关键词: 杜氏利什曼原虫, 无鞭毛体蛋白, 基因克隆, 表达

Abstract: Objective To clone and express the amastin gene of two Leishmania donovani isolates from Sichuan Province of China. Methods Amastin gene was amplified from nuclear DNA of two L.donovani isolates， cloned into pcDNA3.1（+）， and sequenced by the dideoxy chain termination. NIH3T3 cell was transfected by recombinant plasmid. Transient expression of amastin gene was detected by immunofluoresence and stable expression was detected by RT-PCR and Western blot. Results Amastin gene of both isolates was 552 bp. Sequence analysis showed that the similarity was 86% between the two isolates. A high green fluorescence was found on the cell membrane and inside the cell. The NIH3T3 cell was transfected by the recombinant plasmid successfully. Amastin gene was obtained by RT-PCR from the transfected NIH3T3 cells. Western blot analysis showed that there was a protein about Mr 20 000 in lysate of the transfected NIH3T3 cells， indicating that the amastin gene was expressed in the cells. Conclusion Amastin gene of the two L.donovani isolates has been cloned and the gene can be expressed stably in the NIH3T3 cell.

Key words: Leishmania donovani, Amastin, Gene cloning, Expression