目的：通过甲醇营养型毕氏酵母蛋白质表达系统，表达出具有生物活性的人可溶性4-1BBL重组蛋白。方法：PCR方法从XG-4-1BBL转基因细胞中获得4-1BBL的胞外段基因；利用酵母表达载体pPICZαA构建重组质粒pPICZαA-s4-1BBL，经线性化后电转化导入毕氏酵母GS115中，甲醇诱导表达；SDS-PAGE蛋白电泳和Western blot分析发酵上清中hs4-1BBL蛋白表达水平及其特异性；体外T细胞增殖试验（3H-TdR掺入法）观察rhs4-1BBL 蛋白的生物学活性。结果：（1）成功地扩增到4-1BBL的胞外段基因，酶切鉴定及测序结果与已知的基因序列一致；（2）SDS-PAGE蛋白电泳和Western blot结果显示所获重组蛋白的分子量与预期分子量（21 kD）相同，并可被4-1BBL特异性抗体识别；（3）重组蛋白在体外具有协同促进T细胞增殖的效应。结论：成功地在毕氏酵母表达系统中表达出具有生物学活性的人可溶性4-1BBL重组蛋白，为进一步研究其功能奠定了物质基础。

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.

R392.1

国家重大基础研究项目（20001CB51003）资助