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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 ,可以作为候选药物靶标
In order to find a new anti-TB drug target, in vivo induced antigen technology was used to screen the in vivo induced genes of M. tuberculosis. First, we constructed a plasmid expression library of Mycobacterium tuberculosis genomic library with a capacity of 102 × 105CFU. A total of 16 positive clones were obtained by screening the genomic expression libraries by in situ immunoblotting with TB patient serum that had been adsorbed to the lysates of M. tuberculosis and E. coli. The positive clones were sequenced and analyzed by bioinformatics and found that the 16 positive clones may contain 22 open reading frames (ORFs). According to their function, they were divided into seven categories: two lipid metabolism, five signal pathways, two PE / PPE protein families, six intermediate and energy metabolism, one cell wall and cell treatment, four hypothetical proteins and Bovine Mycobacterium orientalis homologous 2, some of which may be related to virulence and can be used as a candidate drug target