摘要
体外培养巴西利什曼原虫 (Lb ,Leishmaniabraziliensis)株无鞭毛体 ,常规方法提取制备基因组DNA。以公开发表的婴儿利什曼原虫 (Li,Leishmaniainfantum)的激活蛋白激酶C受体RACK (receptorforactivatedproteinCkinase)基因核苷酸序列为参照 ,设计并合成利什曼原虫RACK基因序列特异性的引物。以巴西利什曼原虫的基因组DNA为模板 ,利用多聚酶链反应 (PCR)技术 ,扩增获得巴西利什曼原虫RACK的全长编码基因。基因序列测定结果表明 ,巴西利什曼原虫RACK基因序列长度为 10 2 5bp ,开放读码框架由 939bp组成 ,编码产物为 312个氨基酸残基。获得的巴西利什曼原虫的RACK基因与报导的婴儿利什曼原虫的RACK基因编码产物的序列同源性达 99% (310 312 )。本研究克隆了巴西利什曼原虫的RACK基因 ,为应用诱导T细胞免疫应答抗原的编码基因进行巴西利什曼原虫的基因疫苗研究奠定了基础。
Leishmania braziliensis promastigotes were cultured in vitro in Graces medium at 27℃ and the genomic DNA was prepared by routine methods. According to the published RACK DNA sequence from Leishmania infantum parasite,specific primers have been designed and synthesized. Polymerase chain reaction (PCR) was employed to amplify RACK DNA using genomic DNA from Leishmania braziliensis as the template. A 1025 bp DNA fragment was amplified,purified from agarose gel,and subcloned into pCR2 1 T vector. The DNA sequencing result indicated that full length DNA fragment encoding RACK protein of Leishmania braziliensis has been obtained,and the amino acid sequence of RACK protein from Leishmania braziliensis (LbRACK) is 99% (310/312) homology to that of Leishmania infantum (LiRACK). The RACK coding gene of Leishmania braziliensis consists of 939 bp and encodes a protein of 312 amino acid residues. The cloning of RACK from Leishmania braziliensis makes it possible to use this DNA fragment for the study of DNA vaccine against Leishmania braziliensis infection.
出处
《寄生虫与医学昆虫学报》
CAS
2001年第4期193-198,共6页
Acta Parasitologica et Medica Entomologica Sinica
