鸡IL-2基因的克隆、序列分析及蛋白结构预测

Cloning and Sequence Analysis of Gallus IL-2 Gene and Prediction of Protein Structure

根据GenBank中已发表的鸡白细胞介素2(IL-2)mRNA基因序列,利用Premier5.0软件设计一对特异性引物,采用RT-PCR技术,以ConA刺激的鸡外周血淋巴细胞为材料,从总RNA中扩增出鸡IL-2基因。经琼脂糖凝胶电泳显示扩增片段约为432bp,分离纯化片段,克隆入pMD-18T载体,转化DH5α感受态细胞,获得阳性重组质粒,经酶切鉴定,测序结果显示,该基因全长432bp,含有一个432bp的开放阅读框,编码143个氨基酸。生物软件分析结果表明该序列与GenBank中发表的鸡的IL-2的核苷酸序列同源性为98.8%,与黄牛、马、鸡、绵羊、牛、犬、人、山羊、鼠、水牛的核苷酸同源性分别为31.2%、28.2%、27.3%、30.6%、26.2%、31.7%、30.1%、30.8%、26.6%、30.6%。与GenBank中发表的鸡IL-2编码的氨基酸序列同源性为95.8%,与上述动物的氨基酸序列同源性分别为7.6%、7.6%、9.7%、7.6%、6.9%、10.4%、11.8%、7.6%、9.0%、7.6%、10.4%。这表明鸡的IL-2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IL-2增强DNA疫苗的免疫效果奠定了基础。

According to GenBank in the IL-2 cDNA sequences, using Premier 5.0 software design a pairs of specific primer, the local Dorking inject 100 μg/ml Con A, total RNA was extracted from feeding 24 hours lymphocyte from spleen, by RT-PCR cloning colony IL-2 cDNA fragment. The DNA fragments were analyzed use DNAStar software between different species identity for comparison. Sequence analysis showed that IL-2 gene has an open reading frames for 432 nucleotide acids, encoding 143 amino acids and most of signal peptide, compared with published on Genbank, the nucleotide identity was 98.8% 、 31.2% 、 28.2% 、 27.3% 、 30.6% 、 26.2% 、 31.7% 、 30.1% 、 30.8% 、 26.6% 、 30.6%. compared with published on Genbank, the Amino acids was 95.8%, 7.6% 、 7.6% 、 9.7% 、 7.6% 、 6.9% 、 10.4% 11.8%, 7.6%, 9.0%, 7.6%, 10.4% with cattle, horse, cat, sheep, bovine, canis, people, capra, mice, cow of IL-2 sequence comparison The gallus IL-2 gene was successfully cloned, there is a species-specific. And construct a based on further study the biological effects of IL-2 gene in particular the use of IL-2 enhance the immune effect of DNA vaccine.