黄淮山羊Leptin cDNA的克隆及鉴定

Cloning of Leptin cDNA from the adipose of Huanghuai goat

根据GenBank中公布的山羊Leptin基因部分基因组核苷酸序列,设计合成一对引物,以山羊脂肪组织总RNA为模板,采用RT-PCR法,扩增出Leptin成熟蛋白编码cDNA序列,将此扩增产物克隆入pMD18-T载体,进行PCR、双酶切鉴定及序列测定与分析。结果表明,扩增的山羊Leptin基因编码序列长为441bp,编码146个氨基酸。同源性分析表明,山羊Leptin基因成熟蛋白编码cDNA与小鼠、人、猪、牛及绵羊基因相应序列的同源性分别为82.22%、87.76%、92.97%、96.15%和98.64%,氨基酸同源性为84.25%、86.99%、92.47%、97.95%和100.00%,说明Leptin是一组在进化上高度保守的蛋白质。山羊Leptin成熟蛋白cDNA的成功克隆,为进一步研究黄淮山羊Leptin基因全结构、基因表达与调控奠定了基础。

According to the genomic sequence of Leptin of goats which published on GenBank, We designed and synthesized a pair of primers. The cDNA encoding the mature protein of Leptin was amplified from the total RNA abstracted from adipose tissue of Huanghuai goat by RT-PCR technique and cloned into pMD18- T vector. The positive recombinant clones were characterized by PCR, double enzyme digestion and sequencing. The sequence analysis showed that the coding cDNA of goat Leptin mature protein consisted of 441 nucleotides and encoded 146 amino acids. Corresponding sequence comparisons of goat with mouse, human, pig, cattle and sheep demonstrated that the homologies of the nucleotides were 82.22 %, 87.76%, 92.97%, 96. 15%, and 98. 64% respectively, while the homologies for the encoded amino acids were 84. 25%, 86. 99%, 92.47%, 97.95% and 100.00% respectively. It can therefore be concluded that goat Leptin is a highly conservative gene in the evolutionary history. The successive cloning of goat mature protein coding eDNA from the adipose tissue lay a foundation for analyses of its full structure, gene expression and control.