绵羊NICD基因的克隆、真核表达及其序列分析

采用RT-PCR技术扩增绵羊Notch基因胞内段功能结构域(NICD),将其克隆到慢病毒表达载体中,在293T细胞中对其进行真核表达,通过Western blotting鉴定其蛋白表达,进一步对NICD核酸和氨基酸序列进行分析。结果表明,首次克隆获得绵羊NICD基因,该基因序列为2 388bp(GenBank登录号为KF318190.1),编码795个氨基酸,重组蛋白分子质量为110ku左右。序列分析结果显示,核酸序列与牛、马、人和小鼠的同源性分别为96%、90%、87%和83%,进化上与牛的关系最近;蛋白序列与牛、马、人和小鼠的同源性分别为96%、90%、87%和84%,预测二级结构可能存在26个α-螺旋、13个β-折叠、66个转角和64个无规则卷曲。

Molecular Cloning, Eukaryotic Expression and Sequence Analysis of NICD Gene in Sheep

Intracellular domain of Notch (NICD) was amplified by RT-PCR from sheep tissue, the PCR products of sheep NICD was cloned into lentiviral expression vector pLEX-mcs. The plasmid was transformed into 293T cell for eukaryotic expression, and western blotting was used to detect NICD protein. Further, the nucleic acid and protein sequences of NICD were analyzed by bioinformatics method. The results revealed that the NICD sequence in sheep was 2 388 bp, encoding 795AAs (GenBank Accession Number: KF318190.1). The protein size of eukaryotic expression is 110 ku. Nucleic acid sequence homology with cattle, horse, human and mouse were 96%, 90%, 87% and 83% respectively. Protein sequence homology with cattle, horse, human and mouse were 96%, 90%, 87% and 84% respectively, and had the closest evolutionary relationship with cattle. Secondary structure prediction indicated that there were probably 26 alpha helices, 13 beta sheet, 66 angles and 64 random coils.