两种方法纯化的抗体筛选华支睾吸虫模拟抗原表位的比较

Comparison of simulated epitopes by different methods of purification of IgG antibodies screened for antibodies to Clonorchis sinensis

目的比较用不同方法纯化的IgG从噬菌体随机12肽库中筛选出的华支睾吸虫模拟抗原表位。方法用饱和硫酸铵沉淀法和饱和硫酸铵沉淀法加层析法(两步法)纯化提纯的华支睾吸虫病患者血清IgG筛选华支睾吸虫模拟抗原表位,扩增随机挑取噬菌斑并进行序列分析和免疫学鉴定。结果硫酸铵沉淀法纯化IgG筛选的20个噬菌体克隆,6个有不同的DNA插入序列,Western blot显示6个克隆均可与华支睾吸虫患者血清反应,斑点免疫金银染色法有3个克隆可被华支睾吸虫患者血清识别。两步法纯化IgG筛选的20个噬菌体克隆,9个有DNA插入序列,其中有2个序列相同;Western blot和斑点免疫金银染色法显示9个克隆均可与华支睾吸虫患者血清反应。结论两种方法纯化的IgG均筛选到了可与华支睾吸虫患者血清结合的模拟抗原表位,用两步法纯化IgG筛选的模拟抗原表位更具有潜在的诊断价值。

Objective To compare simulated epitopes of Clonorchis sinensis screened from phage 12-mer peptide library with IgG purified by two different methods.Methods Three rounds of immunoscreening to a random 12-peptide phage display library were performed with purified IgG from the sera of patients infected with C.sinensis.Purification was by traditional saturated ammonium sulfate precipitation and a staged（two-step method） of ammonium sulfate precipitation.Blue clones were randomly selected and amplified and then subjected to DNA sequence analysis and immunoassay.Results Six different DNA sequences with 20 phage clones were screened by IgG purified by saturated ammonium sulfate precipitation.Western blot revealed that 6 clones all bound to sera from patients infected with C.sinensis,while dot-IGSS revealed that 3 clones were recognized by sera from patients infected with C.sinensis.Nine clone insert sequences from 20 plaque clones were screened by IgG purified by the two-step method;2 insert sequences were identical.As a result of Western blotting and dot-IGSS,9 clones were identified with sera from patients infected with C.sinensis.Conclusion Mimotopes combined with sera from patients infected with C.sinensis were screened using IgG purified by ammonium sulfate precipitation and a two-step method.Results suggested that mimotopes screened with IgG purified by the two-step method possess a greater potential diagnostic value.