摘要
为寻找新型抗结核药物靶标 ,采用体内诱导抗原技术筛选结核分枝杆菌的体内诱导基因。首先构建了结核分枝杆菌基因组质粒表达文库 ,库容量为 1 0 2× 1 0 5CFU。再用经过结核分枝杆菌和大肠杆菌的裂解产物吸附过的结核病人血清 ,通过原位免疫印迹来筛选基因组表达文库 ,共获得 1 6个阳性克隆。对阳性克隆进行测序和生物信息学分析 ,发现该 1 6个阳性克隆可能包含 2 2个开放阅读框 (ORF)。按照功能将其分为 7类基因 :脂类代谢 2个、信号途径 5个、PE/PPE蛋白家族 2个、中间产物与能量代谢 6个、细胞壁与细胞处理 1个、假想蛋白 4个和与牛型分枝杆菌定向进化同源的 2个 ,其中部分基因可能与毒力相关 。
In order to search new candidates of pharmaceutical target,in vivo induced antigen technology(IVIAT)was used to screen in vivo induced (ivi) genes of Mycobacterium tuberculosis(M.TB).Genomic expression library of M.TB was first constructed with an inducible plasmid pKK223-8;the titer of the library was 1.02×10~5CFU.Sera from ten tuberculosis patients were pooled and absorbed against in vitro-grown M.TB and Escherichia coli,and used to probe the genomic expression library.16 positive clones were identified by immunological screen,including 22 ORF: two encoding lipid metabolism proteins,five information pathways proteins,two PE/PPE proteins,six intermediary metabolism and respiration proteins,one cell wall and cell processes protein,four conserved hypothetical proteins and two conserved hypothetical proteins with an orthologue in Mycobacterium bovis.Parts of these genes can be used as candidates of pharmaceutical target because they may be relate with virulence.
关键词
结核分枝杆菌
体内诱导抗原技术
药物靶标
毒力
Mycobacterium tuberculosis
in vivo induced antigen technology(IVIAT)
therapeutic target
virulence
