关键词: 杜氏利什曼原虫, 动基体DNA, 克隆

Abstract: To identify the heterogeneous DNA sequences in the kinetoplast DNA (kDNA) mini-circles, we digested the kDNA from various species and isolates of Leishmania into fragments with several restriction endonucleases and those fragments were southern hybridized with whole kDNA. By this test, the AluI fragments were shown to possess the species-and strain-specific sequences. We inserted these kDNA (from L. d. Sichuan human isolate) fragments into Smal site of plasmid pUC 18. The blunt ligation reaction was carried out with T4 DNA ligase supplemented by T4 RNA ligase. Tens of recombinants were obtained and at least 16 recombinants were shown to have the sequences of kDNA by colony hybridization with whole kDNA. The inserted kDNA sequences can be cut off from vector by Bam HI and EcoR I digestion. The recombinant DNA had no homolgous sequences with human genomic DNA, Leptospiral DNA, Romanomermis DNA and L. donovani genomic DNA. The clone pLKl-14, which is the smallest one among all the clones obtained, onlyhybridized to the L.d. Sichuan human isolate from which it was originated. When pLKl-14was digested by BamHI and EcoRI, a 180bp fragment comprising about 20 bp of pUC 18sequence, could be produced which corresponded to the 120±30 bp fragment of kDNA digested by Alul and was shown to be isolate specific. The clone of pLKl-10 hybridized to L. donovani isolates from hill and desert foci, might be used as a specific probe in the distinction of L.d. isolates from hill foci and plain foci. The clones pLKl-1, pLKl-2, and pLKl-15 were present in all isolates of visceral Leishmania but not in L. major and lizard Leishmania tested. These sequences might be used as specific probes in the diagnosis of visceral leishmaniasis and might be useful in epidemiological studies for identification of vectors and reservoirs.