Production And Functional Analysis Of Recombinant Human Soluble BAFF And Its Mutants

B cell activating factor belonging to the TNF family (BAFF) is a new member of TNF family. BAFF can regulate proliferation and function of T and B lymphocyte by binding to its receptors. It has a crucial role in both humoral and cellular immunity. Studies have revealed that lack of BAFF can lead to immunodeficiency while overexpression of BAFF closely related with the progression of many kinds of autoimmune disorders and tumor. Therefore, we may develope a new strategy based on above-mentioned theory to treat relevant diseases. For this reason, we cloned the cDNAs encoding the human soluble BAFF(sBAFF) and its mutants and constructed the prokaryotic expression vectors. Then the expression vectors were transformed into E.coli DH5αand the corresponding proteins were expressed in the presence of IPTG. Next, the proteins were purified with Ni2+-NTA chromatography and their immunological functions were assayed. The major results are as follows: 1. The cDNA encoding the human sBAFF was cloned. A 451bp cDNA amplified by RT-PCR was consistent with the sequence encoding human BAFF134-285 amino acids reported in GenBank. 2. Use the pUC19/sBAFF as the template, four mutant sBAFF cDNAs, in which the sequnce encoded 217-285 aa (VHVFGDEL) of BAFF was replaced by GG, SS, SG and GS were cloned by one step opposite direction PCR. 3. The recombinant vectors pQE-80L/sBAFF, pQE-80L/GG , pQE-80L/SS ,pQE-80L/SG and pQE-80L/GS were successfully constructed after the sBAFF cDNA and its mutants were inserted into pQE-80L respectively. 4. The recombinant vectors were transformed into E. coli DH5αrespectively, and the corresponding proteins were expressed in the presence of IPTG. SDS-PAGE and Western blot analysis showed that the recombinant sBAFF protein was about 20kDa and mainly expressed in inclusion bodies, and the four mutant proteins were about 19kDa and mainly located in cytoplasm. 5. The expressed sBAFF was purified by Ni2+-NTA chromatography and refolded by using dialysis against urea buffer. The four mutant proteins were purified by Ni2+-NTA chromatography under the native condition and dialysed against PBS. SDS-PAGE analysis showed that the target proteins were highly purified. 6. The biological activity of the produced proteins was assessed by MTT assay. The result showed that sBAFF protein could strongly stimulate not only proliferation of human peripheral lymphocyte but also proliferation of Raji cell line. While Consistently, we confirmed that the four mutant proteins could suppress the biological activity of sBAFF. In summary, the functional recombinant human sBAFF and its mutant proteins were successfully produced, which may pave a way for further study on mechanism of BAFF and developing novel therapeutic agents for BAFF-associated diseases based on BAFF.