鸡myostatin基因成熟区段DNA的克隆及其在甲醇酵母中的融合表达

Cloning and fusion expression of mature DNA region of chicken myostatin gene in Pichia pastoris

根据GenBank中鸡myostatin(AF019621)成熟区段的cDNA序列设计了1对引物,利用PCR技术扩增了萧山鸡myostatin的成熟区段,构建了重组真核表达载体myostatin-pPIC9K,并在甲醇酵母中融合表达了myostatin蛋白。结果发现,萧山鸡myostatin基因存在2处碱基变异:T204→C204(编码的氨基酸没有变),C205→T205(编码的氨基酸由Pro变成Ser),从而产生了1个EspⅠ或Bpu1102Ⅰ限制性内切酶酶切位点。核苷酸序列同源性分析表明,myostatin基因成熟区段DNA在不同物种间具有高度的保守性。SDS-PAGE鉴定结果表明,表达的目的蛋白(26 ku)具有生物学活性。

A pair of primers was designed according to the mature cDNA region of chicken myostatin gene reported in GenBank（Gallus gallus ,AF019621）. With the primers,the mature DNA region of myostatin gene was amplified from Xiaoshan chicken,cloned and sequenced. Then the gene was subcloned into pPIC9k to construct an expression vector myostatin-pPIC9K and expressed in Pichia pastoris. Sequence analysis revealed that there were 2 mutations [T^204→C^204 and C^205→T^205 （Pro→Ser）] in the cloned sequence. The mutations produced an Esp Ⅰ and a Bpull02 Ⅰ restriction site. Identity analysis among different species at nucleotide level showed that the cloned mature DNA region was quite conserved. SDS-PAGE identification showed that the expressed myostatin protein was 26 ku in size and possessed biological activity.