杜氏利什曼原虫激活蛋白激酶C受体的基因克隆化与序列分析

Cloning and sequence analysis of a receptor of activated protein C kinase (RACK) conding gene from Leishmania donovani 1S

目的 克隆杜氏利什曼原虫 (LeishmaniadonovaniLd) 1S株激活蛋白激酶C受体 (RACK ,receptorofactivatedpro teinCkinase)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的研究奠定基础。方法 体外培养杜氏利什曼原虫 1S株无鞭毛体 ,常规方法提取制备基因组DNA。以硕大利什曼原虫 (Leishmaniaamjor)的RACK基因的核苷酸序列为参照 ,设计并合成利什曼原虫RACK基因序列特异性的引物。以杜氏利什曼原虫的基因组DNA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RACK的全长编码基因。结果 基因序列测定结果表明 ,杜氏利什曼原虫 1S株RACK基因序列长度为 981bp ,开放读码框架由 831bp组成 ,编码产物为 2 76个氨基酸残基。获得的杜氏利什曼原虫 1S株的RACK基因与来源于硕大利什曼原虫的RACK基因序列同源性达 98% (2 6 4 / 2 6 7)。结论 本研究克隆了杜氏利什曼原虫的RACK基因 。

Aim To clone receptor of activated protein C kinase (RACK) coding gene from Leishmania donovani 1S parasite.Method The Leishmania donovani 1S promastigotes were cultured in vitro in Grace's medium at 27℃.The genomic DNA was prepared by a routine method.According to the published RACK DNA sequence from Leishmania major parasite,specific primers have been designed and synthesized.Polymerase chain reaction(PCR) was employed to amplify RACK DNA using genomic DNA from Leishmania donovani 1S as the template.Results 981 bp DNA fragment was amplified and purified from agarose gel and subcloned into pCR2.1 T vector. The DNA sequencing result indicated that we have got full length DNA fragment coding for RACK protein of Leishmania donovani 1S,and that the coding sequence is 98% homology to RACK gene of Leishmania major(LmRACK).The RACK coding gene of Leishmania donovani 1S consists of 831 bp and encodes a protein of 276 amino acid residues.Conclusion The cloning of RACK from Leishmania donovani 1S made it possible to use this DNA fragment in the study of DNA vaccine against Leishmania donovani infection.