绵羊Noggin基因的克隆、序列分析与原核表达

Molecular Cloning,Sequence Analysis and Prokaryotic Expression of Noggin Gene in Sheep

根据同源序列克隆原理设计一对引物,以绵羊脑组织总RNA的反转录产物为模板,利用RT-PCR扩增绵羊Noggin基因cDNA全长编码区,克隆到pMD-18T载体。测序验证后,提取pT-Noggin质粒经BamHⅠ和XhoⅠ双酶切回收目的条带,亚克隆到pET41a原核表达载体,转化BL21(DE3)菌,经IPTG诱导表达,SDS-PAGE和Western blot鉴定表达产物。序列分析表明,绵羊Noggin基因的cDNA全长编码区(GenBank登录号为FJ751853)为699bp,编码232个氨基酸,与其他哺乳动物氨基酸序列同源性达到95%以上。SDS-PAGE结果显示,诱导表达的融合蛋白大小为60ku,与预期蛋白大小一致。Western blot检测结果显示,该蛋白为His融合蛋白。说明成功克隆了绵羊Noggin基因的编码区序列,构建了原核表达载体,在大肠杆菌中成功表达出目的融合蛋白。

A pair of primer was designed according to the Noggin homologous sequence. The full- length coding sequence of the sheep Noggin was amplified using the first strand cDNA, which was generated from sheep brain tissue RNA, the PCR products of sheep Noggin were cloned into pMD- 18T and confirmed by digestion with BamH I and Xho I. Noggin was then sub-cloned into the pET41a expression vector from a correct clone and then transformed into BL21 （DE3） E. coli, which could be induced by IPTG. The expressed products were analyzed with SDS-PAGE and Western blot. The sequence analysis revealed that the Noggin coding sequence in sheep was 699 bp, encoding 232 AAs （GenBank Accession Number： FJ751853）. The predicted protein sequence shared 95% identity with other mammals. SDS-PAGE analysis showed that the expressed protein in E. coli BL21 （DE3） was about 60 ku. Western blot revealed that the expressed protein was a His tag fusion protein. Clo- ning and successful expression of the sheep Noggin built the foundation for further study on its bio- logical function.