杜氏利什曼原虫无鞭毛体蛋白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].
作者信息
Li Jin-Fu, Chen Jian-Ping, Yang Zhi-Wei, Tian Yu, Ma Ying, Hu Xiao-Su
机构信息
Department of Parasitology, School of Preclinical and Forensic Medicine, Sichuan University, Chengdu, China.
出版信息
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2007 Mar;23(3):217-9.
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 RT-PCR.
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.
目的
构建杜氏利什曼原虫无鞭毛体蛋白基因的重组真核表达质粒，并检测该基因在NIH3T3细胞中的表达。
方法
从杜氏利什曼原虫分离株的核DNA中扩增无鞭毛体蛋白基因，并克隆到真核表达载体pcDNA3.1(+)中。重组质粒命名为pcDNA3.1-无鞭毛体蛋白。用pcDNA3.1-无鞭毛体蛋白转染NIH3T3细胞。通过免疫荧光和逆转录-聚合酶链反应检测无鞭毛体蛋白基因的瞬时和稳定表达。
结果
发现细胞膜和细胞内有高绿色荧光。表明pcDNA3.1-无鞭毛体蛋白成功转染了NIH3T3细胞。
结论
成功构建了杜氏利什曼原虫无鞭毛体蛋白基因的重组真核表达质粒，且能在NIH3T3细胞中稳定表达。
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