[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 (IC50 = 303 microg/mL, 220 microg/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 IC50 s of 93 microg/mL and 102 microg/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.
Huang
Jian-Song
JS
Department of Biochemistry and Molecular Biology, College of Medicine, Zhejiang University, Hangzhou 310006, China.
Zhan
Jin-Biao
JB
Zou
Yuan
Y
Feng
Wei-Hong
WH
D003001:Cloning, Molecular; D019427:HIV Integrase / Q000187:drug effects; D006367:HeLa Cells; D006801:Humans; D029604:Phytolacca americana / Q000235:genetics*; D018515:Plant Leaves / Q000235:genetics; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000494:pharmacology; D054789:Ribosome Inactivating Proteins, Type 1 / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology
D011994:Recombinant Proteins; D054789:Ribosome Inactivating Proteins, Type 1; D019427:HIV Integrase; C024275:pokeweed antiviral protein; C514801:p31 integrase protein, Human immunodeficiency virus 1