猪粒细胞-巨噬细胞集落刺激因子基因的克隆、序列分析及蛋白结构预测

GM-CSF在机体免疫系统中发挥重要的免疫调节作用.用PHA与LPS体外联合刺激猪外周血单个核细胞,采用RT-PCR技术成功克隆了猪GM-CSF基因.序列分析表明,该序列与NCBI/GenBank上登载的序列有两个碱基不同;将猪与人、牛、羊、狗、猫、鼠等动物的GM-CSF基因序列相比较,其一致性分别为82.1%、85%、88.1%、82.8%、83.7%、70.5%,在氨基酸水平上一致性为70.3%、70.9%、78.1%、70.3%、71.9%、54.4%.然后利用生物信息学和分子生物学软件,对猪GM-CSF的结构进行预测,结果表明,猪GM-CSF为一结构松散的球状蛋白分子.猪GM-CSF基因的成功克隆及其编码蛋白结构的预测为开发高效、低毒且性质稳定的猪GM-CSF产品奠定了基础.

Cloning and sequence analysis of porcine GM-CSF gene and prediction of protein structure

GM-CSF plays very important role in immune system regulation.We isolated porcine peripheral lymphocytes cells and co-stimulation it with PHA and LPS.We then extracted total RNA from the cells and amplified GM-CSF gene using RT-PCR.The obtained fragment was ligated into pGEM-T easy vector and sequenced.The result showed that the sequence had 2 nucleotides difference with the sequence of GM-CSF gene published in GenBank.sequence analysis shows 82?% identity with human,85?% with bovine,88?% with ovine,83?% with canine,84?% with feline,and 71?% with murine GM-CSF,and 70.3 %,70.9 %,78.1 %,70.3 %,71.9 % and(54.4?%) at the GM-CSF mature protein amino acid level.The protein structure prediction showed that the GM-CSF protein is a loose globe protein.These results pave the way for future developments on the GM-CSF product.