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利用大肠杆菌表达野生型和突变型重组人白介素13(IL-13),以获得具有生物学活性的重组蛋白。采用PCR方法从质粒pET22b-hIL-13上扩增人成熟IL-13的编码序列。用定点突变引物扩增IL-13突变体(IL-13m)编码基因。将IL-13和IL-13m编码基因分别克隆到原核表达载体pET28a(+)中,构建质粒pET28a(+)-IL-13和pET28a(+)-IL-13m,然后将质粒转化到E.coli BL21(DE3)中,构建重组体,IPTG诱导重组蛋白表达。重组蛋白采用镍柱(Ni-NTA)纯化,纯化后复性,并分析其生物学活性。结果成功得到IL-13和IL-13m重组蛋白,经SDS-PAGE分析,在相对分子质量约14.6 kD的位置出现明显蛋白条带,经Western blot证实为重组hIL-13蛋白;重组蛋白主要以包涵体形式存在。纯化复性后,重组蛋白具有生物学活性。最后成功获得了具有生物学活性的野生型和突变型重组人IL-13,为后续研究奠定了基础。
Wild-type and mutant recombinant human interleukin-13 (IL-13) were expressed in E. coli to obtain a biologically active recombinant protein. The coding sequence of human mature IL-13 was amplified by PCR from plasmid pET22b-hIL-13. The IL-13 mutant (IL-13m) encoding gene was amplified using site-directed mutagenesis primers. The IL-13 and IL-13m coding genes were cloned into the prokaryotic expression vector pET28a (+) respectively to construct the plasmids pET28a (+) - IL-13 and pET28a (+) - IL-13m. In BL21 (DE3), recombinant was constructed and IPTG induced recombinant protein expression. Recombinant protein was purified by Ni-NTA column, purified and refolded, and its biological activity was analyzed. Results The recombinant protein of IL-13 and IL-13m was successfully obtained. SDS-PAGE analysis showed a significant protein band at a molecular weight of 14.6 kD, which was confirmed by Western blot as a recombinant hIL-13 protein. Body form exists. After purification and renaturation, the recombinant protein has biological activity. Finally, wild-type and mutant recombinant human IL-13 with biological activity were successfully obtained, which laid the foundation for subsequent research.