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目的利用计算机建模获得单克隆抗体2F2可变区三维结构,再与日本脑炎病毒(JEV)E蛋白进行模拟分子对接,了解JEV E蛋白上与2F2作用的关键氨基酸位点,为阐明2F2的中和作用机制奠定基础。方法利用BLASTP软件在蛋白质数据库(PDB)中搜索2F2同源蛋白。以同源蛋白为模板,用Modeller软件对2F2可变区主链、侧链进行建模,组装出完整的2F2可变区三维结构模型并进行一系列分子动力学优化,得到能量最低的单抗可变区三维结构。最后利用Discovery Studio软件模拟2F2可变区与JEV E蛋白的分子对接。结果获得2F2可变区三维结构模型。模拟分子对接结果显示JEV E蛋白上与2F2可变区相互接触、作用的氨基酸位点有9个,即Ⅰ区的13位谷氨酸、16位丝氨酸、37位天冬氨酸和300位苏氨酸,Ⅲ区的336位赖氨酸、347位天冬氨酸、354位亮氨酸、387位精氨酸和388位甘氨酸残基。其中Ⅰ区13位谷氨酸、16位丝氨酸和37位天冬氨酸可能是中和表位必需的氨基酸。Ⅲ区的387位精氨酸和388位甘氨酸位于优势中和抗原表位区域,336位赖氨酸与已报道的337位中和表位非常接近。结论通过计算机建模确定JEV E蛋白上有9个与单克隆抗体2F2作用的主要氨基酸位点,为利用E蛋白突变体阐明2F2单抗的中和机制提供了依据。
OBJECTIVE: To obtain three-dimensional structure of 2F2 variable domain of monoclonal antibody by computer modeling and then to simulate molecular docking with Japanese Encephalitis virus (JEV) E protein to understand the key amino acid sites of JEV E protein interacting with 2F2. To elucidate the role of 2F2 Neutralization mechanism to lay the foundation. Methods BLASTP was used to search 2F2 homologous proteins in the protein database (PDB). Using the homologous protein as a template, Modeller software was used to model the main chain and side chain of 2F2 variable domain. The complete 2F2 variable region 3D structure model was assembled and subjected to a series of molecular dynamics optimization to obtain the lowest energy monoclonal antibody Variable area three-dimensional structure. Finally, Discovery Studio software was used to simulate the molecular docking of 2F2 variable region with JEV E protein. Results Two-dimensional structure model of 2F2 variable region was obtained. Mock docking results showed that the JEV E protein interacted with the 2F2 variable region and interacted with 9 amino acid residues, that is, the 13th glutamic acid, the 16th serine, the 37th aspartic acid and the 300th sucrose in the Ⅰ region Lysine at position 336, aspartate at position 347, leucine at position 354, arginine at position 387, and glycine residue at position 388. Among them, glutamine at position 13, serine at position 16, and aspartate at position 37 may be essential amino acids for the neutralization epitope. The 387-arginine and 388-glycine in region III are located in the dominant neutralizing epitope region and the 336 lysine is very close to the reported 337-position neutralizing epitope. Conclusion Nine major amino acid sites on JEV E protein interacting with 2F2 were identified by computer modeling, which provided a basis for elucidating the mechanism of neutralization of 2F2 mAb by using E protein mutants.