重组人C22orf37融合蛋白的克隆、表达、纯化及鉴定
Cloning, expression, purification and identification of recombinant human C22orf37 fusion protein
叶星明 1姜昌丽 1张英起1
作者信息
- 1. 第四军医大学药学系生物技术中心,陕西,西安,710033
- 折叠
摘要
目的:构建人C22orf37基因的原核表达载体,表达并纯化C22orf137重组蛋白.方法:应用PCR方法从人外周血白细胞cDNA文库中获得人C22orf37基因,成功构建人C22orf37融合表达载体,诱导表达His-C22orf37融合表达蛋白并对表达产物进行SDS-PAGE电泳分析和Western Blot鉴定.应用镍螯合层析法纯化重组融合蛋白,并对纯化产物进行纯度分析.结果:成功构建了人C22orf37重组融合载体pQE30-C22orf37,工程菌pQE30-C22orf37/DH5α经IPTG诱导后的菌体蛋白经SDS-PAGE电泳分析,在Mr约18 000处出现了一条新生的蛋白条带,Western Blot结果显示该条带能够与His抗体特异性结合.应用Ni-NTA层纯化出目的蛋白,纯化后的蛋白经Primer Premier软件分析纯度约80%.结论:成功构建了人C22orf37的融合蛋白原核表达载体并成功表达与纯化了人C22orf37的融合蛋白,为下一步的C22orf37 mAb的制备和C22orf37蛋白表达的组织分布及功能研究奠定了基础.
Abstract
AIM:To clone,express and purify C22orf 37 fusion protein in E. coli. METHODS: A 510 bp of human C22orf37 gene fragment was obtained by PCR method from human leukyocyte cDNA library and cloned into pQE30 vector,a His fusion expression vector. The recombinant plasmid was transformed into E. coli DH5α and induced to express fusion protein His-C22orf37 with IPTG. The products expressed in E. coli were analyzed by SDS-PAGE and identified by Western blot. The interesting protein was purified by Ni-NTA affinity chromatography and the purity of the purified protein was analyzed using the Primer Premier software. RESULTS:The recombinant plasmid pQE30-C22orf37 was constructed and the fusion protein was expressed in DH5a at a high level. SDS-PAGE showed that its molecular mass was about 18 kD. Recombinant protein was purified by Ni-NTA purification column and the purity of the fusion protein was about 80%. The His-C22orf37 fusion protein showed a good binding ability to anti-His antibody specifically. CONCLUSION: Our successful expression and purification of His-C22orf37 protein lay a basis for the production of anti-C22orf37 monoclonal antibody and further studies of the tissue distribution and function of C22orf37.
关键词
人C22orf37基因/原核表达/重组融合蛋白质类/分离和提纯Key words
human C22orf37 gene/prokaryotic expression/recombinant fusion proteins/isolation & purification引用本文复制引用
出版年
2009
NETL
NSTL
维普
万方数据