抗IDH2 K280乙酰化修饰特异性抗体的制备与鉴定

Preparation and identification of specific antibodies against acetylation modification of IDH2 K280

摘要:

目的 制备识别异柠檬酸脱氢酶 2（IDH2）第 280位赖氨酸残基（K280）乙酰化修饰的抗体，用于检测 IDH2在该位点的乙酰化修饰。方法 利用在线数据库对IDH2蛋白的理化性质、跨膜结构、信号肽、蛋白结构及乙 酰化位点进行生物信息分析。依据IDH2氨基酸序列合成3条包含K280的多肽，其中2条多肽的K280经乙酰化修 饰处理。随后将这 2 条乙酰化多肽分别与钥孔血蓝蛋白（KLH）偶联，并免疫家兔以制备能够特异性识别 IDH2 K280 位乙酰化修饰的抗体。采用 ELISA、Dot blot、Western blot、免疫组化对抗体效价和特异性进行检测。结果 生物信息学分析结果显示，IDH2 K280位乙酰化修饰和非乙酰化修饰蛋白在相对分子质量、理论等电点、带正电荷 残基总数、不稳定指数、疏水性上有差异；三级结构预测结果表明IDH2在280号位点上乙酰化修饰和非乙酰化修饰 突变体侧链在几何构型和长度上存在细微差异。合成的2条乙酰化修饰多肽和1条非乙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∶1 458 × 103、1∶1 458 ×103和 1∶54 ×103。制备的识别IDH2 K280乙酰化修饰的抗体识别IDH2 K280乙酰化修饰性多肽的效价是识别非乙酰化多肽的效价的27倍；识别IDH2 K280乙酰化修饰性多肽的杂交信号强度是识别非乙酰化多肽的效价的 20倍；IDH2乙酰化抗体能特异性识 别 IDH2 K280乙酰化修饰位点。结论 生物信息分析提示 IDH2 K280乙酰化修饰可能影响 IDH2蛋白的结构；制 备的IDH2 K280乙酰化抗体能特异性识别IDH2 K280乙酰化修饰。

Abstract:

Objective To prepare antibodies that specifically recognizing the acetylation modification at lysine 280 (K280) of isocitrate dehydrogenase 2(IDH2), facilitating the detection of IDH2 acetylation at this site. Methods Bioinformatics analysis was performed on the physicochemical properties, transmembrane structure, signal peptide, protein structure and acetylation sites of the IDH2 protein using online databases. Three peptides containing the lysine residue at position 280 were synthesized based on the amino acid sequence of IDH2, and two of these peptides were acetylated at the K280 site. Subsequently, these two acetylated peptides were conjugated to keyhole limpet hemocyanin (KLH) and used to immunize rabbits, aiming to generate antibodies specifically recognizing the acetylated modification at K280 of IDH2. The antibody titer and specificity were evaluated by ELISA, Dot blot, Western blot and immunohistochemistry. Results The results of bioinformatics analysis revealed significant differences in physicochemical properties between the IDH2 protein with acetylation modification at position K280 and its nonacetylated counterpart, including molecular weight, theoretical isoelectric point (pI), total number of positively charged residues, instability index, and hydrophobicity. The predictive analysis of the tertiary structure revealed slight differences in the geometric configuration and side chain lengths between acetylated and non-acetylated mutants of IDH2 at position 280. Antibodies were successfully generated by immunizing rabbits with two synthesized acetylated polypeptides and one non-acetylated polypeptide. The antibody titers of the immune sera, as determined by ELISA, were 1∶1 458 × 103 , 1∶1 458×103 , and 1∶54×103 respectively. The titer of the antibody specific to the IDH2 K280 acetylation modification was 27 times higher for the acetylated polypeptide compared to the non-acetylated polypeptide. The hybridization signal intensity of this antibody for recognizing the IDH2 K280 acetylated polypeptide was 20 times higher compared to its recognition of the non-acetylated polypeptide, which suggested that the IDH2 acetylation antibody specifically recognized the IDH2 K280 acetylation modification site. Conclusion Bioinformatic analysis suggested that IDH2 K280 acetylation modification affects the structure of IDH2 protein. The prepared IDH2 K280 acetylation antibody specifically recognizes the IDH2 K280 acetylation modification, which provides a basis for further investigation of the mechanism of IDH2 acetylation.