Study On Therapeutic B Lymphocyte Stimulator Vaccine

B Lymphocyte Stimulator(BLyS), a novel member of TNF (tumor necrosis factor) family, was firstly found by Swedish and American scientists in 1999. BLyS is a type-II transmembrane protein composed of 285 amino acid residues. The human BLyS gene maps to chromosome 13q32-34. The expression of BLyS protein is restricted to cells of myeloid origin, including macrophages and dendritic cells. BLyS exists as transmembrane and soluble form in human body and both of them have natural bioactivity.Currently, three receptors for BAFF have been identified: BCMA, TACI and BAFF-R. These receptors lack signal sequences and thus are classified as type III membrane proteins. Of these receptors, BCMA and TACI can also bind APRIL, another TNF super family member, while BAFF-R is highly specific. Unlike with their ligand, these receptors are mainly expressed and displayed on B cellsurface, which makes the bioactivity of BLyS------co-stimulation ofB cell maturation and proliferation more selective.Studies showed that BLyS had the function of regulating humoral immune responses, playing a critical role in B cell survive and function regulation, elongating lifespan and stimulating proliferation of B cell. Because of its strong effect on B cells, over-expression of BLyS will lead to autoimmune diseases especially SLE (system lupus erythematosus). But the detail mechanism of BLyS was still unclear.TT, with the molecular weight of 150 kDa, was synthesized by Clostridium tetani. It is a potential inhibitory factor to central nervus system. No. 830-843 amino acid residues of TT is a T cell antigen peptide, which can stimulate Th response in body. Because thymus-dependent antigen(TD-Ag) can stimulate high level immune response, T cell antigen peptide is often fused into antigens in vaccine research.The present study was aimed at obtaining a therapeutic BLyS vaccine for autoimmune diseases. Considering the immunogenicity of BLyS itself maybe not so strong and the natural molecule may be harmful to autoimmune diseases, we tried to construct a recombinant vaccine (TT-BLyS) by fusion-expressing extra-cellular domain of BLyS (134-285 amino acid residues) and TT epitope. The interested gene was cloned into pKKH vector and expressed in an insoluble form. Because the protein was densely aggregated, it can be washed with acute conditions. The purity was about 80% after wash and could be highly purified by size-exclusion chromatography in 7 M guanidine hydrochloride. High titer of immunoglobulin could be induced when TT-BLyS was used to immune rabbits, mice and rats. In vitro and in vivo experiments showed that the induced antibody could neutralize the activity of natural BLyS. When being used in...