CHO Eukaryotic Expression And Function Research Of Human Soluble B Lymphocyte Stimμlator

The B Lymphocyte Stim lator(BLyS) also known as BAFF,TALL-1,THANK, zTNF4,is the most recent addition to the tumor necrosis factor family(TNF) ligands.BlyS induces both in vivo and in vitro B cell proliferation,differentiation and immunoglob lin secretion.Meanwhile,it also strongly suppresses the growth of tumor cell lines .The over-production of BLyS is associated with the development of certain autoimmune disease,such as systemic lupus erythaematosus(SLE) ,rheumatoid arthritis,myasthenia gravis, Sj greh's syndrome. Therefore,BlyS,its receptor or related antagonists may find medical utility in the treatment of B cell disorders associated with autoimmunity,neoplasia,or immunodeficiency syndromes.In this study,EPO signal peptide sequence and hsBLyS gene were linked by SOE method.The fusion product was cloned into eukaryotic plasmids.pcDNA3, pcDNA3.1, pEFneo,respectively.Meanwhile, the EPO signal peptide sequence was mutated so as to form a restriction enzyme cut site :Bin I .Thus the recombinant plasmid can be used as secreting plasmid expressing other gene.The recombinant expression plasmid and marker plasmid:pSV-dhfr were co-transfected into CHO-dhfr- cells.After screened and amplified by selection medium and MTX respectively,we constructed a CHO cell line which secreting express hsBLyS stably.The supernatant containing hsBLyS co-stimulated B cell proliferation highly with anti-IgM by MTT analysis.Flow cytometry results showed only in the function of costim lation,can B cell entered the s phase of cell cycle. Apoptosis was identified by DNA electrophoresis and Flow cytometry .The results showed the supernatant containing hsBLyS induced a does and concentration-dependant increase in apoptosis cells.