GABARAP克隆表达及其抗体的制备与应用

Cloning and Expression of Human GABARAP and Preparation of Anti-GABARAP Antibodies

摘要:

GABARAP(GABAA-receptor-associated protein)是最新发现的与GABAA受体g2亚基胞内区有相互作用的蛋白, 目前的研究结果表明它可能是通过与微管相互作用辅助GABAA受体向细胞膜上运输并使受体在膜上聚集。本文中用PCR方法扩增出编码人GABARAP(hGABARAP)的cDNA片段, 然后分别克隆到真核表达载体pcDNA6/HA和谷胱甘肽转移酶(GST)融合蛋白表达载体pGEX4T2中。将后者导入大肠杆菌BL21中, 经过IPTG诱导后用谷胱甘肽偶联的Sep- harose-4B柱子纯化出融合蛋白GST-hGABARAP。以此蛋白作为抗原免疫家兔制备抗GABARAP的抗血清, 并用GST- hGABARAP耦联的NHS-activated Sepharose 4柱子纯化抗体。纯化的抗体可以用于真核细胞中过量表达hGABARAP的Western和免疫染色检测。结果表明, 过量表达的hGABARAP在真核细胞核和细胞质中都有表达, 部分集中于核周 区域。

Abstract:

GABARAP, a microtuble-associated protein, is identified to interact with GABAA receptor. Anchoring of the GABAA receptor to GABARAP helps to cluster the receptor at the synaptic termini and to mediate fast synaptic transmission. GABARAP may mediate interaction of gephyrin with the GABAA receptor to stabilize clusters by forming multimeric structures. Furthermore, GABARAP and gephyrin may play more of a role in receptor sorting and transport to the cell surface than in anchoring to the cytoplasm, because at inhibitory synapses GABARAP appears to associate with transport vesicles rather than the cell surface. The association of GABARAP with NSF (N-ethylmaleimide sensitive factor), a protein involved in intracellular vesicle transport, supports this hypothesis. We cloned cDNA encoding full-length human GABARAP by nested PCR and inserted it into eukaryon expression vector pcDNA6HA and GST fusion protein expression vector pGEX4T2. The recombinant plasmid pGEX4T2-hGABARAP was transformed into E. coli BL21, from which GST-hGABARAP fusion protein was purified after IPTG induction by affinity chromatography with glutathione Sepharose-4B column. The antiserum against GABARAP was generated by immunizing rabbits with the purified GST-hGABARAP and was purified with GST-hGABARAP coupled NHS-activated Sepherose 4 column. The purified polyclonal antibody was effective for Western blotting and immunostaining. The hGABARAP was located both in the cytoplasm and nucleus with an abundant distribution around the peripheral nucleus.