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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978bp,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-m RNA 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m RNA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m RNA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。
The aim of this study was to examine the tissue-specific expression of CREPT and to clone the coding region of the CREPT gene and to construct a recombinant vector for DF-1 (DF-1) And to prepare polyclonal antibodies, providing a theoretical basis for preliminary study on the biological function of CREPT gene of Gallus gallus in regulating cell cycle. The chicken genital ridge cDNA was used as a template to amplify the CDS region of CREPT gene, and the protein structure prediction and tissue expression profiling of the gene were performed. The gene was linked to pcDNA3.1 and pEGFP-N1 vector to obtain recombinant expression vector pcDNA3.1-CREPT and pEGFP-CREPT, transfected chicken DF-1 cells, and gene immunoassay polyclonal antibody CREPT gene was detected The qRT-PCR was used to detect the expression of related genes in DF-1 cells after overexpression of CREPT gene. Bioinformatics analysis showed that the CREPT coding region of chicken had a total length of 978bp and encoded a protein of 325 amino acids. The amino acid sequence contained a regulation of nuclear pre-m RNA domain and a curly domain. ) CDS sequence as a template successfully, the CREPT gene of G.domesticus was 978 bp in length and the sequencing result was correct, which was consistent with that of the original chicken CDS. The homology was 100%. The tissue expression profile showed that CREPT gene in testis and ovary (P <0.01), while the expression of m RNA in skeletal muscle (P <0.05) and spleen (P <0.05) was lower than that in the control group (P <0.01) Recombinant expression vector pcDNA3.1-CREPT and pEGFP-CREPT; transfected with the fusion expression vector pEGFP-CREPT showed that CREPT expression in DF-1 cytoplasm; polyclonal antibody sera were prepared by gene immunization, immunogen fluorescent assay , IFA) showed that the optimal titer of the prepared polyclonal antibody was 1:10. Western blot confirmed that the pcDNA3.1-CREPT eukaryotic expression vector was successfully expressed and the antiserum was effective. The qPCR results showed that pcDNA3.1- The CREPT vector overexpresses the CREPT gene on DF-1 and causes fineness Cycle regulatory genes p15RS, change (P15 related gene on G1 / S progression) transcription factor 4 (transcription factor 4, TCF4), cyclin D1 (cyclinD1) and β- catenin (b-catein) expression. In this study, we successfully cloned chicken CrePT gene and prepared a specific polyclonal antibody, the gene was expressed in DF-1 cytoplasm, CREPT gene expression can down-regulate the expression of p15RS gene (P <0.01), up-regulated TCF4 (P <0.05), cyclinD1 (P <0.01) and b-catein (P <0.01). The results of this study provide theoretical basis for further study on the biological function of CREPT gene in chicken.