發(fā)布時間：2018-05-12 16:52

  本文選題：鈉泵 + 鈉泵α亞單位　； 參考：《南方醫(yī)科大學(xué)學(xué)報》2015年02期

【摘要】：目的采用多肽制備技術(shù)和免疫學(xué)技術(shù)制備抗鈉泵α2亞單位截斷性片段及其抗體,為檢測和研究鈉泵α2亞單位的組織學(xué)分布及其功能研究提供實(shí)驗(yàn)基礎(chǔ)。方法根據(jù)NCBI-Genebank獲得大鼠鈉泵α2亞單位M1~M2膜外區(qū)截斷性片段目的多肽(LAAMEDEPSNDN),采用9-氟甲氧羰基(Fmoc)固相合成法合成目的多肽,并采用碳化二亞胺法制備出多肽與孔戚血藍(lán)素復(fù)合物,免疫新西蘭大白兔,免疫4次后獲得抗血清,測定其效價。隨后采用protein A純化Ig G抗體,并將其用于大鼠主動脈血管平滑肌細(xì)胞鈉泵α2亞單的組織學(xué)檢測。結(jié)果(1)人工合成的大鼠鈉泵α2亞單位截斷性片段長13氨基酸殘基(LAAMEDEPSNDN-C),理論相對分子質(zhì)量:1408.48,質(zhì)譜實(shí)測相對分子質(zhì)量:1407.90;高效色譜分析純度HPLC純度85.5%;(2)ELISA法檢測免疫后兔子的抗血清效價均大于1∶512 000,蛋白A親和純化獲得親和純化抗體濃度為0.965 mg/ml,按照1∶1000稀釋(終濃度1μg/ml),ELISA檢測抗體效價為1∶256 000;(3)該抗體按照1∶100~1∶200稀釋可用于免疫細(xì)胞學(xué)檢測。結(jié)論成功制備高效價抗鈉泵α2亞單位截斷性片段抗體可用于鈉泵α2亞單位的ELISA和免疫細(xì)胞化學(xué)實(shí)驗(yàn),為鈉泵α2亞單位的組織細(xì)胞學(xué)檢測和功能研究提供新的實(shí)驗(yàn)基礎(chǔ)。
[Abstract]:Objective to prepare the truncated antisodium pump 偽 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 偽 2 subunit. Methods the extracellular fragment of rat sodium pump 偽 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 偽 2 subunit in rat aortic vascular smooth muscle cells. Results 1) synthetic rat sodium pump 偽 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 偽 2 truncated fragment antibody can be used in the ELISA and immunocytochemistry experiments of sodium pump 偽 2 subunit, which provides a new experimental basis for the detection of sodium pump 偽 2 subunit and the function of sodium pump 偽 2 subunit.
【作者單位】： 西安交通大學(xué)醫(yī)學(xué)院第二附屬醫(yī)院心內(nèi)科;
【基金】：國家自然科學(xué)基金(30500204) 教育部新世紀(jì)優(yōu)秀人才支持計劃(NCET-13-0464)~~
【分類號】：R392

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相關(guān)期刊論文 前1條

1 張明娟;楊軍;強(qiáng)磊;王蓉;宋亞燔;牛小麟;;大鼠鈉泵α2亞單位M1~M2膜外區(qū)多肽體外活性鑒定[J];生理學(xué)報;2008年02期

【共引文獻(xiàn)】

相關(guān)期刊論文 前1條

1 劉志文;連易水;李蟬;張s，

本文編號：1879388