抗钠泵α2亚单位截断性片段多肽抗体的制备与鉴定  

Preparation and characterization of polyclonal antibodies against rat sodium pump alpha 2 subunit M1-M2 extra membrane fragment

作　　者：张明娟[1] 张美程 朱参战[1] 张超英[1] 段宗明[1] 

机构地区：[1]西安交通大学医学院第二附属医院心内科,陕西西安710004

出　　处：《南方医科大学学报》2015年第2期168-173,共6页Journal of Southern Medical University

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)~~

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。Objective To prepare polyclonal antibodies against sodium pump alpha 2 subunit M1-M2 extramembrane fragment（NKAα2 EM1） for studying the pathogenesis of hypertension. Methods According to the Gen Bank data, the amino acid sequence of NKAα2 EM1 was obtained and the target peptide（LAAMEDEPSNDN） was synthesized using a peptide synthesizer with Fmoc method and purified with high-performance liquid chromatography. The synthesized peptide was then coupled to KLH for immunizing New Zealand white rabbits for 4 times to obtain the antiserum. The Ig G antibodies against the synthetic peptide, after affinity purification with Protein A, were used for detecting NKAα2 EM1 expression in rat aortic vascular smooth muscle cells by enzyme- linked immunosorbent assay and immunocytochemistry（ICC）. Results The synthesized peptide fragment, which consisted of 13 amino acid residues including one derivatized cysteine residue in the Nterminal（LAAMEDEPSNDN- C）, had a theoretical relative molecular mass of 1408.48 D with a measured relative molecular mass of 1407.90 D and a purity exceeding 85.5%. The titer of the antiserum was more than 1：512 000, and the purified Ig G antibody concentration was 0.965 mg/ml after purification with Protein A. At a 1：1000 dilution（final concentration of 1 μg/ml）,the titer of the purified Ig G antibody was more than 1： 256 000. The purified Ig G antibody could be used at 1：100 to 1：200dilutions for for immunocytological examination of formalin- fixed cells. Conclusion The anti- NKAα2 EM1 polyclonal antibodies obtained can be used in ELISA and immunocytochemistry for detecting the sodium pump alpha 2 subunit in formalin-fixed tissue or cells to facilitate investigation of the relationship between sodium pump and hypertension.

关 键 词：钠泵 钠泵α2亚单位 多肽 抗体 

分 类 号：R392[医药卫生—免疫学]