人水通道蛋白4胞外区的原核表达及纯化
Prokaryotic expression and purification of the extracellular of human aquaporin 4

目的:利用原核表达系统融合表达人水通道蛋白4(AQP4)胞外区,并进行纯化及鉴定。方法:根据大肠埃希菌密码子偏嗜性及后续实验需要设计并人工合成AQP4胞外区序列,退火形成双链后克隆至pET32a(+)载体,构建原核重组表达质粒,转化大肠杆菌BL21(DE3),IPTG诱导表达。表达的融合蛋白经Ni2+亲和层析柱纯化后,进行SDS-PAGE和Western blot鉴定。结果:重组表达质粒经双酶切和测序鉴定构建正确; 表达的融合蛋白pET32a(+)-AQP4胞外区相对分子质量约为26 000,可与鼠抗组氨酸单克隆抗体特异性结合,可以可溶性形式存在于上清液中,纯化的融合蛋白纯度达70%以上。结论:成功构建了重组表达质粒pET32a(+)-AQP4胞外区,并成功表达融合蛋白。
To fusion and express the extracellular of human aquaporin 4(AQP4)in prokaryotic cells, and purify and identify the expressed products.Methods: The sequences of the extracellular of human AQP4 were designed according to the E. coli preferred codons and the necessary of the experiment, synthesized, annealed and cloned into vector pET32a(+). The recombinant plasmids were transformed to E. coli BL21(DE3)for expression under induction of IPTG. The expressed fusion proteins were purified by nickel ion affinity chromatography, and identified by SDS-PAGE and Western blot.Results: Restriction analysis and sequencing proved that the recombinant plasmid was constructed correctly.The relative molecular mass of the fusion protein was 26 000.The fusion protein showed specific binding to mouse anti-His monoclone antibody and existed in the supernatant in a soluble form.The purity of the fusion protein reached more than 70%.Conclusion: The recombinant expression plasmid has been successfully constructed and expressed