奶牛生物钟基因CLOCK的真核表达载体构建、生物信息学分析及其组织表达谱

Eukaryotic expression vector construction,bioinformatics analysis and tissue expression profile of the dairy cow CLOCK gene

旨在克隆奶牛昼夜运动输出周期(circadian locomotor output cycles kaput,CLOCK)基因的蛋白编码区序列(coding sequence,CDS),构建该基因的真核表达载体,检测CLOCK基因在奶牛不同组织的相对表达丰度,并预测分析奶牛CLOCK蛋白的高级结构、理化性质及其功能特征。以奶牛肝脏组织cDNA为模板,通过PCR扩增奶牛CLOCK基因CDS区片段,利用同源重组法将其连接至pcDNA3.1-Puro-N-3HA载体;经酶切与测序鉴定后,将鉴定正确的质粒命名为pcDNA3.1-Puro-N-3HA-bCLOCK;将pcDNA3.1-Puro-N-3HA空质粒和pcDNA3.1-Puro-N-3HA-bCLOCK重组质粒分别转染至HEK293T细胞,通过蛋白质免疫印迹(Western blot)检测CLOCK蛋白的过表达效率;提取奶牛的心、肝、脾、肺等10个组织的总RNA并反转录为cDNA,以此为模板通过实时定量PCR(real-time quantitative PCR,qPCR)检测CLOCK基因在奶牛不同组织的表达变化;同时,利用生物信息学软件对奶牛CLOCK基因及其编码蛋白进行功能预测分析。琼脂糖凝胶电泳结果显示,成功克隆了奶牛CLOCK基因全长CDS区片段。酶切与测序结果显示,pcDNA3.1-Puro-N-3HA-bCLOCK真核表达载体构建成功。Western blot结果表明,该真核表达载体在HEK293T细胞中成功表达HA-CLOCK融合蛋白。qPCR结果表明,CLOCK基因在心、肝、脾、肺等10个组织中均有表达,其中在心脏表达最高,在小肠表达最低。生物信息学分析结果表明,奶牛CLOCK基因的CDS区与绵羊、山羊、骆驼的相似性较高;奶牛CLOCK蛋白由845个氨基酸组成,分子质量为95.12 kDa,无跨膜结构域与信号肽,为亲水性蛋白,二级结构中富含α-螺旋;奶牛CLOCK蛋白的三级结构与绵羊、人和小鼠的差异极小。结论:本研究成功克隆了奶牛CLOCK基因全长CDS区片段并构建了其真核表达载体,qPCR检测CLOCK基因在奶牛不同组织的表达谱,通过生物信息学预测分析CLOCK蛋白的结构与功能特性,为进一步探究奶牛CLOCK基因的生物学功能提供理论依据。

The aim of this study was to clone the coding sequence(CDS)of the circadian locomotor output cycles kaput(CLOCK)gene in dairy cows and construct the eukaryotic expression vector of the gene,and to detect the relative expression abundance of the CLOCK gene in different tissues of dairy cows and predict and analyze the advanced structure,physical and chemical properties and functional characteristics of the CLOCK protein in the animals.The CDS region of the cow CLOCK gene was amplified by PCR using dairy cow liver cDNA as template and was ligated into the pcDNA3.1-Puro-N-3HA vector using the homologous recombination method.After restriction digestion and sequencing,the correct plasmid was named pcDNA3.1-Puro-N-3HA-bCLOCK.The empty plasmid pcDNA3.1-Puro-N-3HA and the recombinant plasmid pcDNA3.1-Puro-N-3HA-bCLOCK were transfected into HEK293T cells,respectively.The overexpression efficiency of the CLOCK protein was detected by Western blot.Total RNA was extracted from the heart,liver,spleen,lung and other ten tissues of dairy cows and reversely transcribed into cDNA,which was used as template to detect the expression of the CLOCK gene in different tissues of dairy cows by qPCR.Meanwhile,bioinformatics software was used to predict the function of the dairy cow CLOCK gene and its coding protein.The results of the agarose gel electrophoresis showed that the full-length CDS fragment of the CLOCK gene was cloned successfully.The results of the restriction enzyme digestion and the sequencing showed that the eukaryotic expression vector pcDNA3.1-Puro-N-3HA-bCLOCK was successfully constructed.The Western blot results showed that the eukaryotic expression vector successfully expressed the HA-CLOCK fusion protein in the HEK293T cells.The results of qPCR showed that the CLOCK gene was expressed in the heart,liver,spleen,lung and other ten tissues,with the highest expression in the heart and the lowest expression in the small intestine.The results of the bioinformatics analysis showed that the CDS region of the CLOCK gene of the dairy cows was highly similar to that in sheep,goat and camel.The dairy cow CLOCK protein was composed of 845 amino acids of a molecular weight of 95.12 kDa.It had no transmembrane domain or signal peptide.It was a hydrophilic protein withα-helices in its secondary structure.The tertiary structure of the dairy cow CLOCK protein showed little difference from that of sheep,humans and mice.In this study,the full-length CDS fragment of the dairy cow CLOCK gene was cloned and its eukaryotic expression vector was constructed successfully.The expression profile of the CLOCK gene in different tissues of dairy cows was detected by qPCR.The structure and functional characteristics of the CLOCK protein were predicted and analyzed by bioinformatics,which laid a preliminary foundation for further exploration of the biological function of the CLOCK gene in dairy cows.