利什曼原虫无鞭毛体蛋白的基因克隆化与序列分析

摘要

Objective To clone amastin coding genes from different strains of Leishmania parasites. Methods Using amastin cDNA sequence as the reference, dbEST data base established by National Center Biotechnology Information (NCB1), USA, was searched by BLAST tool. A 309 bp DNA fragment of Leishmania major was found and used as the probe for the screening of a DNA library. The amastin gene of Leishmania major Abdou was cloned and sequenced. Specific primers were designed and amastin genes for Leishmania mexicana WR972, Leishmania brizeliensis and Leishmania amazonensis joseph were amplified by polymerase chain reaction. Results The amastin genes from 4 strains of Leishmania parasites were cloned and sequenced. It was found that all 4 amastin genes contained unique open reading frame of 552 bp and encoded amastin protein of 183 amino acid residues. Conclusion The amastin genes of 4 strains of Leishmania parasites were successfully cloned.%目的克隆4株利什曼原虫表面无鞭毛体蛋白(amastin)的编码基因，并进行序列分析。方法根据锥虫(T. cruzi)与利什曼原虫亲缘关系相近的原则，首先以锥虫无鞭毛体蛋白的基因为参考，对GenBank中的dbEST数据库检索，获得硕大利什曼原虫(L.major)一段309核苷酸片段，根据其序列合成探针，对硕大利什曼原虫基因组DNA文库筛选，首先获得硕大利什曼原虫无鞭毛体蛋白编码基因，再以硕大利什曼原虫无鞭毛体蛋白编码基因序列为依据，合成特异性引物，以多聚酶链反应(PCR)扩增获得亚马逊利什曼原虫(L.ama.)、巴西利什曼原虫(L.bra.)和墨西哥利什曼原虫(L mtx.)的无鞭毛体蛋白基因。结果克降了4株利什曼原虫无鞭毛体蛋白编码的基因。均为国际上首次克隆化基因，已被美国GenBank收录。结论实现了4株利什曼原虫无鞭毛体蛋白编码基因的克隆化。