Assembly Of The Recombinant MBL Oligomers In Vitro Based On The Characteristic Of Streptavidin Tetramer

The inbeing of innate immunity is to identify molecular-pattern possessed specially by microorganism and it's the first-line of host to defense infection.At present,it has been established Mannan-binding lectin(MBL) is a key molecule of innate immune system.MBL,the member of collectins which belongs to C-type lectin superfamily,is a serum protein secreted by liver cells.And MBL can through the pattern-recognition function to selectively recognize sugars presented on pathogens.On binding to pathogens,MBL may activate the complement cascade via lectin pathway,leads to the killing of microbes by exerting lysis effect and indirect opsonizing function.At the same time,MBL has a direct opsonization when binding to lectin receptors on the phagocytic cells,and it also can mediate the MBL dependent cell-mediated cytotoxicity reaction.MBL also play an important role on surfactant defenses of mucous membrane.The overall polypeptide structure of MBL from N-terminal domain to C-terminal domain includes four domains,a cysteine-rich N-terminal domain,a collagen-like region(CLR),a neck region and a C-terminal carbohydrate-recognition domain (CRD).Integrity MBL is a multimeric protein composed by three of homogeneity trimer subunits and as many as six subunits.Only the high oligomeric forms of MBL have the biologic activity and can fix complement.CRD is recognition function domain of MBL,can selectively recognize sugars presented on pathogens.And CLR is effect function domain.MBL activates the complement and bind to C1qR location on the domain.MBL can form compound with MBL-associated serine proteases (MASPs) through its CLR.After its CRD domain recognize and binding to sugars presented on pathogens,MBL can activity the zymogens of MASPs,so activate the complement cascade via lectin pathway to play the function of anti-infection by innate immunity.Have access to sufficient quantities of the biological activity of MBL protein is the premise of study MBL.However,the content of MBL in human serum is few (about 1 mg / L),extraction difficult and expensive.Because the structure of MBL protein is complex,eukaryotic expression production is low.However,prokaryotic expression system unable to form oligomers assembly protein,because it is lack of specific translation after processing of the eukaryotic cells.So it has seriously hampered the research of MBL molecular.Accordingly,the issue adopt of prokaryotic expression system,and insert the peptide labelling in the N-terminal of MBL(rhMBLâ–³N-strep-tagâ…¡).Use the characteristic of the collagen-like protein,which is has the automatic assembly ability to form dimers or three dimers, Then assembly on the recombinant MBL protein oligomers through the link of between tags peptide and Streptavidin.Through compare with the native MBL,analysis of its binding to ligand and MASP to activated complement lectin way,the combination of phagocytic cells gelled receptor activity,to evaluate its biological activity and immunological activity.â… Expression and identification of the recombinant human MBL N-terminal deletant with Strep-tagâ…¡fusion proteinUse the Primer premier 5.0 software,design and synthesis the primer.Moreover, insert the Strep-tagâ…¡peptide in restriction site of the upstream primer.A length of about 640 bp of MBLâ–³N-strep-tagâ…¡gene fragments was amplified by PCR from pGEM-MBL plasmid which containing human wild-type MBL length cDNA encoding,then inserted into pET43.1a prokaryotic expression vector.To construct a recombinant expression vector by EcoRâ… and BamHâ… digested after about 7525 bp and the 640 bp fragment.After the sequencing right sequence,transformed into E. coli BL21(DE3),use the IPTG-induced reorganization pET43.1a MBLâ–³N-strep-tag â…¡expression plasmid to expressed the MBLâ–³N-strep- tagâ…¡fusion protein.The soluble expression of MBLâ–³N-strep-tagâ…¡fusion proteins were purified by Ni ~+-NTA agarose column chromatography,to access to the fusion protein was purified. One components of Mr 97 000 in the purified recombinant product were detected by SDS-PAGE.Western blot analysis showed that the purified protein binding to mouse anti-human MBL-CRD antibody.ELISA showed that protein react with both mannan and two kinds of Masps,and a good affinity binding to Strepravidin.â…¡Assembly of the recombinant MBL oligomers in vitroBoth of the protein MBL-CLR and CRD which were expressed by prokaryotic expression system has the automatic assembly ability to form dimers or three dimers, and rhMBLâ–³N-strep-tagâ…¡single-chain protein contains both the CLR and CRD part.So it has been dialysis in the buffer,automatic assembly of a three dimers,which is the subunit of recombinant MBL protein.Reduction and non-reduction SDS-PAGE and Westblot results,we sure that about Mr 50,000 protein bands is the subunit of recombinant MBL.Elisa showed that the ability of the subunit of recombinant MBL binding to mannan has been a certain increase compared with the single-chain protein. But the ability of binding to Strepravidin,the subunit recombinant MBL is has been noticeably reduced.Improvement of the buffer,joined Strepravidin,from SDS-PAGE showed that Mr97 000,Mr125 000 and Mr220 000 three bands.We speculate that are the recombinant two,three and four polymer MBL proteins.â…¢Preliminary identification of the recombinant of MBL oligomersThe purpose of this study is to compare the different between the recombinant MBL protein polymer and native MBL protein in the biological activity and immune function.The recombinant MBL can combine mannan on the yeast cell wall lead to a phenomenon that it is the lectin,but the titer level of activity is low than the natural MBL.Ligand binding assay results showed that the recombinant MBL binding to Mannan and two kinds of MASPs.But the ability of ligand binding is low than the natural MBL.Complement C4d deposition experiment was indirectly prove the recombinant of MBL coated with the mannan,which can lead to complement activation;However,this activity is weak than the natural MBL.Besides it can be combine with the THP1 cells,which is piont out the recombinant MBL can bind to phagocytic cells gelled receptor.The experimental based on the charateristic of Streptavidin tetrammer,assembly of the recombinant MBL protein polymer in vitro.Some test testify that the recombinant MBL protein be provided with the basic biological activity as the natural MBL.As a result of the tcchnical constraints,it can not been get a large number of high-purity the recombinant MBL protein oligomers,so it is unable to get further study its immunological function.However,the preliminary experimental results of the issue,it seems certain guiding significance in access to the complex structure of protein.If we can find a better safety and effective of the molecule-protein,which it can be the preparation for get the large number of protein oligomers in vitro and to provide a scientific basis for the development of drug-related research to draw on.