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

作　　者：张迎娜[1] 高峰[2,3] 方华[2,3] 赵雪[2,3] 张婧[2,3] 杜英[1] 潘卫东[1] 

机构地区：[1]郑州大学基础医学院微生物学与免疫学教研室,郑州450001 [2]河南省医药科学研究院神经免疫学研究室,郑州450052 [3]郑州市神经免疫学重点实验室,郑州450052

出　　处：《郑州大学学报（医学版）》2015年第5期601-606,共6页Journal of Zhengzhou University(Medical Sciences)

基　　金：国家自然科学基金资助项目30570625;河南省省属科研单位社会公益项目预研专项资金资助项目

摘　　要：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。Aim：To fusion and express the extracellular of human aquaporin 4（AQP4） in prokaryotic cells, and puri-fy and identify the expressed products .Methods：The sequences of the extracellular of human AQP 4 were designed accord-ing 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 West-ern 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 mon-oclone 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 .

关 键 词：水通道蛋白4 原核细胞 融合蛋白 

分 类 号：R744.52[医药卫生—神经病学与精神病学]