Cloning and expression of pokeweed antiviral protein-II gene from the summer leaves of Phytolacca amercana.
- Author:
Jian-Song HUANG
1
;
Jin-Biao ZHAN
;
Yuan ZOU
;
Wei-Hong FENG
Author Information
1. Department of Biochemistry and Molecular Biology, College of Medicine, Zhejiang University, Hangzhou 310006, China.
- Publication Type:Journal Article
- MeSH:
Cloning, Molecular;
HIV Integrase;
drug effects;
HeLa Cells;
Humans;
Phytolacca americana;
genetics;
Plant Leaves;
genetics;
Plasmids;
Recombinant Proteins;
biosynthesis;
isolation & purification;
pharmacology;
Ribosome Inactivating Proteins, Type 1;
genetics;
isolation & purification;
pharmacology
- From:
Chinese Journal of Biotechnology
2006;22(4):592-597
- CountryChina
- Language:Chinese
-
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 (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.
