the eukaryotic expression vector which obtained its expression in DF-1(D fibroblast-1) cells and polyclonal antibodies to CREPT protein. The CDS zone of CREPT was amplified from the cDNA of chicken germinal ridge, protein structure anticipation and tissue expression were conducted for further understanding. The cloned chicken CREPT gene was connected into pcDNA3.1 and pEGFP-N1 eukaryotic expression vector to construct recombinant vectors pcDNA3.1-CREPT and pEGFP-CREPT, which were lately transfected to DF-1 cells to obtain polyclonal antibodies by gene-base immunization and analyze its antiserum titer. The qRT-PCR approach was used to identify the expression level of relative genes after over-expression of CREPT gene in DF-1 cells. The results of bioinformation anticipation showed that the coding length of the cloned chicken CREPT contains 978 bp sequence and codes 325 amino acids including a RPR domain (regulation of nuclear pre-mRNA domain) and a CCD (coiled-coil domain). The cloned Rugao yellow chicken CREPT CDS sequence was consistent with sequence from NCBI GenBank(G. gallus, DQ372940), the sequencing results were in agreement with the CDS sequence of the chicken (G. gallus), and the homology was 100%. Tissue transcription specificity analysis showed that transcription of CREPT was higher in testis, ovary and brain (P＜0.01), whereas the transcription of CREPT was lower in skeletal muscle (P＜0.05), intestines (P＜0.01) and spleen (P＜0.05). The recombinant vector (pcDNA3.1-CREPT and pEGFP-CREPT) was constructed. The polyclonal antiserum was prepared by genetic immunization, IFA and transfected results showed the CREPT located in cytoplasm. Best antiserum titer reached 1∶10, identified by IFA (immunogen fluorescent assay). Western blot demonstrated pcDNA3.1-CREPT eukaryotic expression vector works well in DF-1 cells and showed a high antiserum titer. qPCR detection results show that pcDNA3.1-CREPT could over-express CREPT gene in DF-1 cells (P＜0.01) and induce changes of expression of gene p15RS (P15 related gene on G1/S progression, P＜0.01), TCF4 (transcription factor 4, P＜0.05), cyclin D1 (P＜0.01) and b-catenin(P＜0.01), which play important roles in cell cycle regulations. These results indicated that the chicken CREPT was successfully cloned and specificity polyclonal antibodies were obtained. The major expression area of CREPT in DF-1 was cytoplasm. We can also conclude that over-expression of CREPT gene down-regulates the p15RS expression and up-regulates TCF4, cyclinD1 and b-catenin expression in DF-1 cells. This study provides theoretical basis for further elucidating the biological function of CREPT gene in chicken.
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Received: 18 September 2016
Published: 13 January 2017
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