Cloning And Expression Of Pokeweed Antiviral Protein-Ⅱ Gene Form The Summer Leaves Of Phytolacca Amercana

[Objective]Pokeweed antiviral proteins have been isolated and purified from pokeweed plant (Phytolacca Americana) in different tissues and at different stages of plant development They belong to the type I ribosome-inactivating proteins (RIPs), consisting of a single polypeptide chain and possessing specific N-glycosidase activity. PAP-II is a natural occurring protein isolated from the summer leaves of the pokeweed (phytolacca amercana) plant. Several isoforms of PAP were isolated from spring leaves (PAP-I), roots (PAP-R) of the pokeweed plant and seeds (PAP-S) of the pokeweed plant Pokeweed antiviral protein II inactivate both eukaryotic and prokaryotic ribosomes by removing A4324 from 28S rRNA and A2660 from 23S rRNA and interferring the elongation factor-2 catalyzed GTP hydrolysis, resulting in arrest protein synthesis at the translation. Present studies showed that PAP-II RIPs inhibit intracellular replication of several viruses, such as HIV and also have been used in cancer therapy as the active moiety of immunotoxins that target cancer cells. In this article, we report that PAP-II gene has been successfully cloned from the summer leaves of Phytolacca Americana and expressed in E. coll [Methods]The cDNA sequence, encoding PAP-II was cloned from the summer leaves ofphytolacca amercana by RT-PCR. Hind. Ⅲ and Nde I site were introduced at the upstream and downsteam primer. The nucleotide sequences of the target DNA amplification fragment were sequenced after T-A cloning. The fragment of PAP-II was digested with restriction endonucleases Nde I and Hind III and was subcloned into the expression vector pET-28a-c(+). E. coli strain BL21 transformants, containing the recombinant plasmid were grown in LB at 37℃ to an optical density of 0.7~0.9 then IPTG was added to induce expression. SDS-PAGE analysis showed that recombinant PAP-II gene was expressed in E.coli. PAP-II expression was in the form of inclusion bodies. The fusion protein was purified after a series of steps including cell sonication, solubilization of inclusion body, renaturation and purification through BBST NTA resin column. The non-radioactive ELISA-based HIV-1 integrase assay was performed to investigate the activity of the recombinant pokeweed antiviral protein-II and RTA inhibiting HTV-1 integrase. The cytotoxicity of pokeweed antiviral protein C for HEP-G2 cells and Hela cells was measured through MTT assay in HEP-G2 cells and Hela cells following fluid-phase endocytosis. [Results]In comparison with the reported pokeweed antiviral protein-II sequence, the nucleotide sequence homology of the cloned pokeweed antiviral protein-II gene was 100 %. The plasmid encoding pET-PAP-II extracted from a single colony was digested with Nde I and BamR I. The expression product was identified by 12% SDS-PAGE, it mainly existed in inclusion bodies. The renatured and purified protein showed a single band on 12 % SDS-PAGE. The non-radioactive ELISA-based HTV-1 integrase assay showed that the recombinant pokeweed antiviral protein-II and RTA were able to inhibit HTV-l integrase to some extent(IC5o=3O3 ug/mL, 220 ug/mL respectively). MTT assay showed mat cytotoxicity of pokeweed antiviral protein II for HEP-G2 cells and Hela cells was in a dose-dependent manner with IC50 of 93 ug/mL and 102 ug/mL, respectively. [Conclusion]The expression system of pokeweed antiviral protein-II has been successfullyestablished. The rinding of integrase inhibitory activity and the discovery of cytotoxicity provided more insights into the anti-HTV and the anti-tumor activities of PAP-H.