Human MBL.docVIP

 PAGE \* MERGEFORMAT 15 Human MBL Author: Cai Min, Zuo-Ming, Zhao Na, ZHANG Li-yun, Cheng-liang [Keywords:] mannan-binding lectin;, collagen-like region;, prokaryotic expression Construction of the expression vector pET32a/His MBLCLR and its expression in E.coli [Abstract] AIM: To construct pET32a/His MBLCLR recombinant prokaryotic expression plasmid and to express mannanbinding lectinCLR (MBLCLR) protein in E.coli METHODS: The human MBLCLR gene was amplified by PCR from pGEMMBL plasmid, and was inserted into prokaryotic expression vector pET32a . After identified by restriction mapping and sequencing, the recombinant plasmid pET32a/His MBLCLR was transformed into E.coli BL21 (DE3) cells. The expressed product was purified by Immobilized Metal Affinity Chromatography (IMAC) and identified by SDSPAGE, Western blot and indirect enzymelinked immunosorbent assay (ELISA) using the antibody from BALB / c mice immunized with the recombinant human MBL protein.RESULTS: The cDNA fragment of 180bp was amplified from pGEMMBL plasmid and the recombinant expression vector pET32/His MBLCLR was constructed. The recombinant plasmid was consistent with those expected by restriction maps and sequence. Three components of Mr 30000, 60000 and 120000 in the purified recombinant product were detected by SDSPAGE and all the components could be recognized by anti6His antibody in Western blot assay. The three components were correspondingly with the band of the monomer and oligomer of the fusion protein. The purified recombinant product could react with the antibody against the recombinant human MBL protein in the indirect ELISA. CONCLUSION: The prokaryotic expression strains that efficiently express recombinant human MBLCLR and the recombinant human MBLCLRTrx fusion protein were obtained successfully, which will help the further structurefunction research of MBL molecule. [Keywords] mannanbinding lectin; collagenlike region; prokaryotic expression [Abstract] Objective: In the E.