Human DC-SIGN Protein Clone And Primary Immunology Function Study

The full name of DC-SIGN is dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintergrin,also called CD209,expressed on the surface of immature dendritic cells(DCs).The main functions of DC-SIGN are capturing antigens and intercellular adhesion.Many pathogens such as HIV,BCG,HCV,MCV,Ebola virus,Leishmania and so on combine with DC-SIGN , then enter into dendritic cells.Mycobacterium tuberculosis is among these pathogens,combined tightly with DC-SIGN by its cell wall component-mannose-capped lipoarabinomannan,then mediating infection of DCs,interrupting signal exchanging,which causes the maturation inhibition of immature DCs.Mtb incubates in Non-lysosomal acidic compartment in DCs,through some uncertain mechanism,escaping from the surveillance of immune system and drug attack.This becomes the main reason for recurrence of TB.At first we intended to take C57BL/6J mouse as the animal model for DC-SIGN study.After culturing the mouse dendritic cells in vitro for several days and analyzing by flow cytometry,we found that the quantity of DC-SIGN expressed by mouse dendritic cells became lower and lower and was unable to be detected on the third day.In the articles before 2007,scientists abroad found that the components of mouse DC-SIGN related proteins were rather complex.There were many homologous compounds and the differences of function and structure between human and mouse were enormous.So they pointed out that the value of mouse being an animal model for DC-SIGN study was still need to be discussed.We decided to turn our aim to hunman being cells.First,we separated DCs from human peripheral blood,cultured,then extracted total RNA.We got a 1240bp long gene fragment of DC-SIGN,recombined it with plasmid pEGFP-C1 to form a 5938bp long recombined plasmid called DS-pEGFP-C1,whose expression sign was green fluorescent protein(GFP).Real-Time PCR quantitated the copy of mRNA was 4.52×1011copies/ml in transfected cells.GFP marker connected with DC-SIGN at the amino-end,which we supposed would not affect the antigen binding function of DC-SIGN.At last we did the primary test of DC-SIGN function binding with BCG.Result:our study successfully constructed a eukaryotic vector of human DC-SIGN and EGFP fusion protein,and identified the protein in cell line COS7. Real-time PCR test showed the mRNA was 4.52×10¹¹copies/ml and laser scanning confocal microscope confirmed it was expressed and could uptake BCG.Conclusion : We have constructed a recombinated vector DS-pEGFP-C1 expressing DC-SIGN and EGFP fusion protein.It is unaffected for DC-SIGN combining with BCG,even with GFP marker at the amino-end.Recombinant plasmid can be used in screening the best gene segment for RNAi,which becomes a piece of basic work to the whole study.