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Expression of Recombinant Human Soluble 4-1BBL in Yeast Pichia Pastoris and It′s Costimulating Activity on T Cells
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Abstract:
Objective: Methylotropic yeast pichia pastoris system was used to express recombinant human soluble 4-1BBL protein with biological activity.Methods:According to the nuclear acid sequence coding human soluble 4-1BBL, we cloned the genes with PCR from XG-4-1BBL transfection cell line,then the gene fragment for extracellar domain was subcloned into the PUCm-T vector and sequence of s4-1BBL cDNA was confirmed by sequencing. The s4-1BBL gene was inserted into the pPICZαA , which was transformed into Pichia pastoris GS115 by linearized electroportion.The recombinant protein was identified by the assay of SDS-PAGE and Western-blot. Costimulating activity of rhs4-1BBL on T cell proliferation in vitro was evidenced by 3H-TdR incorporation assay.Results: The s4-1BBL cDNA was successfully obtained and insected into pPICZαA. The protein molecular weight of hs4-1BBL in the yeast supernamant was about 21 kD by SDS-PAGE analyses,and the specificitity was identified by western blot. Finally, rhs4-1BBL protein could costimulate the proliferation of T cells in vitro.Conclusion: The rhs4-1BBL protein was efficiently expressed in Pichia pastoris (GS115)and showed natural biological activities. And it may provide a valuable materials for further study of 4-1BB/4-1BBL.
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Clc Number:
R392.1
