抗钠泵α2亚单位截断性片段多肽抗体的制备与鉴定

发布时间：2018-05-12 16:52

本文选题：钠泵 + 钠泵α亚单位　；参考：《南方医科大学学报》2015年02期

【摘要】：目的采用多肽制备技术和免疫学技术制备抗钠泵α2亚单位截断性片段及其抗体,为检测和研究钠泵α2亚单位的组织学分布及其功能研究提供实验基础。方法根据NCBI-Genebank获得大鼠钠泵α2亚单位M1~M2膜外区截断性片段目的多肽(LAAMEDEPSNDN),采用9-氟甲氧羰基(Fmoc)固相合成法合成目的多肽,并采用碳化二亚胺法制备出多肽与孔戚血蓝素复合物,免疫新西兰大白兔,免疫4次后获得抗血清,测定其效价。随后采用protein A纯化Ig G抗体,并将其用于大鼠主动脉血管平滑肌细胞钠泵α2亚单的组织学检测。结果(1)人工合成的大鼠钠泵α2亚单位截断性片段长13氨基酸残基(LAAMEDEPSNDN-C),理论相对分子质量:1408.48,质谱实测相对分子质量:1407.90;高效色谱分析纯度HPLC纯度85.5%;(2)ELISA法检测免疫后兔子的抗血清效价均大于1∶512 000,蛋白A亲和纯化获得亲和纯化抗体浓度为0.965 mg/ml,按照1∶1000稀释(终浓度1μg/ml),ELISA检测抗体效价为1∶256 000;(3)该抗体按照1∶100~1∶200稀释可用于免疫细胞学检测。结论成功制备高效价抗钠泵α2亚单位截断性片段抗体可用于钠泵α2亚单位的ELISA和免疫细胞化学实验,为钠泵α2亚单位的组织细胞学检测和功能研究提供新的实验基础。
[Abstract]:Objective to prepare the truncated antisodium pump 伪 2 subunit fragment and its antibody by polypeptide preparation technique and immunological technique, so as to provide an experimental basis for the detection and study of the histological distribution and function of sodium pump 伪 2 subunit. Methods the extracellular fragment of rat sodium pump 伪 2 subunit (M1~M2) was obtained by NCBI-Genebank. LAAMEDEPSNDN was synthesized by solid phase synthesis of 9-fluoromethoxycarbonyl (FmocN), and the complex of polypeptide and hemocyanin was prepared by carbodiimide method. New Zealand white rabbits were immunized with antiserum for 4 times and their titers were determined. Ig G antibody was purified by protein A and used for histological detection of sodium pump 伪 2 subunit in rat aortic vascular smooth muscle cells. Results 1) synthetic rat sodium pump 伪 2 subunit truncated fragment with long 13 amino acid residues: LAAMEDEPSNDN-Con, theoretical relative molecular weight: 1408.48, mass spectrometry measured relative molecular weight: 1407.90. High performance chromatographic analysis of purity of HPLC by Elisa The titers of antiserum were all greater than 1: 512000, the antibody concentration of affinity purification of protein A was 0.965 mg / ml, and the antibody titer was 1: 256000 ~ 3 according to 1: 1000 dilution (final concentration 1 渭 g / ml ~ (-1) / ml ~ (-1). The antibody could be used in 1: 100 / 100 ~ 1: 200 dilution. Immunocytology examination. Conclusion the high titer anti-sodium pump 伪 2 truncated fragment antibody can be used in the ELISA and immunocytochemistry experiments of sodium pump 伪 2 subunit, which provides a new experimental basis for the detection of sodium pump 伪 2 subunit and the function of sodium pump 伪 2 subunit.
【作者单位】：西安交通大学医学院第二附属医院心内科;
【基金】：国家自然科学基金(30500204) 教育部新世纪优秀人才支持计划(NCET-13-0464)~~
【分类号】：R392

【参考文献】