NMDAR1蛋白膜外抗原结构域的重组表达、纯化和免疫反应原性鉴定

Expression, purification and immunoreactivity characterization of extracellular antigenic domains of NMDAR1 protein

摘要:

构建编码NMDAR1蛋白膜外片段的原核表达重组质粒，在大肠杆菌中诱导表达、纯化并鉴定其免疫反应原性。根据人NMDAR1基因序列，利用Phyre 2软件预测蛋白的三级结构并分析其结构域。设计引物用RT-PCR方法扩增编码NMDAR1膜外蛋白不同结构域的核酸片段，并插入原核表达载体pCold-SUMO构建重组质粒。转化DH5α感受态细胞，菌落PCR鉴定，阳性单克隆进行测序验证。鉴定正确的重组体转化大肠杆菌BL21(DE3)，IPTG诱导目的蛋白的表达和纯化，Ni-NTA柱亲和层析和凝胶过滤层析纯化蛋白，酶切切除融合蛋白6His-SUMO标签，用AKTA Purifier进行凝胶过滤层析，收集纯化蛋白。利用SDS-PAGE鉴定蛋白纯度，并用Western blotting进行免疫反应性鉴定。克隆获得NMDAR1膜外部分的三段DNA序列，分别是NR1-M1 (编码19–393 aa)、NR1-S1 (编码394–544 aa) 和NR1-S2 (编码663–800 aa)。其中NR1-S1和NR1-S2片段之间以G (甘氨酸) 和T (苏氨酸) 作为接头连接成为复合片段。经菌落PCR筛选和测序鉴定，成功构建了重组质粒pCold-SUMO-M1和pCold-SUMO-S1-GT-S2。SDS-PAGE鉴定结果表明重组质粒在大肠杆菌中经诱导可表达可溶性NR1-M1及NR1-S1-GT-S2蛋白。对表达产物进行亲和层析和凝胶过滤层析获得了高纯度的目标蛋白。Western blotting证实纯化的目的蛋白能与相应抗体发生特异性结合反应。本研究成功构建了NMDAR1蛋白膜外抗原结构域的原核表达系统，并获得了具有免疫反应性的NR1-M1及NR1-S1-GT-S2纯化蛋白。该蛋白有望用于NMDAR1蛋白的功能研究及自身抗体的检测。

Abstract:

This study aimed to construct prokaryotic recombinant plasmids for expression of the extracellular domains of NMDAR1 protein, purify and characterize the immunoreactivity of the recombinant proteins. Based on the mRNA sequence of human NMDAR1 gene, we predicted the structure of the antigenic domains in the extracellular part of the protein using the “phyre2” software. Primers were designed to amplify the nucleic acid fragments encoding the NMDAR1 extracellular antigenic domains by RT-PCR. The amplified gene fragments were cloned into pCold-SUMO vector to construct the recombinant plasmids which were transformed into Escherichia coli DH5α. The positive colonies harboring the recombinant plasmids were picked and verified by PCR and DNA sequencing. Then, the recombinant plasmids were transformed into E. coli BL21(DE3) strain and induced by IPTG for protein expression. The recombinant proteins were purified by Ni-NTA affinity chromatography. The target proteins were further purified by removing the 6 His-SUMO tag using enzyme excision followed by gel filtration chromatography using AKTA purifier. The purity of the recombinant proteins were evaluated by SDS-PAGE and the immunoreactivity were characterized by Western blotting. Three DNA fragments encoding the extracellular domains of NMDAR1 protein, including NR1-M1 (encoding 19–393 aa), NR1-S1 (encoding 394–544 aa) and NR1-S2 (encoding 663–800 aa), were amplified by RT-PCR. The NR1-S1 and NR1-S2 were linked with G (arginine) and T (threonine) amino acid as a combined fragment. The NR1-M1 and NR1-S1-GT-S2 fragments were cloned into pCold-SUMO vector and two recombinant plasmids, pCold-SUMO-M1 and pCold-SUMO-S1-GT-S2, were generated and expressed in E. coli. SDS-PAGE analysis showed that the recombinant plasmids expressed soluble NR1-M1 and NR1-S1-GT-S2 proteins in bacterial. After affinity chromatography and gel filtration chromatography, we obtained high purity target proteins. Western blotting assay showed that the recombinant proteins NR1-M1 and NR1-S1-GT-S2 can bind specially with their corresponding antibodies, suggesting the recombinant proteins retained antigenic reactivity. We constructed a prokaryotic expression system for expressing the NMDAR1 protein extracellular parts that had immunoreactivity successfully, and the purified proteins can be used for studying NMDAR1 function and testing the autoantibodies.