间日疟原虫海南株SSUrRNA编码基因的克隆及其序列分析

Cloning and sequencing SSUrRNA-encoding gene of Plasmodium vivax Hainan strain at blood stage

目的克隆间日疟原虫海南株红内期小亚基核糖体核糖核酸(SSUrRNA)编码基因片段,并进行序列特征分析。方法采用聚合酶链反应技术从海南间日疟患者血样DNA中扩增出间日疟原虫SSUrRNA基因片段,纯化后与pGEM-Teasy质粒连接构建重组子并转化大肠杆菌JM109;阳性克隆以双酶切和PCR鉴定后,进行序列测定,采用BLAST软件分析其特征。结果 PCR扩增出间日疟原虫红内期SSUrRNA基因特异性片段大小约为998 bp;阳性克隆重组质粒经双酶切及PCR鉴定与预期结果一致;核酸序列测定显示插入的SSUrRNA基因扩增片段,含有998个核苷酸,与GenBank中的间日疟原虫Sal 1株、Pv2008/TR/DEL株及El Salvador株相同序列进行比对,其同源性均大于99%。结论成功克隆间日疟原虫海南株红内期SSUrRNA编码基因序列,该序列在不同地理株间相对稳定保守。

Aim To clone and analyze the specific sequence of SSUrRNA-encoding gene fragment of a P.vivax isolate at blood stage from Hainan Province in China.Methods The SSUrDNA fragment was amplified by PCR from the DNA extractes of the P.vivax infection blood sample from Hainan Province.After purification,the gene fragment was ligated with plasmid pGEM-Teasy to construct a recombinant plasmid,which was transformed into E.coli JM109.Positive bacteria clones were identified by PCR and double enzymes digestion methods.The sequence of inserted SSUrDNA fragment was also determined and analyzed.Results The amplified SSUrDNA fragment was about-998 bp in length,as aligned with the P. vivax corresponding sequences of Sal 1,Pv2008/TR/DEL,and El Salvador strains deposited in the GenBank database,the indentity of nucleotides was more than 99%respectively.Conclusion The P.vivax SSUrRNA gene fragment sequence at blood stage was cloned,which was relative conserved among different P.vivax isolates.