岭南黄鸡肌生长抑制素基因成熟肽的克隆及酵母表达质粒的构建

根据Genebank上登录的鸡的myostatin基因cDNA全长序列以及成熟肽序列设计一对引物,并分别在两引物前设计两个酶切位点EcoRⅠ和KpnⅠ,克隆岭南黄鸡肌肉生长抑制激素的成熟肽蛋白编码基因,然后将特异性片段连接到pMD18-T载体,经酶切、PCR鉴定后,构建了岭南黄鸡真核单纯表达载体pPICZαA-MSTN-m,经测序鉴定,结果表明所克隆的myosta-tin成熟肽基因与Genebank上发表的鸡（AF019621）、猪（AY208121）和家鹅（AF440862）的MSTN-m核酸序列同源性分别可达到99%、92%、86%,但翻译后的成熟蛋白氨基酸序列与鸡、猪和家鹅的同源性可以分别达到100%、99.1%及99.1%。

Molecular Cloning of Mature Peptide of Lingnan Yellow Chicken Myostatin Gene and Its Expression in Pichia pastoris

Based on the published cDNA nucleotide sequence of chicken myostatin gene and the nucleotide sequence of mature peptide in the Genebank,a pair of primers were designed and synthesized to clone the gene coding Lingnan yellow chicken myostatin mature protein.In the front of the primers,two sites of restrictive enzyme digestion,EcoRⅠ and KpnⅠ were designed.Then the myostatin mature peptide DNA fragment was cloned into pMD18-T vector.Restriction enzyme digestion and PCR amplification confirmed the inserted fragment was myostatin mature peptide nucleotide sequence.The sub-cloned myostatin mature peptide gene has been successfully inserted to eukaryotic expression vector pPICZαA.DNA sequencing confirmed the structrue of recombinant expression plasmid was correct,which respectively had the identities of 99%,92%,86% with the myostatin gene mature peptide nucleotide sequence of chicken(AF019621),swine(AY208121) and domestic goose(AF440862) published in the Genebank.But the amino acid sequence of mature protein translated had the identities of 100%,99.1%,99.1% with that of chicken,swine and domestic goose respectively.