CAJ | 학술논문

[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. coli BL21(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.
[목적]표체화순화우음리자교환단백1(AE1)화생전탄산경납협동전운단백(NBCe1)적친수결구역.[방법]근거AE1화NBCe1적친수결구역설계인물,통과PCR확증우AE1화NBCe1적친수결구역,통과원핵표체계통,이E. coli BL21(DE3)위표체숙주,경IPTG유도,표체중조적우AE1화NBCe1적친수결구역융합단백,병채용금속오합층석법대목적단백진행순화.15% SDS-PAGE분석목적단백적표체、분포화순도.[결과] PCR확증료우AE1화NBCe1적친수결구역;IPTG유도후,성공적표체료목적단백;목적단백주요존재대장간균적포장중,가이피교호적순화.[결론]우AE1화NBCe1적친수결구역단백적표체위제비항체화연구막재체단백적조절궤리제공료조건.
[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(NBCe1), which were associated with the transfer of HCO_3~-. [Method] The hydrophilic domains of bovine AE1 and NBCe1 were amplified respectively by PCR and inserted into prokaryotic expression vector pET-28a. 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 hydrophilic domains of bovine AE1 and NBCe1 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 hydrophilic domains of bovine AE1 and NBCe1 provides a major exit route for preparation of antibodies and the regulatory mechanisms of carrier proteins.