【摘要】 根据报道的cDNA序列,用RT-PCR的方法从美洲商陆夏季的叶片中克隆美洲商陆抗病毒蛋白-Ⅱ(pokeweedantiviralproteinⅡ,PAP-Ⅱ)基因。将PAP-Ⅱ基因克隆至表达载体pET-28a(+)并在大肠杆菌中表达,SDS-PAGE电泳分析结果表明,PAP-Ⅱ蛋白在BL21(DE3)菌中获得表达,表达产物以不溶性包涵体形式存在,经过溶解包涵体、复性和BBSTNTA树脂柱亲和层析纯化,获得高纯度的PAP-Ⅱ蛋白。用非放射性基于ELISA方法检测经过复性纯化后PAP-Ⅱ蛋白和蓖麻毒素A链(RTA)在体外对HIV-1整合酶有较强的抑制活性,其IC50分别约为303μg/mL,220μg/mL。用MTT法分析PAP-Ⅱ蛋白的生物学活性,复性纯化后蛋白对HEP-G2和Hela细胞有细胞毒作用,IC50分别为93μg/mL,102μg/mL,说明了PAP-Ⅱ蛋白能抑制肿瘤细胞的生长。构建的PAP-Ⅱ表达系统所表达的蛋白经复性后具有生物学活性,为进一步研究PAP-Ⅱ的抗HIV-1机制和抗肿瘤作用奠定了基础。更多还原

【Abstract】 The cDNA sequence encoding pokeweed antiviral protein-Ⅱ was cloned from the fresh summer leaves of phytolacca amercana by RT-PCR. The recombinant PAP-Ⅱ was subcloned into the expression vector pET-28a(+) and expressed in E.coli BL21 after IPTG induction. SDS-PAGE analysis showed that the expressed PAP-Ⅱ existed in the form of inclusion bodies. The purified fusion protein was obtained after a series of steps including cell break, inclusion body solubilization, protein refolding and purification through BBST NTA resin column. The non-radioactive ELISA-based HIV-1 integrase assay showed that the recombinant pokeweed antiviral protein-Ⅱ and RTA were able to inhibit HIV-1 integrase to some extent(IC- 50 =303μg/mL, 220μg/mL respectively). MTT assay showed that cytotoxicity of pokeweed antiviral protein Ⅱ for HEP-G2 cells and Hela cells was in a dose-dependent manner with IC- 50 s of 93μg/mL and 102μg/mL, respectively. The results suggested that pokeweed antiviral protein-Ⅱ is a potent anti-tumor candidate. The finding of integrase inhibitory activity and the discovery of cytotoxicity provide more insights into the anti-HIV and the anti-tumor activities of PAP-Ⅱ.更多还原