HIV-1核衣壳蛋白NCp7抑制剂体外筛选方法的建立

目的表达HIV-1核衣壳蛋白(nucleocapside proteinp7,NCp7),并建立抑制剂筛选方法。方法从pBRU2/ADP202质粒经PCR得到HIV-1NCp7编码序列,克隆到pet28a质粒中构建HIV-1NCp7表达载体。阳性克隆转染E.coliBL21DE3,以IPTG诱导,细胞裂解液经Q SepharoseH.P和SP Sepharose H.P纯化后纯度达到90%以上。将具有锌离子逐出活性的H2O2和2,2′-联硫基二吡啶(2,2′-di-thiodipyridine,AT-2)与NCp7作用,用锌离子特异的荧光染料检测。用该方法检测KIZ-CM系列化合物。结果可以检测H2O2和AT-2的锌离子逐出活性,经该方法筛选,KIZ-CM10,18,19等化合物具有一定的锌离子逐出活性。结论在国内首次建立了HIV-1NCp7锌离子逐出方法,该方法可以用于NCp7抑制剂的筛选。

Establishment of a screening method for HIV-1 NCp7 inhibitors

Aim This study aimed to express HIV-1 NCp7(nucleocapside protein p7)and establish a method screening for inhibitors.Methods NCp7 protein coding sequence was amplified from plasmid HIV-1 pBRU2.The PCR product was inserted into the pet28a expression vector.Positive clone was selected and transformed into E.coli BL21 DE3.Cells were cultured in LB medium and induced with IPTG.NCp7 was purified by Q sepharose H.P.and followed by SP sepharose H.P.chromatography.The purity of the NCp7 protein reached above 90% after purification.Zinc ejection activity of H2O2 and AT-2 was analyzed by TSQ zinc ion indicator.Further,the zinc ejection activity of KIZ-CM series compounds was also analyzed.Results Zinc ejection activities of H2O2 and AT-2 could be measured and some of KIZ-CM compounds might eject the zinc ion from NCp7 protein.Conclusions The method can be applied to screen for inhibitors of HIV-1 NCp7 protein.