| Mannan-binding lectin(MBL),a member of the collectin superfamily(C-type lectin with a collagen-like domain),is a plasma glycoprotein mainly secreted by liver cells.By carbohydrate-recognition domain(CRD),MBL can recognize the carbohydrate structures on pathogenes surface.Forming compounds with two MBL-associated serine proteases(MASP-1,MASP-2),MBL can activate the complement cascade via lectin pathway independent of antibody or C1q,which leads to the microorganisms lysis.Also MBL can opsonize the phagocytosis by enhancing the attachment of microbes to phagocytes.MBL can recognize a wild range of pathogenes and play an important role in innate immunity.The frequencies of mutations in MBL gene are very high in crowds.Three known mutations in MBL structure gene can change the spatial structures of MBL protein,which leads to the function decreasing of binding to glycon ligands and MASPs,the function almost disappear of activating complements.In clinic,the person insufficient of MBL is susceptible to varieties of infections acute or chronic recurrently.The concentration of human MBL in plasma is very low,which makes the large-scaled extraction from blood cost highly.Therefore,by technology of genetic engineering,cell culturing and et al,high-level expression of human MBL in vitro will be important for further studies and clinic therapy.By electro-perforation,our research team have successfully transfected recombinant eukaryotic expression vectors:PcDNA3.1+-MBL and PcDNA4/His MaxC-MBL into CHO,HEK293 and HEPG2 cells.G418-resistant cell clones and Zeocin-resistant cell clones were selected.And by RT-PCR and ELISA,the expressive efficiencies of the recombinant vector PcDNA3.1+-MBL in CHO and HEK293 cells showed obviously higher than other combinant of vectors and celles. So we selected the CHO cells transfected with PcDNA3.1~+-MBL by G418 for two months and then obtained the stablely and highly expressive monoclonal cell lines by ELISA assay.Then cultured the cells in large-scale,purified the protein products and researched the biological characteristics.1.Establishing sandwich ELISA systemUse the anti-MBL-CRD mAb as capture antibody,the HRP labled anti-MBL-CLR pAb as detecting antibody and the rhMBL presented by professor Jens Chr.Jensenius as standard protein to select the highly expressive cells lines.The sensibility of this system is 0.1mg/L.2.Screening the monoclonal cell lines stably and highly expressing the recombinant human MBLOur research team have constructed CHO cells contain recombinant eukaryotic expression vector PcDNA3.1~+-MBL.We resused the cells,monocloned in 96 orifice and selected them by 800 mg/L of G418.The culture solution was changed after 3～5 days to sustain the concentration of G418 for 1 months.Then by sandwich ELISA assay we obtained 6 monoclonal cell lines highly expressing MBL,and the concentration of MBL in the supernatant was all above 3 mg/L.The stable expression of mRNA was analyzed by RT-PCR.3.Purifying the recombinant human MBL and researching its biological characteristicsThe recombinant proteins were initially purified by ammonium sulfate precipitation, and further purified through the anti-MBL-CRD mAb chromatography.Then We obtained 2mg recombinant protein from 1L culture supernant.Analyzed by Western blot and ELISA,the purified recombinant proteins can react with anti-MBL mAb, anti-MBL-CRD mAb and anti-MBL-CLR pAb.Analyzed by SDS-PAGE and Western blot,it was showned that the protein appears mainly above Mr 200 000,which indicates the 3～6 oligomers.Analyzed by ELISA,the recombinant MBL showned effectively binding to mannan and MASPs.The C4d deposition assay indicated the recombinant MBL and plasma-derived MBL can both mediate the activiation of complements.Moreover,by C4d deposition assay,the recombinant protein conserved in different temperatures for 6 months showned nearly equal function of mediating complements activiation,which proved the stability of recombinant MBL is good.
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