中国生物工程杂志

PAP-C23, a kind of deleted mutant of pokeweed antiviral protein (PAP) gene, was amplified from mature pokeweed antiviral protein gene by PCR, then the amplified fragment was cloned into the expression vector PET101, and then the recombinant plasmid was transformed into Escherichia coli BL21(DE3) strain. Induced with IPTG, the recombinant gene was expressed. SDS-PAGE analysis showed that the recombinant protein existed in the form of inclusion.The recombinant protein amount may accounted for 29.6% of the total inclusion proteins by optimizing the expression conditions. After recycled by cutting the gel which has been stained by KCL solution, the recombinant protein was grinded fully with a pestle in ice-bath and dissolved in phosphate buffered saline(PBS), and then added into same volume Freund's adjuvant incomplete. After emulsified completely, the mixed solution was used to inject rabbits, to prepare polyclonal antibody. Indirect enzyme linked immunosorbent assay(ELISA) showed that the titer of the antiserum was 1:1000. Western blot analysis showed that the antiserum could specifically react with expressed recombinant protein and nature PAP isolated from Phytolacca amercana, which also indicated that the recombinant PAP gene was expressed correctly in Escherichia coli.