东方田鼠骨髓基因池的构建及抗日本血吸虫抗性相关基因的筛选  

Construction of Gene Pool of Microtus fortis and Screening Resistance Associated Genes to Infection of Schistosoma japonicum

作　　者：秦志强[1] 胡维新[1] 邬国军[1] 许冰[1] 申群喜[1] 龚强[1] 吴驰[1] 

机构地区：[1]中南大学湘雅医学院分子生物学研究中心,中国湖南长沙410078

出　　处：《生命科学研究》2004年第4期333-338,共6页Life Science Research

基　　金：国家自然科学基金资助项目(30070403);国家自然科学基金资助项目(30400256)

摘　　要：利用表达克隆法,从东方田鼠骨髓细胞中克隆出抗日本血吸虫抗性相关基因.首先提取高质量的mRNA,逆转录成cDNA,将cDNA与哺乳动物细胞瞬时表达载体pcDNA1.1/Amp连接,建立表达cDNA文库;将cDNA文库分成A-H8个基因池,转染HEK293细胞,48h后收集培养上清,获得条件培养基.将条件培养基与日本血吸虫童虫一起培养,观察杀虫效应,取对童虫抑制作用最强的基因池E再分成8个亚基因池E1-8,分别转染HEK293细胞,并将条件培养基与血吸虫童虫一起培养,获得具有明显抑制血吸虫童虫活性的亚基因池E77,按上述方法反复进行筛选,直到获得有抑制作用的单个克隆,该技术的建立为克隆东方田鼠抗日本血吸虫抗性相关基因以及研究其作用机制奠定基础.In order to screen and clone the resistance associated genes to Schistosoma japonicum infection in Microtus fortis, mRNA was isolated from bone marrow cells of Microtus fortis and cDNA was synthesized, which was linked to a kind of special adaptors and amplified by polymerase chain reaction(PCR).The cDNA library was constructed by ligating cDNA with mammalian expression vector pcDNA1.1/Amp through EcoRⅠsticky ends. The total clones of expression library were transferred in stiu to nylon membrane, which was divided into eight equals(A H) to set up gene pool and cultured in LB medium. Corresponding mixed plasmids of A H gene pools were extracted. Using Lipofect 2000 Reagent, transient expression of gene pool was performed in HEK293. The empty vector plasmid were used as negative control, and the supernatant of cell culture was collected 48 hours after transfection, which was called as conditional media. The schistosomula then were cultured in conditional media up to 96 hours. The number of schistosomula was counted and death rate was calculated. The inhibition of schistosomula with some gene pools was significant compare with the negative control. The gene pool with the strongest activity to schistosomula was divided to eight subpools for next screening. Repeated the screening above, the gene pool and subpools E, E7, E77 were collected. The second subpool E77 has the stronger activity than others. Therefore, it can be concluded that the method which may lay foundation to clone resistance associated gene and to reveal the resistance mechanism of Microtus fortis to Schistosoma japonicum infection.

关 键 词：东方田鼠 基因池 表达克隆法 日本血吸虫 抗性相关基因 

分 类 号：Q786[生物学—分子生物学]