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

目的:构建人C22orf37基因的原核表达载体,表达并纯化C22orf37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.

AIM： To clone, express and purify C22orf37 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 DHSα and induced to express fusion protein His-C22orf37 with IPTG. The products expressed in E. coli were analyzed by SDSPAGE 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 monoelonal antibody and further studies of the tissue distribution and function of C22orf37.