新疆巴什拜羊BPI基因cDNA全长的克隆及序列分析

Cloning and sequence analysis of Bactericidal/PermeabilityIncreasing protein gene in Xinjiang Baishibai sheep

【目的】克隆巴什拜羊杀菌/通透性增强蛋白(BPI)的cDNA序列,为研究该蛋白的结构和功能奠定基础。【方法】采集巴什拜羊外周血液,从中分离得到中性粒细胞后提取RNA。根据GenBank中牛BPI基因的cDNA序列设计兼并引物,用RT-PCR方法扩增巴什拜羊BPI基因部分序列,再根据已知的巴什拜羊BPI基因部分序列设计RACE引物,分别扩增出5′和3′端目的片段,分别回收克隆的目的基因片段,再与pMD18-T载体连接,分别转化到大肠杆菌DH5α中,筛选阳性克隆,将重组菌测序后进行拼接,获得巴什拜羊BPI基因全长cDNA序列,并对序列进行分析。【结果】克隆的巴什拜羊BPI基因cDNA序列全长1 922bp,其中开放阅读框为1 452bp,共编码483个氨基酸;BLAST分析表明,巴什拜羊BPI基因的核苷酸序列与绵羊、牛、虎鲸、野猪、猕猴、人、家兔、小鼠、非洲爪蟾核苷酸的一致性分别为99%,91%,78%,74%,64%,61%,58%,53%和50%;氨基酸进化分析显示,巴什拜羊首先与绵羊聚为一类,其次与牛聚为一类,该聚类分析结果与生物学分类结果表现一致。【结论】通过RACE方法成功地克隆了1 922bp的巴什拜羊BPI基因cDNA全长序列,且基因进化树聚类结果与生物学分类结果相一致。

【Objective】To reveal the bactericidal/permeability-increasing protein(BPI)in Bashibai sheep and improve the study of its structure and function,the BPIcDNA was cloned from Bashibai sheep by homologous cloning and rapid amplification cDNA ends(RACE).【Method】This study used Bashibai sheep as the research object.First,merger primers were designed according to the cDNA sequence of Bos tautus BPI gene from the GenBank and BPI gene segment was amplified by RT-PCR.The sequencing results were compared with known results.Second,according to BPI gene sequence,new primers were designed to extend the segments from the 5′and 3′ends,respectively.Then the cloned fragments were recycled and ligated into pMD18-T vector before being transformed into DH5α.At last,masculine clones were detected and sequence analysis was conducted.【Result】The porcine BPI cDNA cloned was 1 922 bp in length(GenBank accession No:KF523344)and the open reading frame was 1 452 encoding 483aminoacids.BLAST analysis shows that homology of BPI amino acids sequence between Bashibai sheep and those of Ovis aries,Bos taurus,Orcinus orca,Sus scrofa,Homo sapiens and Oryctolagus were 99%,91%,78%,74%,64%,61%,58%,53% and 50%,respectively.Molecular phylogenetic tree analysis shows that Bashibai sheep assembled to Ovis aries,followed by bos,which was identical to the biological classification.【Conclusion】Sheep Bashibai BPI cDNA with length of 1 922 bp was successfully cloned using RACE method and the phylogenetic tree result was consistent with the biological classification.