重组人白细胞介素21在毕赤酵母中的分泌表达及活性分析

Expression and analysis of the recombinant human interleukin-21 (rhIL-21) in Pichia pastoris

摘要:

为了获得有活性的重组人白细胞介素21(rhIL-21), 本研究建立了在毕赤酵母 (Pichia pastoris) 中分泌表达rhIL-21的技术。首先, 通过RT-PCR从人外周血淋巴细胞中扩增IL-21cDNA, 克隆到酵母表达载体pPIC9K中, 构建了重组酵母表达载体pPIC9K-hIL21cDNA; 然后, 线性化的载体转化毕赤酵母表达菌株GS115, 经抗性梯度筛选获得了多拷贝重组酵母菌; 加入甲醇诱导培养后, SDS-PAGE和Western blotting 检测到培养液中有rhIL-21的分泌表达, 相对分子量约为16 kD, ELISA结果显示摇瓶表达量可达229.28 mg/L; 表达上清经阳离子交换介质SP Sepharose Fast Flow纯化后, 目的蛋白纯度达到95%。细胞增殖试验结果显示该rhIL-21联合刀豆蛋白A(Con A)对人淋巴细胞的增殖具有显著的促进作用。本研究首次成功在毕赤酵母表达系统中分泌表达了有生物活性的rhIL-21, 为相关疾病免疫治疗的研究奠定了基础。

Abstract:

Interleukin-21 is a type I cytokine mainly produced by activated CD4+ T cells that acts as a regulator of immune system. In this work, hIL-21cDNA was amplified from human peripheral blood lymphocytes by RT-PCR, and then inserted into pPIC9K. The recombinant vector pPIC9K-hIL21cDNA was linearized by Sac I, and transformed into Pichia pastoris strain GS115 by electroporation. Transformants were selected by G418 and confirmed by PCR. The recombinant protein was expressed and secreted into the supernatant after inducing by methanol. SDS-PAGE analysis indicated the molecular weight of rhIL-21 was about 16 kD. ELISA results show that the yield of rhIL-21 reach 229.28 mg/L, rhIL-21 was purified from culture supernatants, and it was purified to about 95% purity with ion-exchange chromatography. When co-stimulate with Con A, rhIL-21 can promote the proliferation of human lymphocytes. This is the first expression of bio-active rhIL-21 in Pichia pastoris. It lays a foundation for further research in immunotherapy and cancer therapy.