恶性疟原虫红内期SSUrRNA编码基因的克隆与序列分析

目的 克隆恶性疟原虫海南株红内期小亚单位核糖体核糖核酸SSUrRNA编码基因(SSUrDNA)片段，并进行序列分析。方法根据献报道恶性疟原虫基因序列设计1对特异性引物，采用聚合酶链反应(PCR)方法从海南恶性疟患血样核酸提取物中扩增出恶性疟原虫SSUrDNA目的基因片段，纯化后与PUC19m-T质粒载体连接构建重组子，并导入大肠杆茵JM109；阳性克隆经双酶切鉴定后，双脱氧末端终止法测定序列，并采用ExPASy Proteomicstools软件分析。结果 恶性疟原虫SSUrDNA扩增片段大小约为431bp；阳性克隆重组质粒作双酶切及PCR扩增均得到预期大小的基因片段；测定的SSUrDNA插入片段核酸序列与巴西恶性疟原虫IMTM／7G8相同基因序列对比。未发现碱基的插入或缺失去，同源性为99．3％；第353位碱基由C取代了G，第371位碱基由T取代了C，其余序列相同。结论 成功克隆了恶性疟原虫海南株红内期SSUrDNA基因片段，该序列在不同恶性疟原虫虫株间相对保守。

Cloning and sequencing of SSUrRNA encoding gene fragment of Plasmodium falciparum from Hainan strain

Objective To clone and analyze the sequence of blood stage specific SSUrRNA gene fragment of a P. falciparum isolate from Hainan Province in China. Methods The SSUrDNA gene fragment was amplified by PCR from the extracts of the P. falciparum infection blood sample from Hainan Province. After purification, the gene fragment was ligated with plasmid PUC19m-T vector to construct a recmbinant plasmid,which was transformed into E. coli JM109.Positive bacteria clones were screened and identi-fid by PCR method and digestion with double restriction enzyme Pst I and BamH I. The sequence of inserted SSUrDNA gene fragment was also determined and analyzed. Results The 431 bp SSUrDNA gene fragmen was amplified from the extracts of the P .falciparum infection blood sample and cloned into the plasmid PUC19m-T.The sequence of cloned SSUrDNA gene fragment was 99.3% i-dentical as aligned with that of P. falciparum IMTM 22/7G8 clone (Brazil) . Conclusion The SSUrDNA gene fragment of P. falciparum isolate from Hainan Province was cloned ,which was relatively conserved among different P. falciparum isolates.