论文部分内容阅读
本文使用化学合成的吡咯并乙氨嘧啶作为双氢叶酸还原酶(DHFR)的抑制剂,使用点突变技术模拟乙胺嘧啶和环鸟嘌呤抗性恶性疟原虫株。 0.9kb的SphⅠ/BamHI pET-pfDHFR231的片段被亚克隆入M13mp18,其单链DNA作为直接点突变的模板。用放射性同位素在M13mp18中产生突变,并与质粒PET-3a连接,再进行质粒重组并用双脱氧终止法证实构建者无其它突变的存在,人DHFR基因由PCR扩增获得,扩增的DNA用BamHI酶解后插入pUC19中,以此来检验试验的正确性。构建的多种PET-pf DHFR在大肠杆菌系统中表达,人重组的DHFR与E.coliDHFR均用氯甲蝶呤亲和层析法加以纯化,
In this paper, pyrroloethyl pyrimidine was synthesized as an inhibitor of dihydrofolate reductase (DHFR) and point-mutagenesis was used to simulate pyrimethamine and cycloguanine-resistant P. falciparum strains. The 0.9 kb fragment of SphI / BamHI pET-pfDHFR231 was subcloned into M13mp18 with the single-stranded DNA as a template for direct point mutation. A radioisotope was used to generate mutations in M13mp18 and ligated to plasmid PET-3a. Plasmid recombination was performed and no other mutations were confirmed by dideoxy termination. The human DHFR gene was amplified by PCR. The amplified DNA was digested with BamHI After digestion inserted into pUC19, in order to test the correctness of the test. The constructed multiple PET-pf DHFRs were expressed in E. coli system. Both human recombinant DHFR and E. coli DHFR were purified by methotrexate affinity chromatography,