目的：本课题组前期发现凋亡诱导因子（apoptosis-inducing factor,AIF）蛋白在非吸烟女性肺腺癌患者中存在第89位赖氨酸残基琥珀酰化修饰水平增高。本课题组拟制备识别AIF第89位赖氨酸残基琥珀酰化修饰的抗体,用于检测AIF在该位点琥珀酰化修饰。方法：利用在线数据库对AIF蛋白的理化性质、跨膜结构、信号肽、蛋白结构及琥珀酰化位点进行生物信息分析。根据AIF氨基酸序列合成包括89位赖氨酸残基（K89）位点的三条多肽,其中两条含AIF K89的多肽赖氨酸残基进行了琥珀酰化修饰。用三条合成的多肽偶联KLH,然后分别免疫家兔,制备特异识别AIF K89位琥珀酰化修饰的抗体。用ELISA、Dot blot、Western blotting和免疫组化对抗体效价和特异性进行检测。结合胶内酶解和高分辨生物质谱技术分析与AIF相互结合的蛋白。结果：生物信息学分析结果显示,AIF K89琥珀酰化修饰促使AIF 79~99氨基酸残基形成的α-螺旋结构发生变化：由一个较长的α-螺旋变成两个间隔短的较短的α-螺旋。合成的两条琥珀酰化修饰多肽和一条非琥珀酰化的多肽免疫家兔均成功获得相应的抗体;ELISA检测免疫血清抗体效价,分别是1∶486 k、1∶162 k和1∶18 k。制备的识别AIF K89琥珀酰化修饰的抗体识别AIF K89琥珀酰化修饰性多肽的效价是识别非琥珀酰化多肽的效价的27倍;抗体Dot blot检测,该抗体识别AIF K89琥珀酰化修饰性多肽的杂交信号强度是识别非琥珀酰化多肽的效价的20倍;Western bloting结果显示AIF琥珀酰化抗体能特异性识别AIF K89琥珀酰化修饰位点。质谱鉴定与AIF结合的蛋白有XIAP/BIRC4 、EIF3G和PRELID1等。结论：生物信息分析提示AIF K89琥珀酰化修饰影响AIF蛋白的结构。制备的AIF K89琥珀酰化抗体能特异性识别AIF K89琥珀酰化修饰,为进一步研究AIF琥珀酰化的机制提供了基础。
Abstract
Objective Our research team has previously found that AIF protein exhibits increased levels of lysine 89 succinylation modification in non-smoking female lung adenocarcinoma patients. Our team aims to prepare an antibody that can recognize the succinylation modification of lysine 89 residue on AIF, which will be used to detect AIF succinylation at this site. Methods Bioinformatics analysis was conducted using online databases to analyze the physicochemical properties, transmembrane structure, signal peptide, protein structure, and succinylation sites of AIF protein. Based on the amino acid sequence of AIF, three peptides were synthesized, including the lysine 89 residue (K89) site. Among them, two peptides containing AIF K89 were succinylated. The three synthesized peptides were coupled with KLH and then immunized in rabbits to prepare antibodies specifically recognizing AIF K89 succinylation modification. The antibody efficacy and specificity were evaluated using ELISA, Dot blot, Western blotting, and immunohistochemistry. Gel digestion and high-resolution mass spectrometry were used to analyze the proteins interacting with AIF. Results Bioinformatics analysis revealed that succinylation modification of AIF K89 led to a change in the α-helical structure formed by amino acid residues 79-99 of AIF: it transformed from a longer α-helix into two shorter α-helices with a gap. The two synthesized succinylated peptides and one non-succinylated peptide successfully generated corresponding antibodies in rabbits. The antibody titers in the immune sera detected by ELISA were 1: 486, 000, 1: 162, 000, and 1: 18, 000, respectively. The prepared antibody recognizing AIF K89 succinylation modification exhibited 27 times higher efficacy in recognizing succinylated peptides compared to non-succinylated peptides, as determined by antibody Dot blot. Western blotting results showed that the AIF succinylation antibody specifically recognized the AIF K89 succinylation modification site. Mass spectrometry identified proteins interacting with AIF, including XIAP/BIRC4, EIF3G, and PRELID1. Conclusion Bioinformatics analysis suggests that succinylation modification of AIF K89 affects the structure of AIF protein. The prepared AIF K89 succinylation antibody can specifically recognize AIF K89 succinylation modification, providing a foundation for further investigation into the mechanism of AIF succinylation.
关键词
AIF /
琥珀酰化 /
肺腺癌 /
互作蛋白 /
抗体制备
Key words
AIF /
succinylation /
lung adenocarcinoma /
interacting protein /
antibody preparation
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基金
广东医科大学生物化学与分子生物学课程建设项目 （4SG22095G）
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