Expression of Recombinant Human Soluble 4-1BBL in Yeast Pichia Pastoris and It′s Costimulating Activity on T Cells / 中国肿瘤生物治疗杂志

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 pPICZ?A , 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 3 H TdR incorporation assay. Results: The s4 1BBL cDNA was successfully obtained and insected into pPICZ?A. 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.