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为了实现HIV-1整合酶蛋白核心区(central core domain of integrase,IN-CCD)的可溶性表达,并建立以IN-CCD为靶点的抑制剂体外筛选方法,从包含F185K突变HIV-1 IN基因的质粒中经PCR扩增得到含有F185K突变的IN-CCD基因,克隆到pET28b载体上构建重组质粒pIN-CCD,转化pIN-CCD至E.coli BL21(DE3)中经IPTG诱导、表达,Ni-亲和层析纯化,获得IN-CCD蛋白。修饰DNA底物,以链亲和素包被的磁珠为载体捕获DNA产物,结合酶联免疫吸附测定法(ELISA)检测IN-CCD的去整合活性,并筛选以IN-CCD为靶点的抑制剂。结果表明重组蛋白IN-CCD实现了高效可溶性表达,纯化后蛋白纯度达95%。建立的ELISA可以检测IN-CCD的去整合活性,且方法特异性和灵敏度好,可以实现高通量抑制剂筛选。从100个样品中筛选得到5个具有初步抑制IN-CCD去整合活性的样品。
In order to achieve the soluble expression of HIV-1 core region integrase (IN-CCD) and establish an in vitro screening method using IN-CCD as a target, The recombinant plasmid pIN-CCD was cloned into pET28b vector and transformed into E.coli BL21 (DE3) for expression in E.coli BL21 (DE3) Purification by affinity chromatography gave IN-CCD protein. DNA substrate was modified, and streptavidin-coated magnetic beads were used as capture vector to capture DNA. The de-integration activity of IN-CCD was detected by enzyme-linked immunosorbent assay (ELISA) Inhibitors. The results showed that the recombinant protein IN-CCD to achieve highly efficient soluble expression, purity of 95% protein purity. The established ELISA can detect the de-integration activity of IN-CCD, and the specificity and sensitivity of the method can be used to screen high-throughput inhibitors. Five samples from 100 samples were screened for their initial inhibitory IN-CCD deconjugation activity.