Production And Functional Analysis Of Recombinant Human Soluble B-lymphocyte Stimulator

B-lymphocyte stimulator(BLyS) is a new member of TNF family. BLyS can regulate proliferation and function of T and B lymphocyte by binding to its receptors. Studies have revealed that lack of BLyS can lead to immunodeficiency while overexpression of BLyS always involves in progression of many kinds of autoimmune disorders.Therefore, we may developed a new strategy based on above-mentioned theory to treat relevant diseases.For this reason,we cloned the cDNAs encoding the extracellular regions of human B-lymphocyte stimulator and constructed the prokaryotic expression vectors .Then the recombinant E.coli cells were induced by IPTG to express the corresponding proteins. Next, the proteins were purified with Ni-NTA chromatography, and their immunological functions were assayed. This research aims at paving the way for further study of novel therapeutic agents.The results are as follows:1. The cDNAs encoding the extracellular regions of BLyS were cloned. 627bp cDNA and 525bp cDNA amplified by RT-PCR were consistent with the sequence encoding human BLyS78-285 and 112-285 amino acids reported in GenBank. 2. The recombinant vectors pQE-80L/ BLyS78 and pQE-80L/ BLyS112 were successfully constructed after the target DNA fragments were inserted into pQE-80L respectively. 3. The recombinant vectors were transformed into E.coli DH5α respectively.The expression was induced by IPTG and optimal conditions of the induction were achieved. SDS-PAGE analysis showed that the about 26kDa and 21kDa recombinant fusion proteins were highly expressed as inclusion bodies.The expressed proteins were identified by Western Blotting. 4. The expressed proteins were purified and renatured through Ni-NTA chromatography and multistep dialysis. SDS-PAGE analysis showed that the target proteins were highly purified.5. The biological activity of the produced proteins was assessed through [3H]Thymidine incorporation assay. The proteins could strongly stimulate not onlyproliferation of human peripheral lymphocyte but also proliferation of mouse lymphocyte. Moreover, they could inhabit the growth of tumor cells.The functional recombinant human soluble BLyS proteins were successfully produced, which may pave the way for further study of mechanism of BLyS and novel therapeutic agents based on BLyS.