促红细胞生成素原核表达载体构建分离纯化

Vector construction,expression and purification of erythropoietin

目的构建和表达人源性促红细胞生成素(EPO),为促红细胞生成素发挥功能与结构差异研究做准备。方法通过NCBI查找人源性促红细胞生成素全基因序列并合成,构建原核表达载体pTWIN1-rhEPO,转化至大肠杆菌BL21(DE3),获得高效表达重组转化子菌株。结果成功构建人源性促红细胞生成素原核表达载体,经酶切和DNA序列测序分析,结果表明该基因序列与合成序列完全相同。终浓度0.5mmol/L ITPG诱导表达,SDS-PAGE电泳分析,4小时时蛋白表达量最高。结论构建和表达重组载体pTWIN1-rhEPO,分离纯化出目的蛋白,为进一步研究促红细胞生成素组织修复功能及突变体相关功能研究奠定基础。

Objective To construct and express the human-derived erythropoietin in order to study the differences in structure and function for the further research. Methods The rhEPO sequence was identified through NCBI and was synthesized, constructed Prokaryotic expression vector pTWINI-rhEPO, transformed into E. coli BL21 （DE3）, to obtain efficient expression of recombinant transformant strains respectively. Results Successfully constructed human-derived erythropoietin prokaryotic expression vector, as a result of digestion and DNA sequencing analysis showed that the gene sequence was identical with the synthetic sequence. The final concentration of 0.5mmol/L IPTG was used to induce protein expression, SDS-PAGE electrophoresis analysis, protein expression was highest at 4h. Conclusion Constructed and expressed the Prokaryotic expression vector pTWINI-rhEPO, utilized chitin affinity chromatography to separate and purify of rhEPO, for further study of erythropoietin tissue repair functions and the mutants＇ related functions do research foundation.