Construction of the expression vector pET32a/His MBLª²CLR and its expression in E.coli

    CAI Xueª²min, ZUO Daª²ming, ZHAO Na, ZHANG Liª²yun, CHEN Zhengª²liang

    Department of Immunology, Southern Medical University, Guangzhou 510515, China

    \[Abstract\] AIM: To construct pET32a/His MBLª²CLR recombinant prokaryotic expression plasmid and to express mannanª²binding lectinª²CLR (MBLª²CLR) protein in E.coli METHODS: The human MBLª²CLR gene was amplified by PCR from pGEMª²MBL plasmid, and was inserted into prokaryotic expression vector pET32a. After identified by restriction mapping and sequencing, the recombinant plasmid pET32a/His MBLª²CLR was transformed into E.coli BL21 (DE3) cells. The expressed product was purified by Immobilized Metal Affinity Chromatography (IMAC) and identified by SDSª²PAGE, Western blot and indirect enzymeª²linked 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 pGEMª²MBL plasmid and the recombinant expression vector pET32/His MBLª²CLR 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 SDSª²PAGE and all the components could be recognized by antiª²6His 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 MBLª²CLR and the recombinant human MBLª²CLRª²Trx fusion protein were obtained successfully, which will help the further structureª²function research of MBL molecule. ......