重组人白介素1受体Ⅰ型细胞外基因在毕赤酵母的表达

Expression of recombinant extracellular region of human interleukin-1 receptor type I in Pichia pastoris

目的构建白介素1受体Ⅰ型细胞外基因(可溶性白介素1受体Ⅰ型,sIL-1RⅠ)的pPICZαA-sIL-1RⅠ重组表达载体,利用毕赤酵母表达技术表达sIL-1RⅠ蛋白。方法采用RT-PCR技术扩增基因sIL-1RⅠ,经酶切连接构建重组质粒pPICZαA-sIL-1RⅠ,转化E.coliStb13;阳性克隆经测序成功后,把pPICZαA-sIL-1RⅠ重组质粒电转入毕赤酵母菌株GS115,PCR对阳性克隆进行鉴定,以及对GS115转化子表型筛选。确定表型后,对重组菌株进行甲醇诱导蛋白表达。提取菌体蛋白和上清液进行Westernblotting检测以鉴定蛋白表达属于菌内表达还是分泌表达。结果 pPICZαA-sIL-1RⅠ重组质粒构建成功;pPICZαA-sIL-1RⅠ成功电转入酵母菌GS115;转化子表型筛选结果为甲醇诱导缓慢型Muts;对培养上清和菌体蛋白进行Westernblotting检测结果:上清没有检测到蛋白,在菌体内检测到蛋白。结论成功运用毕赤酵母表达体系表达sIL-1RⅠ蛋白,为sIL-1RⅠ的研究和应用提供实验基础。

Objective To construct pPICZαA-soluble interleukin-1 receptor type I （sIL-1RI） recombinant expression vector containing the gene fragment encoding the extracellular domain of sIL-1RI for its expression in Pichia pastoris. Methods sIL-1RI gene was amplified by RT-PCR and inserted into the yeast expression vector pPICZαA by digestion ligation. The recombinant plasmid pPICZαA-sIL1RI was transformed into E.coli Stb13, and the positive clones were analyzed by PCR and DNA sequencing. The pPICZαA-sIL1RI recombinant plasmid was electroporated into GS115 cells and the transformants were analyzed by PCR. After phenotype identification, the recombinant strains were induced by methanol to express the target protein, which was analyzed by Western blotting of the cell extract and supernatant. Results The recombinant plasmid pPICZαA-sIL-1RⅠ was constructed successfully, and the results of Western blotting showed that yeast induced by methanol expressed a protein of about 39 kD. Conclusion sIL-1RI protein has been successfully expressed in P.pastoris expression system, which provides the basis for further study of sIL-1RI.