Expression,Purification And Characterization Of Recombinant Mouse Soluble Baff Secreted From The Yeast Pichia Pastoris

Object To clone，express and purify the mouse soluble B lymphocyte activationfactor (msBAFF) which has the ability to stimulate mouse B lymphocyte proliferationin vitro，and provide valuable materials for research into mice BAFF as a potential Bhybridomas growth stimulators and adjuvant.Method Peripheral blood mononuclear cells of BALB/c mice were separated，and then the total RNA of these cells was extracted and reverse transcripted，the targetgene was amplified and cloned from the cDNA products template. The PCR productwas subcloned into the vector pMD18-T. The constructed recombinant plasmid wasdigested with two restriction enzymes，and then the target DNA fragment was ligatedto the corresponding sites of the expression vector pPICZαA. Then the ligationproduct was transformed into the competent cells of E. coli DH5， and therecombinant plasmid was extracted from positive clone and determinated by usingrestriction enzymes and DNA sequence analysis. The recombinant expression vectorpPICZαA-msBAFF was digested with restriction enzymes and transformed to theyeast host strain GS115. Zeocin was utilized to select transformants. Expressionproduct induced with methanol was determinated with SDS-PAGE. Purified msBAFFwas obtained by ammonium sulfate precipitation，dialysis and Q Sepharose XL anionexchange column chromatography. The purified msBAFF was characterized by B cellactivating test. Cell proliferation was measured by the CCK8kits using a microplatereader. Mice were co-immunized with antigen and msBAFF, the antibody titers ofthese mice were analysis for in vivo immunological effects of the recombinantmsBAFF.Results The msBAFF DNA fragment was cloned and confirmed by DNA sequencing. The recombinant expression vector pPICZαA-msBAFF was constructedand finally transferred to Pichia pastoris GS115. The msBAFF was expressed withinducing condition. The molecular weight of the expressed protein was approximate20kDa as determined by SDS–PAGE. Most of the recombinant msBAFF were elutedwith0.3M NaCl from Q Sepharose XL ion exchange chromatography revealed bySDS–PAGE analysis. The data of B cell proliferation assay showed that B lymphocyteproliferation could be co-stimulated by the purified recombinant msBAFF andanti-IgM. The vivo test showed that higher concentrations of msBAFF inhibited theproducing of serum antibody.Conclusion The msBAFF gene have been cloned and constructed to secretoryPichia pastoris expression vector， and the recombinant msBAFF protein wasexpressed and purified. Co-stimulation assays indicated that purified msBAFF fromyeast Pichia Pastoris，could stimulated B lymphocyte proliferation in vitro. The vivotest showed that higher concentrations of msBAFF inhibited the producing of serumantibody.