作　　者： ; ; ; ; ; ; ; ;

机构地区： 华南农业大学动物科学学院

出　　处： 《畜牧兽医学报》 2007年第5期432-436,共5页

摘　　要： 以人CTSD mRNA全长序列为基序,在db EST库中搜索同源性大于80%、重叠大于40个碱基的猪EST下载经Seqman软件装配后,利用RT-PCR扩增到猪CTSD基因部分cDNA序列,进一步通过RACE技术获得cDNA全长（GenBank登录号为DQ018727）,猪CTSD基因cDNA全长2032 bp包括93 bp 5′UTR区、706 bp 3′UTR区和1 233bp ORF,编码410个氨基酸残基。其编码区与人、鼠、牛、羊CTSD编码区同源性分别为87.9%、78.2%、86.6%和85.0%,其氨基酸序列与人、鼠、牛、羊氨基酸序列同源性分别为86.6%、80.1%、86.0%和84.7%。疏水性分析和信号肽预测发现猪CTSD氨基酸序列存在至少7个疏水性区域,信号肽位点为第1-20个氨基酸残基。在NCBI进行Blast P搜索结果显示猪CTSD氨基酸结构中含有Asn（Asparagine,天门冬酰胺）结构域,与天冬氨酸蛋白酶3D结构同源性高达99.7%,具有真核生物天冬氨酸蛋白酶的典型结构。以猪多组织RNA池为模板,以1026 bp部分cDNA序列作为杂交探针进行Northern杂交,得到一条2000 bp左右特异条带,从而验证了RACE产物为全长cDNA并揭示该基因只有一个转录本。为了研究猪CTSD基因在体内不同组织内表达的特点,采用半定量RT-PCR对CTSD基因在猪10个组织中的表达进行研究,结果表明在10个组织中均有表达,且表达量与-βactin内参接近。 The human CTSD full-length mRNA wasused to blast dbEST database, porcine ESTs that showed above 80% similarity and over 40 overlapped base pairs were assembled by Sequman software. Then partial cDNA sequence was amplified by RT-PCR, further the full-length cDNA was obtained by RACE （GenBank accession number： DQ018727 ）. Porcine CTSD full-length cDNA was 2 032 bp containing a 93 bp 5＇-untranslated region,a 706 bp 3＇-untranslated region and a 1233 bp open reading frame coding 410 amino acid. Porcine CTSD cDNA ORF region showed 87.9%,78.2%, 86.6% and 85.0% alignments to those of human, mouse, bovine and sheep CTSD. And porcine CTSD amino acid sequence showed 866% ,80.1% ,86.0% and 84.7% similarities to counterparts of human,mouse, bovine and sheep CTSD. Hydrophobicity profile analysis and signal peptide prediction in bioniformatics websites demonstrated that there were seven transmembrane regions at least and a signal peptide with 20 amino acid residues （1-20）. The deduced 410 amino acid of CTSD was predicted to contain an Asn （Asparagine） domain which was the representative structure of lysosomal aspartic proteinase by blast in NCBI, and three-dimentional structure of porcine CTSD showed 99.7 % alignment to that of mammalian CTSD. Northern hybridization was perfomed with porcine mix RNA pool and 1 026 bp porcine CTSD eDNA probe. The result displayed a approximate 2 000 bp specific band,which revealed the eDNA which was obtained in this research was full-length cDNA and porcine CTSD has one transcript. RT-PCR simi-quantitative expression analysis of porcine ten tissues indicated that CTSD was expressed widely and there was no difference within the four pig breeds, and no difference among the tissues.

关 键 词： 猪 溶酶体组织蛋白酶 电子克隆 快速扩增 末端 全长

领　　域： [农业科学] [农业科学]