可生物素化H-2K~d-BSP融合蛋白的原核表达和纯化

目的构建可生物素化的H-2Kd-BSP融合基因的表达载体,原核表达H-2Kd-BSP融合蛋白,以制备H-2Kd-肽四聚体。方法采用RT-PCR技术从小鼠SP2/0细胞中克隆小鼠MHC-Ⅰ类分子H-2Kd基因的胞外区,拼接上依赖BirA酶的可生物素化序列(BSP)后,插入pET-22b高效表达载体多克隆位点,诱导表达后对表达产物进行纯化,用Western印迹法分析鉴定纯化融合蛋白。结果成功构建pET-H-2Kd原核表达载体,测序证实H-2Kd-BSP融合基因序列正确。pET-H-2Kd原核表达载体可在大肠杆菌BL21中高效诱导表达H-2Kd-BSP融合蛋白,表达量占菌体总蛋白的36%,主要以包涵体形式表达;经反复洗涤纯化,纯化蛋白纯度可达90%以上。纯化蛋白可被H-2Kd分子特异性单克隆抗体SF1-1.1所识别。结论可生物素化H-2Kd-BSP融合蛋白的原核表达和纯化为进一步制备H-2Kd-肽四聚体奠定了实验基础。

Purification and expression of H-2Kd-BSP fusion protein

Objective To obtain a recombinant H-2Kd-BSP fusion protein for preparing H-2Kd-peptide tetramers to detect antigen-specific cytotoxic T lymphocytes(CTLs) in H-2Kd mouse.Methods The extracellular region of H-2Kd was cloned by RT-PCR from mouse SP2/0 cells,and added a 15 amino acid substrate peptide for BirA-dependent biotinylation to the COOH terminus of H-2Kd,then H-2Kd-BSP fusion gene was cloned into pET22b vector.Recombinant proteins of H-2Kd-BSP were obtained by prokaryotic expression system.After purification,Western blot analysis was used to evaluate the expression of the fused protein.Results The fusion protein was successfully expressed in the form of inclusion body and amounted to over 36% of total cell proteins via induction.After H-2Kd-BSP fusion protein was washed and purified,the final purity reached above 90%.Furthermore,the fusion protein can be recognized by SF1-1.1,a monoclonal antibody-specific for H-2Kd molecule.Conclusions This work establishes a convenient approach for purification of large quantity of recombinant H-2Kd-BSP fusion protein and provides the basis for the preparation of H-2Kd tetramers to detect CTLs in H-2Kd mouse.