重组人IL-4的克隆、表达和鉴定

摘要

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。%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 .