Construction Of Human DC-SIGN Transgenic Cells And Preparation Of Anti-human DC-SIGN Monoclonal Antibody

Dendritic cells (DCs) are the most important antigen presenting cells (APCs) in immune system, which have strong antigen processing and presenting capacity. Because of wide distribution, they can capture and process foreign antigens in the first time, and then present them to T cells to induce immune responses or immune tolerance. The functions of dendritic cells are closely related to their surface molecules.DC-SIGN is one member of C-type lectins, which consists of intracellular, transmembrane and extracellular domains, Its molecular weight is 44kDa. There is only one extracellular carbohydrate recognition domain (CRD) which recognizes some monosaccharide and oligosaccharide. DC-SIGN is primarily expressed on dendritic cells and is one important marker of monocyte-derived dendritic cells. While DC-SIGN molecules at the surface of DCs bind antigens, antigens can be endocytosed and presented to T lymphocytes to induce immune responses. Moreover, DC-SIGN can be used as a tool for evading immune surveillance by HIV-1 envelope glycoprotein gp120 which contributes to T cells infection. In addition, DC-SIGN also mediates the transfer of dendritic cells and involves in the regulation of immune responses. Therefore, construction of human DC-SIGN transgenic cells and preparation of anti-human DC-SIGN monoclonal antibody are of great significant in both theoretical studies and clinical applications.This article consists of two parts:Part I The cloning of human DC-SIGN gene and the construction of its transgenic cellsTotal RNA was extracted from dendritic cells derived from human peripheral blood monocytes, which was reverse transcripted into cDNA . The DC-SIGN gene was then amplified by RT-PCR. The target gene was inserted into retrovirus vector pGEZ-Term and confirmed by sequencing. The recombinant retrovirus vector together with its two helper virus vectors were cotransfected into the package cell 293T in the context of LipfectAMINE. The supernatant of 293T was used to infect L929 cells. After 72 hours, L929 cell line stably expressing human DC-SIGN protein was selected in the presence of Zeocin.Part II Preparation and identification of mouse anti-human DC-SIGN monoclonal antibodyThe L929/DC-SIGN transgenic cells were used to immunize BALB/c mice. The immunized splenocytes were fused with murine myeloma cells (SP2/0) by the cell fuse technique. By means of HAT selective culture and repeated screening with L929/DC-SIGN as antibody-screening positive cell and L929/mock as negative control, one hybridoma cell line (4G8) continuously and steadily secreting specific anti-human DC-SIGN was obtained. This hybridoma grew well after long-term culture in vitro and storage in liquid nitrogen.The monoclonal antibody was produced according to ascites-inducing procedure in mouse abdominal cavity. The average yield of ascites was about 3.5 milliliter (ml) per mouse. After the ascites was purified by protein G affinity chromatography, the concentration of the protein was above 4 milligram (mg)/ml and the ascitic titer was over 1:1000 dilution by immunofluorescence analysis. Every 1×106 cells required 0.2～2 microgram (μg) purified monoclonal antibody in indirect immunofluorescence. Karyotype analysis result showed that the number of the chromosomes of the hybridoma was more than these of mouse somatic chromosomes, which suggested that they are fused cells.Fast-strip method analysis displayed that monoclonal antibody 4G8 was mouse IgG1 and the light chain wasκ. Western blot and flow cytometry analysis showed that monoclonal antibody 4G8 exclusively binded DC-SIGN molecules. The competitive inhibition results showed that monoclonal antibody 4G8 and E021819, a commercial anti-DC-SIGN antibody, recognized different epitopes.Flow cytometry analysis showed that DC-SIGN molecules primarily expressed on monocytes derived from dendritic cells. After maturation, dendritic cells still expressed DC-SIGN molecules, but in some lower level; Monocytes, B lymphocytes, T lymphocytes isolated from peripheral blood did not express DC-SIGN molecules; human B lymphoma cell lines Daudi and Raji, human T lymphoma cell line Jurkat, human leukemia cell line K562, human lung cancer cell lines A549 and H1299, human monocyte derived cell lines THP-1 and U937 did not express DC-SIGN molecules.The expressions of DC-SIGN molecules on human monocyte derived cell lines THP-1 and U937 were analyzed by flow cytometry. The results showed that THP-1 cells expressed DC-SIGN molecules after stimulation of PMA and IL-4. The expression was at a higher level after 48 hours, but fell after 72 hours. In comparison, DC-SIGN expression was not detected on U937 cells. All these results confirmed that the cell line THP-1 can be used as a model for monocytes to differentiate into dendritic cells.