摘要本研究旨在检测CREPT(cell-cycle related and expression-elevated protein in tumor)基因的组织表达特异性，克隆其编码区并构建重组载体转染DF-1(D fibroblast-1, DF-1)细胞并制备多克隆抗体，为初步研究鸡(Gallus gallus) CREPT基因参与调控细胞周期的生物学功能提供理论依据。以鸡生殖嵴cDNA为模板扩增CREPT基因CDS区，并对该基因进行了蛋白质结构预测和组织表达谱分析。将该基因与pcDNA3.1和pEGFP-N1载体相连获得重组表达载体pcDNA3.1-CREPT和pEGFP-CREPT之后，转染鸡DF-1细胞，并采用基因免疫法制备CREPT基因多克隆抗体并检测效价，利用qRT-PCR检测过表达CREPT基因后DF-1细胞内相关基因的表达情况。生物信息学分析发现，鸡CREPT编码区序列总长978 bp，编码325个氨基酸，该氨基酸序列中存在一个RPR结构域(regulation of nuclear pre-mRNA domain)和卷曲结构域；以原鸡(G. gallus)CDS序列为模板成功克隆的如皋黄鸡(G. domesticus)CREPT基因长978 bp，测序结果准确，与原鸡CDS序列一致，同源性100%；组织表达谱分析显示，CREPT在睾丸、卵巢和大脑中mRNA表达量较高(P＜0.01)，而在肠组织(P＜0.01)、骨骼肌(P＜0.05)和脾脏(P＜0.05)中的mRNA表达量较低；成功构建重组表达载体pcDNA3.1-CREPT和pEGFP-CREPT；转染融合表达载体pEGFP-CREPT后显示，CREPT表达于DF-1细胞质中；使用基因免疫法制备多克隆抗体血清，免疫荧光检测(immunogen fluorescent assay, IFA)检测显示，制备的多克隆抗体最佳效价为1∶10，Western blot证实，pcDNA3.1-CREPT真核表达载体成功表达且抗血清效果良好，qPCR检测结果表明，pcDNA3.1-CREPT载体能够在DF-1上过表达CREPT基因，并引起细胞周期调控相关基因p15RS(P15 related gene on G1/S progression)、转录因子4(transcription factor 4, TCF4)、细胞周期蛋白D1(cyclinD1)和 β-链蛋白(b-catein)的表达变化。本研究成功克隆了鸡CREPT基因，并制备了具有特异性的多克隆抗体，该基因在DF-1细胞质中表达，CREPT基因的表达能下调p15RS基因的表达(P＜0.01)，上调TCF4(P＜0.05)、cyclinD1(P＜0.01)和b-catein(P＜0.01)的表达，该研究结果为进一步研究鸡CREPT基因的生物学功能提供了理论依据。
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.
