杜氏利什曼原虫蛋白磷酸酶-2C的基因克隆化与序列分析

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

CLONING AND SEQUENCE ANALYSIS OF A PROTEIN PHOSPHATASE 2C CODING GENE FROM LEISHMANIA DONOVANI 1S

Aim To clone protein phosphatase 2C(PP2C)gene from Leishmania donovani 1S parasite.Methods The Leishmania donovani1S promastigotes were cultured in vitro in Grace's medium at 27℃.The genomic DNA was prepared by a routine method.According to the published PP2C DNA sequence Leishmania chagasi,specific primers were designed and PCR was conducted using DNA from Leishmania donovani1S as the template.Results 1317 bp DNA fragment was amplified and purified from agarose gel,and subcloned into pCR2.1 T vector.The DNA sequencing result indicated that we had got full length DNA fragment coding for PP2C protein of Leishmania donovani1S,and the coding sequence was 95% homology to PP2C gene of Leishmania chagasi(LcPP2C).The PP2C coding gene consists of 1221 bp and encodes a protein of 406 amino acid residues.Conclusion The cloning of PP2C from Leishmania donovani1S made it possible to use the DNA fragment in the study of DNA vaccine against Leishmania donovani infection.