猪粒细胞-巨噬细胞集落刺激因子(GM-CSF)基因的克隆和序列分析及其基因表达

Cloning and Sequence Analysis of Porcine GM-CSF Gene and Its Gene Expression

采取三元杂交猪的外周血,提取单核细胞总RNA,采用RT-PCR克隆猪粒细胞-巨噬细胞集落刺激因子(GM-CSF)cDNA(GenBank登录号为DQ108393)。以pcDNA3.1(-)为质粒载体,构建猪GM-CSF基因真核表达质粒pGM-CSF,对克隆片段进行测序,并进行不同物种间同源性的比较。将重组质粒转染COS-7细胞,Western blotting鉴定表达蛋白的特异性。序列分析表明,GM-CSF的cDNA开放阅读框为435 bp,编码144个氨基酸和大部分信号肽,与用作PCR引物设计的参考序列比较,其核苷酸的同源性为97.5%,氨基酸的同源性为95.1%,同绵羊、人、牛、小鼠和犬的GM-CSF序列比较,其核苷酸的同源性分别为86.2%、80.5%、81.7%、69.7%和81.8%,氨基酸的同源性分别为78.5%、71.3%、70.3%、55.9%和71.5%。Western blotting证实目的基因可以在真核细胞中特异性的表达GM-CSF蛋白。以上结果表明,猪的GM-CSF基因被成功克隆,它存在着种属特异性。这为进一步研究该基因的生物学作用特别是利用GM-CSF增强DNA疫苗的免疫效果奠定了基础。

cDNA encoding porcine granulocyte - macrophage colony stimulating factor （ GM - CSF） （GenBank accession number is DQ108393） were cloned from peripheral blood mononuclear cells （PBMC） using the reverse transcription- polymerase chain reaction （RT- PCR）. The obtained fragment was inserted into pcDNA3.1 （ - ） vector and sequenced. Identified recombinant plasmid coated in lipofectamine was transfected into COS- 7 cell lines. Specific protein expressed in the cell lines was detected by Western blotting. The results were as follows：The cloned cDNA of porcine GM -CSF was 435 bp in length and encoded a predicted mature protein of 144 amino acids and the majority of the signal peptide. Compare with reference sequence of PCR primer designing, homology of the porcine GM - CSF coding sequence was 97.5% at the nucleotide level and 95.1% at the amino acid level. Compared with ovine, human, bovine, murine and canine GM - CSF, homology of the porcine GM - CSF coding sequence was, respectively, 86.2%, 80.5%, 81.7%, 69.7% and 81.8% at the nucleotide level, and 78.5%, 71.3% , 70.3% , 55.9% and 71.5% at the amino acid level. Western blotting indicated that the target gene could be expressed in the mammalian cell line COS- 7. In conclusion, cDNA encoding porcine GM -CSF was successfully cloned and sequenced, which provides a material foundation for immunomodulation of GM - CSF on DNA vaccine.