牛阴离子交换蛋白1和生电碳酸氢钠协同转运蛋白亲水结构域的克隆·表达和纯化

Expression and Purification of Hydrophilic Domains of Bovine Anion Exchanger,Member 1 and Electrogenic Sodium Bicarbonate Cotransporter 1

[目的]表达和纯化牛阴离子交换蛋白1(AE1)和生电碳酸氢钠协同转运蛋白(NBCe1)的亲水结构域。[方法]根据AE1和NBCe1的亲水结构域设计引物,通过PCR扩增牛AE1和NBCe1的亲水结构域,通过原核表达系统,以E.coliBL21(DE3)为表达宿主,经IPTG诱导,表达重组的牛AE1和NBCe1的亲水结构域融合蛋白,并采用金属螯合层析法对目的蛋白进行纯化。15%SDS-PAGE分析目的蛋白的表达、分布和纯度。[结果]PCR扩增了牛AE1和NBCe1的亲水结构域;IPTG诱导后,成功的表达了目的蛋白;目的蛋白主要存在大肠杆菌的胞浆中,可以被较好的纯化。[结论]牛AE1和NBCe1的亲水结构域蛋白的表达为制备抗体和研究膜载体蛋白的调节机理提供了条件。

[ Objective] To express and purify the intracellular hydrophilic domains of cattle membrane carrier proteins, anion exchanger, member 1 （AE1）and electrogenic sodium bicarbonate cotransporter 1 （NBCel）, which were associated with the transfer of HCO3^- [ Method ] The hydrophilic domains of bovine AE1 and NBCel were amplified respectively by PCR and inserted into prokaryotic expression vector pET28a. Recombinant plasmids were transformed into the expression strain E. coli BL21 （ DE3 ） and then induced by IPTG. The expressed proteins were purified by Ni^2＋ affinity chromatography and analyzed by 15% SDS-PAGE. [ Result] The bydrophilie domains of bovine AE1 and NBCel were amplified respectively by PCR and expressed in prokaryotic expression system with the induction of IPTG. The proteins were mainly expressed in the cytoplasm of E. coli and high-purity was achieved by Ni^2＋ affinity chromatography. [ Conclusion] The expression of the hydrophilie domains of bovine AEI and NBCel provides a major exit route for preparation of antibodies and the regulatory mechanisms of carrier proteins.