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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。
Objective To clone the small subunit ribosomal RNA (SSUrRNA) gene fragment of Plasmodium vivax in mid - red blood stage and analyze its sequence characteristics. Methods Polymerase chain reaction (PCR) was used to amplify the fragment of SSUrRNA gene of Plasmodium vivax in blood samples from a Japanese malaria patient in Hainan province. After purification, the recombinant plasmid was ligated with pGEM-Teasy plasmid and transformed into E. coli JM109. The positive clones were double digested After PCR and identification, sequence analysis was carried out and its characteristics were analyzed by BLAST software. Results The specific fragment of SSUrRNA gene was about 998 bp in length during PCR amplification. The positive clones were identified by double enzyme digestion and PCR. The results of nucleic acid sequencing showed that the inserted SSU rRNA gene amplified fragment , Containing 998 nucleotides, was aligned with the same sequence of Plasmodium vivax Sal 1, Pv2008 / TR / DEL and El Salvador in GenBank and the homologies were all greater than 99%. Conclusion The sequence of the SSUrRNA coding gene of P. vivax in Hainan was successfully cloned, which was relatively stable and conserved among different geographical isolates.