人白细胞介素11融合蛋白切割位点的改变及成熟蛋白的纯化

将 h IL- 1 1 c DNA克隆入硫氧还蛋白基因融合表达载体 p TRXFUS的 trx A基因 3′末端 ,构建符合读码框的融合基因 ,并在两基因间改变原有的肠激酶切割位点 ,引入蛋白质的羟胺切割位点序列 ,该融合蛋白在大肠杆菌中表达量达 2 0 %以上 .经羟胺切割 ,柱层析等纯化步骤后 ,得到纯度98%以上的成熟 h IL - 1 1 .用 IL- 6依赖细胞株 7TDI及 MTT法测定生物学活性 ,比活达 8× 1 0 6 IU/mg.并对纯品进行了 Western blot,N端 1 5个氨基酸测序及 h IL- 1 1氨基酸组成等分析和鉴定

The Change of Fusion hIL-11 Cutting Site and Purification of rhIL-11

Interleukin eleven (IL 11) is a thrombopoietic growth factor that directly stimulates the proliferation of hematopoietic stem cells and megakaryocyte progenitor and induces megakaryocyte maturation resulting in increased platelet production.The 5′ modified hIL 11 cDNA was cloned to the 3′ end of the gene trxA in plasmid pTRXFUS to construct the trxA and IL 11 fusion expression vector in which the original enterokinase site between the two fused genes was changed to hydroxylamine site.The fusion hIL 11 was expressed in E.coli G1724 and amounted to more than 20% of the total bacteria proteins.Over 98% of the purity of rhIL 11 was obtained through a series of purification steps such as Q Sepharose fast flow,cutting with hydroxylamine,CM Sepharose fast flow.The bioactivity tested by the MTT method in 7TDI cell line was 8×10 6 IU/mg purified rhIL 11.The rhIL 11 has the same biological and immunological activity with Neumega,the standard IL 11.At last the sequence of the N terminal 15 amino acid residues and the amino acid components of the rhIL 11 were analysed.