杜氏利什曼原虫四川分离株无鞭毛体蛋白基因的克隆及真核表达

Cloning of Amastin Gene of Leishmania donovani Isolates from Sichuan Province and its Expression in Eucaryotic System

目的克隆、真核表达杜氏利什曼原虫四川株的无鞭毛体蛋白(amastin)编码基因。方法PCR扩增杜氏利什曼原虫四川汶川人株L．d．SC10H2与四川南坪犬株L．d．SC7的无鞭毛体蛋白基因,将该基因导入pcDNA3．1(+),构建真核表达重组质牲,转染NIH3T3细胞,采用免疫荧光法鉴定重组质粒的瞬时表达;RT-PCR和Western blotting鉴定稳定表达,结果2株杜氏利什曼原虫均扩增出552 bp的无鞭毛体蛋白基因。同源性为86％。转染后在NIH3T3细胞膜和细胞内观察到较强的绿色荧光,表明无鞭毛体蛋白基因在NIH3T3细胞中获得短暂表达。细胞裂解产物经Western blotting,在相对分子质量(Mr)约20000处检测到阳性杂交信号,表明无鞭毛体蛋白基因在NIH3T3细胞内获得了稳定表达。结论获得了我国杜氏利什曼原虫四川分离株L．d．SC10H2和L．d．SC7的无鞭毛体蛋白基因序列,并在NIH3T3细胞中稳定表达。

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 20000 in lysate of the transfected NIH3T3 ceils, indicating that the amastin gene was expressed in the ceils. Conclusion Amastin gene of the two L.donovani isolates has been cloned and the gene can be expressed stably in the NIH3T3 cell.