牛乳及乳制品中过敏原β-乳球蛋白适配体的筛选及表征研究
首发时间:2020-03-06
摘要:本文利用指数富集的配体系统进化技术筛选过敏原β-乳球蛋白(β-lactoglobulin,β-Lg)的适配体,并对适配体结构特性进行初步研究,以期获得过敏原β-乳球蛋白的新型识别分子。利用Capture-SELEX技术,通过13轮的循环富集,经TA克隆测序,对获得的40条序列进行同源性和二级结构分析,利用等温滴定量热技术(ITC)和荧光分析法对挑选出的10条候选序列进行亲和力和特异性分析。实验结果表明,筛选富集获得一条最优适配体序列Lg-18,Kd值为65.00±27.42nmol/L,且适配体Lg-18能够与β-乳球蛋白特异性结合。作为β-乳球蛋白的一种新型识别分子,适配体具有亲和力高、特异性强、稳定性好和成本低的优点,因此在食物过敏原β-乳球蛋白的检测中具有广阔的应用前景。
关键词: β-乳球蛋白 适配体 指数富集的配体系统进化技术 等温滴定量热技术
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Selection and characterization of an ssDNA aptamer against allergen β-lactoglobulin of milk and dairy products
Abstract:In this paper, the aptamers against allergen β-lactoglobulin were selected by exponentially enriched ligand system evolution, and the structural characteristics of aptamers were preliminary studied. Capture-SELEX were used for 13 rounds of cycle enrichment, TA clone sequencing was performed to obtain 40 sequences for homology and secondary structure analysis. Isothermal titration calorimetry (ITC) and fluorescence method were used to assess affinity and specificity of aptamer. select the selected sequences.The results showed that the best of aptamer Lg-18had good affinity and specificity: the Kdwas 65.00±27.42nmol/L. As a new recognition molecule of β-lactoglobulin,Aptamers have the advantages of high affinity,strong specificity, good stability and low cost, so they have broad application prospects in the detection of allergen β-lactoglobulin in of cow milk and dairy products.
Keywords: β-lactoglobulin aptamer SELEX ITC
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牛乳及乳制品中过敏原β-乳球蛋白适配体的筛选及表征研究
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