猪白细胞介素-2重组蛋白的原核表达及其生物学活性分析  

Prokaryotic Expression and Bioactivity of Porcine Interleukin-2 Gene

作　　者：宋世斌[1] 

机构地区：[1]甘肃农业职业技术学院,甘肃兰州730020

出　　处：《畜牧兽医杂志》2015年第3期34-38,共5页Journal of Animal Science and Veterinary Medicine

摘　　要：用已构建好的重组表达载体pET30-IL2转化BL-21E.coli,挑取单克隆,利用诱导剂IPTG诱导表达;通过改变诱导剂的浓度、诱导的时间来摸索最佳诱导条件,在最佳诱导条件下大量表达,并利用镍层析柱纯化重组蛋白,用MTS法测定其生物学活性。结果表明,表达产物经SDS-PAGE分析,表达出25ku的融合蛋白,表达产物主要以包涵体的形式存在,最佳的诱导剂浓度为0.1mM,最佳的诱导时间为4h;对表达产物的包涵体处理后,用镍琼脂糖凝胶FF纯化,所得蛋白的纯度约在90%以上。通过透析除去部分尿素,复性后在体外利用MTS法检测其生物学特性,结果表明其仍具有较好的生物学活性,这为进一步大量制备和研究猪IL-2重组蛋白奠定了基础。In this study, the recombinant expression vector （pET30-- IL--2） was transferred into BL--21 E. coli and pick the monoclones. The pET30--IL--2 was induced to express by IPTG. The optimal condition was confirmed by changing the concentration of IPTG and induction time. The recombinant protein was purified by NI--affinity chromatography and the bioac- tivity was detected by MTS after the gene expressed abundantly in optimal condition. The results showed that the optimal IPTG concentration was 0. 1mM and the best induction time was 4 h. SDS--PAGE analysis showed that the expression product was a 25 ku fusion protein and it was existed in the form of inclusion body. The purity of the treated inclusion body was above 90% after purified by Ni FF affinity chromatography. Get rid of a part of carbamide by dislysis and detect the bioaetivity by MTS in vivo after renaturation. The results showed that it still remain a better biological activity and lay a foundation for pro- duce and study of the recombinant protein of porcine IL--2.

关 键 词：猪白细胞介素2 原核表达 纯化 活性分析 

分 类 号：S828[农业科学—畜牧学]