Expresson of the N-terminus truncated phosphotase D in Escherichia coli and characterization of its anti inflammatory activity / 中国人兽共患病学报

ABSTRACT

To investigate the immunological activities of the recombinant human phosphatase D2 (rhPLD2) in vitro and in vivo, especially its ability to reduce inflammatory reactions, the cDNA fragment encoding rhPLD2 was cloned into prokaryotic expression vector pET30a by RT-PCR and the recombinant protein rhPLD2 expressed in E.coli was purified from the inclusion bodies, while the anti inflammatory activity of rhPLD2 was determined by the amount of eosinophils in bronchoalveolar fluid(BALF) and blood and the expression of IL-5 and MMP-9 in lung tissues of guinea pig model of chronic asthma. It was found that the rhPLD2 recombinant protein was obtained from human Daudi cells by cloning to E.coli, which contained no membrane-binding site and signal peptide. The cDNA sequence encoded 631 amino acid residues (GenBank Accession Number AY178289). The purity of the rhPLD2 approached up to 76% with a bioactivity of 50.9745 units/L (0.9212 g/L). In addition, the anti inflammatory effect of rhPLD2 protein could be demonstrated in the guinea pig model of chronic asthma after treatment with rhPLD2 protein, such as down regulation in the expression of the inflammatory cytokine IL-5. It is concluded that the anti-inflammator activity of the recombinant human truncated PLD2 protein produced from the E.coli plasmid can be demonstrated both in vitro and in vivo.