摘要
目的为了进一步考察促红细胞生成素(EPO)的组织保护作用,构建了高效表达人源性EPO(rhEPO)的原核表达载体,并通过小鼠体内实验考察了其促红细胞生成活性。方法构建原核表达载体pET30b(+)-rhEPO;转化至大肠杆菌BL21(DE3),获得高效表达重组转化子菌株;Ni-NAT亲和层析纯化融合蛋白;利用网织红细胞指数考察融合蛋白在小鼠体内的促红细胞活性。结果成功构建pET30b(+)-rhEPO重组子;实现了在原核生物中的表达;纯化后的融合蛋白达到了电泳级纯;其相对分子质量与理论值相符,原核表达的融合蛋白能够显著提高小鼠体内网织红细胞数,可溶性融合蛋白和包涵体蛋白比活性分别是应用化学科1 059.63、727.94U/mg。结论构建和表达重组载体pET30b(+)-rhEPO,表达的目的蛋白经分离纯化后具有体内生物学活性,为进一步的功能研究奠定基础。
Objective To construct the efficient expression vector of the human-derived erythropoietin,and investigate its in vivo activity through experiment in mice,in order to study the tissues protective function for the further research. Methods Prokaryotic expression vector pET30b（＋ ）-rhEPO was constructed, transformed into E. coli BL21 （DE3）, to obtain low-cost and efficient expression of recombinant transformant strains,fusion protein purificated by Ni-NAT affinity chromatography column,and in vivo activity detected through animal experiment. Results Successfully constructed pET30b（＋）-rhEPO prokaryotic expression vector,and realized the expression in prokaryotes. The purified fusion protein showed expected molecular weight and reached the electrophoretic purity level,the reticulocyte number in rats increased significantly showed that the fusion protein has in vivo activity,the specific activity of soluble fusion protein is 1 059.63 μ/mg, and the insoluble fusion protein is 727.94μ/mg. Conclusion Construction and expression the prokaryotie expression vector pET30b（＋）-rhEPO,and the fusion protein has in vivo activity after separate and purify,which can be as the foundation for further study of erythropoietin functions.
出处
《重庆医学》
CAS
北大核心
2016年第28期3913-3915,共3页
Chongqing medicine
关键词
红细胞生成素
遗传载体
分离和提纯
活性检测
erythropoietin genetic vector
isolation and purification
activity detection
