伊氏锥虫HGPRT基因的RT-PCR扩增及克隆

RT PCR Amplification and Cloning of HGPRT Gene of Trypanosoma evansi

参照布氏锥虫次黄嘌呤鸟嘌呤磷酸核糖转移酶 ( HGPRT)基因的核苷酸序列 ,设计合成了 1对引物 ,引物间距为 63 0 bp,包含完整的 HGPRT基因。以伊氏锥虫湖北水牛株总 RNA为模板进行 RT-PCR扩增 ,琼脂糖凝胶电泳显示 ,获得 1条长约 63 0 bp的特异性条带 ,符合设计要求。扩增片段克隆于 p GEM-T Easy载体 ,经筛选鉴定 ,证明已获得了 HGPRT基因阳性克隆。核苷酸序列分析表明 ,克隆的 HGPRT基因在重组质粒中的连接向位和阅读框架是正确的。二级结构和疏水性分析表明 ,HGPRT基因产物具有复杂的空间结构 。

One pair of PCR primer for Trypanosoma evansi HGPRT gene was designed according to the HGPRT gene sequence of Trypanosoma brucei. With the primers, HGPRT gene(about 630 bp)was successfully amplified by RT PCR. The amplified fragment was cloned into pGEM T Easy vector. The recombinant plasmid was identified by PCR amplification test and nucleotide sequencing. The results showed that HGPRT gene fragment has been cloned with a positive reading frame. The analysis of the secondary structure indicated that the HGPRT gene had a complex space structure which would be a useful antigen.