犬γ-干扰素基因的克隆、序列分析及蛋白质结构预测

Cloning and sequence analysis of canis γ-interferon gene and prediction of its protein structure

根据Gen Bank上犬γ-干扰素基因(IFN-γ)序列(序列号WO318193),利用Primer 5.0软件设计特异性引物,应用RT-PCR技术从100μg/ml Con A刺激24 h后的犬外周血淋巴细胞总RNA中扩增出γ-干扰素基因,测序后应用DNAStar软件对克隆的犬γ-干扰素基因进行序列分析,并与Gen Bank上发表的犬、牛、大熊猫、鸡、牦牛、人、猪、野猪、斑马鱼、土拨鼠等的IFN-γ基因序列进行同源性比较。结果显示,克隆的犬IFN-γ基因大小501 bp,编码166个氨基酸,与犬、牛、大熊猫、鸡、牦牛、人、猪、野猪、斑马鱼、土拨鼠IFN-γ基因的核苷酸序列同源性分别为98.4%、81.6%、26.1%、26.1%、22.7%、22.7%、80.8%、81.0%、28.5%和25.0%;氨基酸序列同源性分别为95.8%、72.5%、5.4%、5.4%、6.6%、6.6%、68.9%、69.5%、5.4%和5.4%。运用DNAstar软件对犬γ-干扰素进行蛋白结构预测的结果显示,犬γ-干扰素蛋白含有10个α-螺旋、9个β-折叠、11个β-转角和5个无规则卷曲。

Based on canis interferon（ IFN） gene sequence on Gen Bank,specific primers were designed using Primer5.0 software,and canis γ-IFN gene was amplified by RT-PCR from the RNA extracted from peripheral blood lymphocytes stimulated with Con A for 24 h. The cloned γ-IFN gene was a size of 501-bp fragment encoding 166 amino acids. The nucleotide sequence of the canis γ-IFN gene shared homologies of 98. 4%,81. 6%,26. 1%,26. 1%,22. 7%,22. 7%,80. 8%,81. 0%,28. 5%,and 25. 0% with canis on Gen Bank,buffalo,ailuropoda,chicken,yak,human,swine,wild boar,zebrafish,and marmot,respectively,and the amino acid sequence encoded by canis γ-IFN gene shared homologies of95. 8%,72. 5%,5. 4%,5. 4%,6. 6%,6. 6%,68. 9%,69. 5%,5. 4%,5. 4%,respectively. The protein structure predicted with DNAstar software reveals that he secondary structure of canis protein γ-IFN consists of ten α-helixes,nine β-folds,eleven β-corners and five random coils.