OBJECTIVETo clone the pokeweed anti-viral protein (PAP) gene, to express it in Pichia pastoris, and to study the inhibitory effect of PAP on U251 in vitro.

METHODSThe cDNA sequence encoding PAP was cloned by Real-time PCR from Phytolacca americana. The recombinant PAP was subcloned into the expression vector pPICZaA and expressed in Pichia pastoris GSI15 after methanol induction. SDS-PAGE analysis showed that the expressed PAP existed in the yeast culture supernatant. The drug cytotoxicity to U251 cells was assessed using MTT assay and the obvious apoptotic nuclei of the tumor cells detected using the method of single cell gel electrophoresis.

RESULTSThe full-length PAP gene was cloned. The recombinant expression plasmid pPICZaA-PAP was constructed successfully. SDS-PAGE analysis showed that the relative molecular mass (M) of the recombinant protein was about 35 kDa. The degradation of the genome of the apoptotic cells induced by PAP was detected using the method of single cell gel electrophoresis. PAP possessed very high ability to inhibit the growth of U251. The anti-tumor activities (IC50) to U251 cells of PAP was 81.0 microg/mL.

CONCLUSIONPAP could be a potent anti-tumor candidate for inhibiting the growth of U251 and inducing its apoptosis.