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.
ABSTRACT
Objective To clone and express the fused gene fragment coding rhoptry protein ROP2 and major surface protein P30 from Toxoplasma gondii as a preparation for the construction of the complex ROP2,P30 antigen by gene engineering.Methods The gene fragment encoding P30 was amplified by PCR from T.gondii RH strain and subcloned into the recombinant plasmid pUC119/ROP2 already constructed.The recombinant plasmid pUC119/ROP2,P30 was digested by SacⅠ/HindⅢ and inserted into the same site of expression vector pET28b.The recombinant plasmid of pET28b/ROP2,P30 was transformed to E.coli and expressed under the induction of IPTG.Results The gene fragment 700 bp encoding P30 was obtained from the total DNA of T.gondii by PCR.The recombinant plasmid pET28b/ROP2,P30 was successfully constructed,which was highly expressed in E.coli,a fusion protein with molecular weight of 69 000.Conclusion The fusion gene encoding the rhoptry protein ROP2 and the major surface protein P30 of T.gondii has been successfully cloned and expressed to be an expected recombinant fusion protein ROP2,P30 with molecular weight 69 000.
ABSTRACT
Objective: Methylotropic yeast pichia pastoris system was used to express recombinant human soluble 4 1BBL protein with biological activity. Methods: According to the nuclear acid sequence coding human soluble 4 1BBL, we cloned the genes with PCR from XG 4 1BBL transfection cell line,then the gene fragment for extracellar domain was subcloned into the PUCm T vector and sequence of s4 1BBL cDNA was confirmed by sequencing. The s4 1BBL gene was inserted into the pPICZ?A , which was transformed into Pichia pastoris GS115 by linearized electroportion.The recombinant protein was identified by the assay of SDS PAGE and Western blot. Costimulating activity of rhs4 1BBL on T cell proliferation in vitro was evidenced by 3 H TdR incorporation assay. Results: The s4 1BBL cDNA was successfully obtained and insected into pPICZ?A. The protein molecular weight of hs4 1BBL in the yeast supernamant was about 21 kD by SDS PAGE analyses,and the specificitity was identified by western blot. Finally, rhs4 1BBL protein could costimulate the proliferation of T cells in vitro. Conclusion: The rhs4 1BBL protein was efficiently expressed in Pichia pastoris (GS115)and showed natural biological activities. And it may provide a valuable materials for further study of 4 1BB/4 1BBL.
ABSTRACT
Objective:To express recombination human interleukin-13(rhIL-13)in methylotropic yeast Kchia pastoris and study its effect on the induction of DC from PBMC in vitro. Methods: An artificial gene with the optional condon usage of Kchia pastoris for IL-13 was designed and synthesized by T4 DNA ligase and PCR.The expression vector pPICZoA-IL-13 was constracted and introduced in Kchia pastoris GS115 by electroporation after linearized with Sacl.Recombinants were selected by plating cells on YPD/Zeocine plates.The recombinant protein secreted by the yeast was identified by 15%SDS-PAGE,ELISA and Western blot.The abilities to induce DC differentiation of IL-13 and IL-4 were compared. Results: Based on the result of the 15% SDS-PAGE and Western blot assay, an about 13 kD recommbinant protein expressed in the yeast supernatant was identified to be recombinant human IL-13. Adherent PBMC cultured in GM-CSF,TNFa and IL-13 displayed morphological characteristic of DC generated in media containing IL-4. They formed cellular clumps and had extensive dendrites sharp. Fluorescence-activated cell sorter analysis showed that they expressed a high level of MHC class II ,CDla,CD80,CD83,CD86. The purity and yield of both DC populations are not significantly different with IL-13 or IL-4. The phagocytosis of immature DC induced by IL-13 is potent. They were also equally potent stimulators of allogeneic lymphocytes in the mixed lymphocyte reaction. Conclusion: rhIL-13 can substitute for IL-4 in the proliferation and development of dendritic cells.