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目的 克隆杜氏利什曼原虫 (Leishmaniadonovani,Ld) 1S株蛋白磷酸酶 2C(PP2C)的编码基因 ,为应用这种编码T细胞抗原的基因进行基因疫苗研究奠定基础。方法 体外培养杜氏利什曼原虫 1S株无鞭毛体 ,常规方法从虫体提取制备基因组DNA。以夏科氏利什曼原虫 (Leishmaniachagasi,Lc)的PP2C基因序列为参照 ,设计并合成利什曼原虫PP2C基因序列特异性的引物。结果 以杜氏利什曼原虫的基因组为模板 ,利用多聚酶链反应 (PCR)技术 ,扩增获得了杜氏利什曼原虫PP2C的全长编码基因。基因序列测定结果表明 ,杜氏利什曼原虫 1S株PP2C基因序列长度为 1317bp ,开放读码框架由 12 2 1bp组成 ,编码产物为 40 6个氨基酸残基。获得的杜氏利什曼原虫 1S株的PP2C基因与来源于夏科氏利什曼原虫的PP2C氨基酸残基序列的同源性为 95 % (387/ 40 6 )。结论 本研究克隆了杜氏利什曼原虫的PP2C基因 ,为应用诱导T细胞免疫应答抗原的编码基因进行杜氏利什曼原虫的基因疫苗研究奠定了基础
Objective To clone the gene encoding protein phosphatase 2C (PP2C) from Leishmaniadonovani (Ld) 1S strain and lay the foundation for gene vaccine research using this gene encoding T cell antigen. Methods Leishmania donovani strain 1S was cultured in vitro. The genomic DNA was extracted from the worms by routine methods. Based on the sequence of PP2C gene of Leishmaniachagasi (Lc), the sequence-specific primers of leishmania PP2C gene were designed and synthesized. Results The genome of Leishmania donovani was used as a template to amplify the full-length coding gene of Leishmania donovani PP2C by polymerase chain reaction (PCR). The results of gene sequencing showed that the sequence of PP2C gene of Leishmania donovani strain 1S was 1317 bp in length and the open reading frame consisted of 12 2 1bp. The encoded product was 40 6 amino acid residues. The homology between the PP2C gene of Leishmania donovani strain 1S and the PP2C amino acid residue sequence from Leishmania pastoris was 95% (387/40 6). Conclusion This study cloned PP2C gene of Leishmania donovani and laid the foundation for gene vaccine research of Leishmania donovani using the gene encoding T cell immune response antigen