鸭脂联素基因全长cDNA的克隆和原核表达的研究

Identification and Prokaryotic Expression of Duck Adiponectin Gene

本研究旨在通过克隆鸭脂联素(Adiponectin)基因序列并进行序列分析,揭示该基因的序列特征、组织特异性表达规律,并对其进行原核表达。采用RT-PCR、5′-RACE和3′-RACE的方法从鸭脂肪组织总RNA中扩增脂联素基因cDNA片段;半定量RT-PCR检测组织表达特异性;构建原核表达载体,建立原核表达体系。从鸭脂肪组织中得到3个脂联素基因cDNA片段,克隆测序后,拼接获得了鸭脂联素基因1374bp全长cDNA序列,其包含一个长度为738bp完整的开放阅读框,编码245个氨基酸;编码区与鹅、鸡脂联素基因序列的同源性分别为94.9%和86.0%,与人、小鼠、狗等物种脂联素基因的同源性在64.2%～67.0%之间;氨基酸序列比较可知,鸭与鹅、鸡的脂联素氨基酸的同源性分别达95.5%和84.0%,与人、小鼠、狗等哺乳动物的同源性在65%左右。半定量RT-PCR研究结果表明脂联素基因在所测组织中均表达,且高度表达于脂肪组织、肌胃、小肠、心和骨骼肌,中度表达于肺、腺胃、卵巢和脾组织中,而在肝、肾和间脑中低度表达。构建得到在其C端含有8个组氨酸的鸭脂联素融合蛋白表达载体pET41a-duck-adp,重组表达质粒转入BL21(DE3)大肠杆菌中表达,经IPTG诱导后并SDS-PAGE检测获得了30ku的鸭脂联素原核表达蛋白。脂联素基因在动物进化中具有一定的保守性,该基因在不同组织中表达水平不同,通过原核表达可获得鸭脂联素的重组蛋白。

The purpose of this study was to clone and characterize the duck Adiponectin gene,and to reveal the tissue expression pattern of Adiponectin mRNA in duck,and to express recombinant duck Adiponectin in prokaryotic cells.The sequence of duck Adiponectin gene was obtained by reverse transcription PCR,5 ′-rapid amplify cDNA end and 3′-rapid amplify cDNA end（5′-RACE and 3′-RACE） methods,and its expression in various duck tissues was detected by semi-quantity RT-PCR.Recombinant plasmid was constructed and transformed into E.coli.to express the recombinant duck Adiponectin in prokaryotic cells.The three Adiponectin cDNA fragments were amplified from total RNA using RT-PCR,5′-RACE and 3′-RACE methods,respectively.After cloning and sequencing the three fragments,a 1 374 bp full length cDNA sequence of duck Adiponectin gene was acquired,which contained a full 738 bp nucleotides open reading frame,encoding 245 amino acids.The deduced Adiponectin amino acid sequence of duck contained 22 glycine-X-Y repeats at the collagen repeats as found in the chicken and mammalian Adiponectin.The duck Adiponectin CDS shared 94.9% and 86.0% homology with goose and chicken,and about 64.2%-67.0% homology with mouse,human and dog.Meanwhile,the putative amino acid sequence of duck Adiponectin shares 95.5% and 84.0% homology with goose and chicken,and about 65% homology with the above mammals.The semi-quantity RT-PCR showed that Adiponectin expressions were detectable in all tissues,and the expression level of Adiponectin gene was high in adipose,muscular stomach,small intestine,heart and skeletal muscle,medium in lung,gland stomach,ovary and spleen,low in liver,kidney and diencephalons.The recombinant plasmid of pET41a-duck-adp was constructed and transformed into E.coli BL21（DE3）.The 30 ku recombinant duck adiponectin was successfully expressed in E.coli.The Adiponectin gene is conserved in animal evolution,and Adiponectin mRNA expresses differently in various tissues.The recombined duck adiponectin could be expressed in prokaryotic system.