| Mannan-binding lectin (MBL), one of members of the collectin (C-type lectin with a collagen-like domain) family in the superfamily of C-type lectins, is a central component of the innate immune system. On binding to specific carbohydrate structures on the surface of a broad range of microorganisms, MBL activates MBL-associated serine proteases and hence the lectin pathway of complement activation in antibody- and C1q-independent way. Activation of the complement may lead to killing of the microorganisms by direct lysis effect of complement system or the phagocytosis by the enhancement of the attachment of microbes to the phagocyte (opsonization). The frequencies of point mutations in MBL structure gene, which can confer low MBL concentrations and resulting in opsonophagocytosis deficiency, are very high in crowd and MBL deficiency appears to be one of the most common immunodeficiencies. Individuals with MBL deficiency present with recurrent, acute or chronic infections by various kinds of pathogens. However, the relationship between structure and functions of MBL and the mechanisms by which MBL structure gene mutations cause opsonic deficiency are to be clarified.MBL is synthesized by liver cell and secreted into the blood, but the concentration of MBL in blood plasma is very low (~1 mg/L), which makes preparing MBL in large-scale impossible and the scientific research using MBL expensive. Therefore, high-level expression of human MBL with the full biological activity will be useful for studies on the immunological role of MBL in humans.Objectives:1 . To express human MBL gene in mammary cells and obtainrecombinant human MBL (rhMBL) protein.2. To compare the expression efficiencies of the eukaryotic expression vector PcDNA4/HisC-MBL and PcDNA3.1+- MBL in CHO, HEPG2 and HEK293 cells.Methods:1. The target sequence in pGEM-MBL plasmid that contains human MBL cDNA was amplified by PCR, inserted into eukaryotic expression vectors PcDNA4/His C and PcDNA3.1+, and the resulting recombinant expression vectors, PcDNA4/His C-MBL and PcDNA3.1-MBL, were identified by restriction mapping and sequencing. By using electroperforation, the recombinant vector PcDNA4/His C-MBL was transformed into CHO and HEPG2 cells and PcDNA3.1+-MBL, into CHO, HEPG2 and HEK293 cells. Zeocin-resistant clones and G418-resistant cell clones were selected and MBL mRNA expression was analyzed by RT-PCR.2. The expressed product of PcDNA4/His C-MBL/CHO was purified by Immobilized Metal Affinity Chromatography (IMAC) from the supernatant of the cultured cells and identified by SDS-PAGE, western-blot assay and enzyme-linked immunosorbent assay (ELISA), and its antigenicity and bioactivity were detected by an indirect ELISA using the anti-serum from Balb/C mice immunized respectively with the recombinant MBL protein and recombinant hCRD.3. The expressive efficiencies of the recombinant vectors PcDNA4/ HisC-MBL and PcDNA3.1+-MBL in different cells was analyzed by RT-PCR and ELISA.Results:1. The cDNA fragment of about 750pb was amplified from pGEM-MBL plasmid and the expression vectors PcDNA4/His C-MBL and PcDNA3.1+-MBL were constructed successfully, which was shown by restricting enzyme digestion and DNA sequencing. After two recombinant plasmid wastransformed into different cells respectively by electroperforation. The mRNA expression of Zeocin- and G418-resistant cell clones was determined by RT-PCR.2. Three components of 29KD, 58 KD and 87KD in the purified recombinant product from PcDNA4/His C-MBL/CHO cells were found by SDS-PAGE and the component of 29KD could be recognized by anti-6xHis antibody in Western-blot assay. The recombinant protein can be recognized by anti-6-Histidine monoclonal antibody and mouse serum against recombinant trimeric carbohydrate-recognition domain (CRD) of human MBL in ELISA, and bind to mannan effectively. The titers of the anti-serum from the immunized mice are 1: 819200 against the recombinant protein and 1: 25600 against both the natural huma...
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