筛选及鉴定IgG型单克隆抗-D噬菌体克隆的实验研究
发布时间：2018-08-21 08:07
【摘要】：目的利用随机噬菌体12肽库筛选出血型D抗原的模拟多肽并验证。方法采用Ig G型单克隆抗-D筛选噬菌体随机12肽库,测定每轮筛选后洗脱物与抗-Rh D的结合情况。对经特异性筛选得到的阳性噬菌体克隆进行DNA序列测定,将推导出的12肽氨基酸序列进行同源性比较,通过凝集抑制试验验证阳性噬菌体与Ig G型单克隆抗-D的反应。结果随着筛选轮次增加,噬菌体洗脱物与抗-Rh D的结合力逐渐增强。经过4轮淘洗后,得到8个亲和力较强且具有较高重合率的保守区域的12肽序列。凝集抑制实验表明具有相同氨基酸序列的阳性噬菌体对Ig G型单克隆抗-D与Rh D阳性红细胞的凝集反应具有抑制作用。结论随机噬菌体12肽库筛选得到的血型D抗原模拟多肽,为Rh D结构与功能研究及研制针对Rh D新生儿溶血病的新型诊断抗原、制备特异疫苗以及探索新的治疗方法提供实验基础。
[Abstract]:Objective to screen and verify the mimic peptide of blood group D antigen using random phage 12 peptide library. Methods the phage random 12 peptide library was screened by Ig G monoclonal anti-D and the binding of the eluate to anti-Rh D was determined after each screening. The DNA sequence of positive phage clones was determined by specific screening. The deduced 12-peptide amino acid sequences were compared with each other. The agglutination inhibition test was used to verify the reaction between the positive phage and IgG monoclonal anti-D. Results with the increase of screening times, the binding ability of phage elution and anti-Rh D increased gradually. After four rounds of washing, the 12 peptide sequences of 8 conserved regions with strong affinity and high coincidence rate were obtained. The agglutination inhibition experiment showed that the positive phage with the same amino acid sequence could inhibit the agglutination of IgG monoclonal anti-D and Rh D positive erythrocytes. Conclusion the mimic peptide of blood group D antigen obtained from random phage 12 peptide library is a novel diagnostic antigen for neonatal hemolytic disease of Rh D, which is used to study the structure and function of Rh D and to develop a novel antigen for diagnosis of Rh D hemolytic disease. Preparation of specific vaccines and exploration of new therapeutic methods provide experimental basis.
【作者单位】： 深圳市血液中心;
【基金】：深圳市卫生计生系统科研项目(201502008)
【分类号】：R446.6
，
本文编号：2195082
[Abstract]:Objective to screen and verify the mimic peptide of blood group D antigen using random phage 12 peptide library. Methods the phage random 12 peptide library was screened by Ig G monoclonal anti-D and the binding of the eluate to anti-Rh D was determined after each screening. The DNA sequence of positive phage clones was determined by specific screening. The deduced 12-peptide amino acid sequences were compared with each other. The agglutination inhibition test was used to verify the reaction between the positive phage and IgG monoclonal anti-D. Results with the increase of screening times, the binding ability of phage elution and anti-Rh D increased gradually. After four rounds of washing, the 12 peptide sequences of 8 conserved regions with strong affinity and high coincidence rate were obtained. The agglutination inhibition experiment showed that the positive phage with the same amino acid sequence could inhibit the agglutination of IgG monoclonal anti-D and Rh D positive erythrocytes. Conclusion the mimic peptide of blood group D antigen obtained from random phage 12 peptide library is a novel diagnostic antigen for neonatal hemolytic disease of Rh D, which is used to study the structure and function of Rh D and to develop a novel antigen for diagnosis of Rh D hemolytic disease. Preparation of specific vaccines and exploration of new therapeutic methods provide experimental basis.
【作者单位】： 深圳市血液中心;
【基金】：深圳市卫生计生系统科研项目(201502008)
【分类号】：R446.6
，
本文编号：2195082
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