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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 30 9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 .mex .)的无鞭毛体蛋白基因。结果 克隆了 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
Objective To clone the coding genes of amastin on the surface of four Leishmania parasite and to analyze the sequence. Methods Based on the similar genetic relationship between Tcruzi and Leishmania protospatii, the dbEST database in GenBank was searched with the gene of trypanosome immunoblastic protein as reference. major) a 30 9 nucleotide fragment was synthesized according to its sequence probe, Leishmania major genome DNA library screening, first Leishmania major flagellar body protein coding genes, and then Leishmania Leishmania Flagellar body protein coding gene sequence as the basis, the synthesis of specific primers, amplified by polymerase chain reaction (PCR) amplified Leishmania (L. Ma.), Leishmania brazil (L.bra.) And Mexico Immunoglobulin gene of L. Mex. Results Four genes encoding Leishmania amastigotes were cloned. Are the first cloned genes in the world, have been included in the United States GenBank. Conclusion Cloning of four leishmania protein coding genes was achieved