日本血吸虫SjPGK基因克隆和序列分析

CLONING AND SEQUENCE OF PHOSPHOGLYCERATE KINASE GENE OF SCHISTOSOMA JAPONICUM

目的 克隆日本血吸虫磷酸甘油酸激酶 (Sj PGK)编码基因片段 ,分析其核苷酸序列。方法 根据曼氏血吸虫磷酸甘油酸激酶 (Sm PGK) c DNA序列设计并合成一对引物 ,以日本血吸虫成虫总 RNA为模板 ,采用逆转录 -聚合酶链反应 (RT- PCR)特异性扩增 Sj PGK基因片段 ,将其克隆入p MD18- T载体中 ,经双酶切分析和 PCR鉴定 ,将阳性克隆进行脱氧核糖核酸序列测定 ;运用BL AST程序 ,将测序结果及其推导的编码氨基酸序列与 NCBI数据库在核苷酸水平和氨基酸水平进行同源性比较。结果 RT- PCR特异性扩增出一条长约 830 bp的条带 ;重组质粒的双酶切和以其脱氧核糖核酸为模板的 PCR均可见一条与 RT- PCR产物相同的条带 ;脱氧核糖核酸序列测定和分析结果表明 :Sj PGK基因片段长为 830 bp,与 Sm PGK的核苷酸同源性为 85 % ,分值为 6 72 ;氨基酸同源性为 94 % ,分值为 4 73。结论 成功地克隆了 Sj PGK编码基因片段 。

Objective To clone and sequence the partial gene of Schistosoma japonicum phosphoglycerate kinase (SjPGK). Methods A pair of primers were designed and synthesized according to the cDNA sequence of Schistosoma mansoni phosphoglycerate kinase (SmPGK) gene. The gene fragment of SjPGK was amplified and isolated from the total RNA of S.japonicum by reverse-transcription polymerase chain reaction (RT-PCR). The gene fragment was cloned into the cloning vector of pMD18-T, The positive clones were acquired and identified with restrictive enzymes and PCR amplification. After being sequenced with DNA auto-sequence analysis instrument,the cDNA sequence of SjPGK was searched for homologue identity with NCBI BLAST program. Results The gene encoding SjPGK was obtained and isolated by RT-PCR .The fragment of SjPGK was about 830 bp.The cDNA sequence of the phosphoglycerate kinase was highly homologous between Schistosoma mansoni and Schistosoma japonicum. The identity of nucleotide sequence was 85% and score 672, and the identity of amino sequence was 94% and score 473. Conclusion The partial gene of encoding SjPGK is cloned into the cloning vector of pMD18-T, which gives the basis for discovering new candidate vaccine molecular for schistosomiasis.