CAJ | 학술논문

目的：构建人IL‐4重组表达载体，表达纯化并鉴定人IL‐4重组蛋白。方法采用巢式PCR技术从健康志愿者外周血单核细胞（PBMC）总RNA中扩增人IL‐4开放阅读框基因，将扩增的IL‐4目的基因与表达质粒pET101/D‐TOPO连接，转化大肠杆菌BL21，进行表达纯化和鉴定。结果采用巢式PCR扩增得到IL‐4开放阅读框大小为460 bp ，测序比对显示序列正确。转化BL21后，通过对不同克隆子表达水平的筛选，筛选到高表达 IL‐4的克隆子，表达和纯化后聚丙烯酰胺凝胶电泳（SDS‐PAGE）显示，其融合蛋白大小约为28×103，与目的蛋白大小一致。Western blot鉴定结果显示表达的蛋白正确，为人IL‐4目的蛋白。结论成功表达纯化到人IL‐4重组蛋白。
목적：구건인IL‐4중조표체재체，표체순화병감정인IL‐4중조단백。방법채용소식PCR기술종건강지원자외주혈단핵세포（PBMC）총RNA중확증인IL‐4개방열독광기인，장확증적IL‐4목적기인여표체질립pET101/D‐TOPO련접，전화대장간균BL21，진행표체순화화감정。결과채용소식PCR확증득도IL‐4개방열독광대소위460 bp ，측서비대현시서렬정학。전화BL21후，통과대불동극륭자표체수평적사선，사선도고표체 IL‐4적극륭자，표체화순화후취병희선알응효전영（SDS‐PAGE）현시，기융합단백대소약위28×103，여목적단백대소일치。Western blot감정결과현시표체적단백정학，위인IL‐4목적단백。결론성공표체순화도인IL‐4중조단백。
Objective Construction of human IL‐4 recombinant expression vector and then conduct the expression ,purification and identification of human recombinant IL‐4 .Methods the open reading frame of IL‐4 was amplified by nest PCR with total RNA from PBMC of healthy volunteer .And then the amplified IL‐4 was inserted into pET101/D‐TOPO ,transformed into BL21 ,ex‐pressed ,purified and indentified .Results The size of amplified open reading frame of IL‐4 was about 460 bp and the sequence was correct .After transformed into BL21 ,the IL‐4 clone with higher expression level was selected by selection of different clones insert‐ed with IL‐4 and the size of expressed ,purified IL‐4 was about 28 × 103 .Western blot results showed that the size of single band was identical with the expected protein .Conclusion Human IL‐4 recombinant protein was got successfully .