伊氏锥虫对安锥赛抗药性的分子基础初探

ANALYSIS OF ANTRYCIDE-RESISTANT MOLECULES OF TRYPANOSOMA EVANSI

为了探讨伊氏锥虫对安锥赛抗药性的分子基础 ,将伊氏锥虫单虫克隆 ,一部分克隆锥虫通过 2 8只免疫抑制小白鼠 ,用安锥赛亚治疗剂量治疗 ,使其产生抗药性 ,为抗药锥虫 ;另一部分克隆锥虫也通过 2 8只免疫抑制小白鼠 ,但不用药物治疗 ,为敏感锥虫 ,即对照。用体外生长抑制法测得抗药和敏感锥虫的 IC5 0 分别为 43.8和0 .16 878μg/ m L。分别提取它们的总 RNA和 m RNA,总 RNA分离出 3条带 ,即从电泳的阴极到阳极分别为 2 6 s、2 1s和 5 s,m RNA主要分布在 2 1s RNA之后。用抑制性消减杂交法 ,以敏感锥虫的 c DNA为试验组 (Tester) ,抗药锥虫的 c DNA为驱动组 (Driver) ,共挑选出 34个阳性克隆。以 c DNA来合成探针 ,用 Southern dot blot鉴定 ,其中18个片段所在的基因是由于抗药性产生而表达量降低 ,这 18个片段长度为 30 0～ 70 0

The antrycide-resistant trypanosome line was from the cloned T.evansi through the 28 immunosupressed mice(cycloposphamide)treated with subsure antrycide by i.p. The sensitive trypanosome line also was from the cloned T.evansi through the 28 immunosuppressed mice treated without drug.Their IC50 were 43.81 and 0.16878μg/mL,respectively,used the in vitro assay.On the other hand,the PolyATtract R mRNA isolation systems was used to elute their total RNA and mRNA. The cDNA of sensitive trypanosome line was as Tester and the cDNA of resistant trypanosome line was as Driver. The SSH technique was preformed to have elected the 34 differentinally expressed cDNA in the tester cDNA.The 18 cDNA fragments were selected from them used the Southern dot blot by the probes from the cDNA.The range of their length was 300~700bp.