[Cloning and expression of pokeweed antiviral protein-II gene from the summer leaves of Phytolacca amercana].

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.