杜氏利什曼原虫amastin基因重组真核表达质粒的构建与表达  

Construction and expression of recombinant eucaryotic expression plasmid of amastin gene of Leishmania Donovani

作　　者：李金福[1] 陈建平[1] 杨志伟[1] 田玉[1] 马莹[1] 胡孝素[1] 

机构地区：[1]四川大学华西基础医学与法医学院寄生虫学教研室,四川成都610041

出　　处：《细胞与分子免疫学杂志》2007年第3期217-219,222,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金资助项目(39870656)

摘　　要：目的:构建杜氏利什曼原虫无鞭毛体蛋白(amas-tin)编码基因的真核表达重组质粒pcDNA3.1-amastin,并研究其在NIH3T3细胞中的表达。方法:提取杜氏利什曼原虫基因组DNA进行PCR扩增。将扩增的无鞭毛体蛋白基因片段导入质粒pcDNA3.1(+)中,构建真核表达重组质粒pcDNA3.1-amastin。以pcDNA3.1-amastin转染NIH3T3细胞,采用免疫荧光染色法和RT-PCR分别鉴定pcDNA3.1-amastin的瞬时表达和稳定表达。结果:在细胞膜和细胞内均观察到较强的绿色荧光,表明pcDNA3.1-amastin成功地转入NIH3T3细胞,并在细胞膜和细胞内获得短暂表达。稳定转染的NIH3T3细胞的总RNA经反转录后,用PCR扩增出无鞭毛体蛋白基因,表明获得了稳定表达。结论:成功地构建杜氏利什曼原虫无鞭毛体蛋白基因的真核表达重组质粒,并且该基因在NIH3T3细胞中获得了稳定表达。AIM： To construct recombinant eukaryotic expression plasmid of amastin gene of Leishmania Donovani and detect expression of the gene in NIH3T3 cells. METHODS： Amastin gene was amplified from nuclear DNA of Leishmania Donovani isolates and cloned into an eukaryotic expression vector pcDNA3. 1 （ ＋ ）. The recombinant plasmid was named pcDNA3.1-amastin. NIH3T3 cell was transfected by pcDNA3. 1-amastin. Transient and stable expression of amastin gene were detected by immunofluoresence and RTPCR. RESULTS： It was found that there was high green fluorescence on the cell membrane and inside the cell. It showed that NIH3T3 cell was transfected by pcDNA3.1- amastin successfully. CONCLUSION： A recombinant eukaryotic expression plasmid of amastin gene of Leishmania Donovani was successfully constructed, and can be expressed stably in the NIH3T3 cells.

关 键 词：杜氏利什曼原虫 无鞭毛体蛋白基因 体外表达 

分 类 号：R382.22[医药卫生—医学寄生虫学]