鸡CREPT基因的克隆及其在鸡DF-1细胞中的表达

发布时间：2018-02-27 18:19

本文关键词： CREPT基因鸡真核表达载体多克隆抗体 DF-细胞表达调控　出处：《农业生物技术学报》2017年02期 　论文类型：期刊论文

【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(P0.01),而在肠组织(P0.01)、骨骼肌(P0.05)和脾脏(P0.05)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(P0.01),上调TCF4(P0.05)、cyclinD1(P0.01)和b-catein(P0.01)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]:The aim of this study was to detect the tissue expression specificity of CREPT(cell-cycle related and expression-elevated protein in tumor, clone its coding region and construct a recombinant vector to transfect DF-1(D fibroblast-1 DF-1 cells and prepare polyclonal antibodies. To provide theoretical basis for the preliminary study of the biological function of chicken Gallus gallus)CREPT gene involved in the regulation of cell cycle, the CDS region of CREPT gene was amplified by using chicken reproductive crest cDNA as template. The protein structure and tissue expression profile of the gene were predicted and analyzed. The recombinant expression vectors pcDNA3.1-CREPT and pEGFP-CREPT were obtained by connecting the gene with pcDNA3.1 and pEGFP-N1 vectors, and then transfected into chicken DF-1 cells. The polyclonal antibody of CREPT gene was prepared by gene immunoassay and its titer was detected. The expression of related genes in DF-1 cells was detected by qRT-PCR after the expression of CREPT gene. The total length of chicken CREPT coding region was 978 BP, encoding 325 amino acids, and there was a RPR domain regulation of nuclear pre-m RNA domain and curl domain in the amino acid sequence. The expression of m RNA in testis, ovaries and cerebrum was higher than that in intestinal tissues (P0.01), skeletal muscle (P0.05) and spleen (P0.05). The recombinant expression vectors pcDNA3.1-CREPT and pEGFP-CREPTwere successfully constructed, and the fusion expression vector pEGFP-CREPT was transfected into the cytoplasm of DF-1. The best titer of the polyclonal antibody was 1: 10 Western blot. The eukaryotic expression vector pcDNA3.1-CREPT was successfully expressed and the antiserum effect was good. The results showed that the pcDNA3.1-CREPT vector could overexpress the CREPT gene on DF-1. The expression changes of cell cycle regulation related gene p15RSSv P15 related gene on G 1 / S progression, transcription factor 4 related factor 4T CF4, cyclin D 1 and 尾 -chain protein B catein) were induced. Chicken CREPT gene was cloned and specific polyclonal antibody was prepared. The expression of this gene in the cytoplasm of DF-1 could down-regulate the expression of p15RS gene, up-regulate the expression of p15RS gene and up-regulate the expression of cyclin D1 and b-catein P0.01). The results provided a theoretical basis for further study on the biological function of chicken CREPT gene.
【作者单位】：扬州大学动物科学技术学院/江苏省动物遗传繁育与分子设计重点实验室;
【基金】：国家自然科学基金(No.31472087、No.31301959和No.31272429) 中国博士后基金(No.2012M511326和No.2014T70550) 江苏高校优势学科建设工程资助项目扬州大学大学生学术科技创新基金(No.x20160682)
【分类号】：Q78;S831

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