血型A抗原模拟多肽的初步研究

发布时间：2018-03-26 03:09

本文选题：噬菌体展示　切入点：血型A抗原　出处：《华中科技大学》2008年博士论文

【摘要】： 第一部分血型A抗原模拟多肽的筛选目的: 筛选血型A抗原的模拟多肽。方法: 用血型A抗体NaM87-1F6对噬菌体随机12肽库进行三轮筛选,随机挑选32个噬菌体克隆,经噬菌体酶联免疫吸附试验(ELISA)、噬菌体微筛选鉴定噬菌体与抗体的结合力。测定特异性噬菌体DNA序列并推导其展示的多肽序列。凝集抑制试验检测噬菌体展示多肽对血型A抗原的模拟作用。结果: 1.经三轮筛选和相应的鉴定后得到7个抗A抗体NaM87-1F6的特异性噬菌体克隆。 2.其中6噬菌体(C5,22,23,26,27,31)展示的多肽序列为EYWYCGMNRTGC(将此多肽序列命名为P5,展示此多肽的噬菌体以C5为代表),另一噬菌体克隆C17展示的多肽序列为QIWYERTLPFTF(将此多肽序列命名为P17)。 3.凝集抑制试验提示展示这两个多肽的噬菌体可以抑制A型红细胞的凝集作用,阴性对照噬菌体原肽库不能抑制这种凝集作用。结论: 实验结果提示,多肽EYWYCGMNRTGC,QIWYERTLPFTF可以模拟血型A抗原的表位。此两种多肽具有建立新型诊断和亲和滤除抗A抗体方法的潜能。第二部分血型A抗原模拟多肽的GST融合表达目的: 构建表达血型A抗原模拟多肽的融合表达载体,鉴定融合表达蛋白模拟血型A抗原的能力。方法: PCR扩增血型A抗原模拟多肽(P5、P17)和谷胱苷肽S-转移酶(GST)编码基因,并连接成P5-GST和P17-GST,双酶切后重组入原核表达质粒pET28b,构建质粒pET28b-P5-GST和pET28b-P17-GST。通过酶切、测序鉴定重组子。以BL21(DE3)为表达菌,在IPTG诱导下表达P5-GST和P17-GST融合蛋白,Ni-NTA柱纯化蛋白。竞争ELISA鉴定融合蛋白对噬菌体展示多肽抗原活性的模拟作用,红细胞凝集抑制实验分析融合蛋白模拟血型A抗原的能力。结果: 1.成功构建P5、P17基因和GST基因融合后的原核表达质粒pET28b-P5-GST和pET28b-P17-GST。 2.P5-GST和P17-GST基因在宿主菌中高效表达并得到纯化。 3.P5-GST和P17-GST蛋白以浓度依赖的方式抑制A型红细胞的凝集作用。结论: 原核表达的多肽融合蛋白P5-GST和P17-GST可模拟天然血型A抗原,具有替代天然A抗原用于发展新型抗A抗体检测方法和滤除材料的潜能。第三部分模拟多肽融合蛋白检测血型抗体初步研究目的: 探讨多肽融合蛋白EYWYCGMNRTGC-GST和QIWYERTLPFTF-GST在血型A抗体检测中的应用价值。方法: 收集多份健康献血者血浆,试管凝集实验对血型抗体进行ABO定型,滴度测定;用基于GST融合表达多肽EYWYCGMNRTGC(P5-GST),QIWYERTLPFTF(P17-GST)的ELISA方法检测血型抗体;统计学分析比较ELISA方法与经典凝集试验检测血型抗体的效能。结果: 1.收集到120份健康献血者血浆,其中90份为抗A抗体阳性,30份抗A抗体阴性。 2.基于P5-GST的ELISA检测血浆抗A抗体实验受试者工作特性曲线的曲线下面积分别为0.873(P0.001),ELISA结果A值与凝集试验抗体滴度间的Spearman相关系数分别为0.728(P0.001)。基于P5-GST的ELISA在阈值为0.280时其敏感度、特异度分别为80%和76.7%。基于P17-GST的ELISA未能显示出具有检测血型抗体A的效能。结论: 基因工程多肽EYWYCGMNRTGC-GST融合蛋白具有一定的血型A抗体检测能力,为开发新型的血型抗体检测方法提供了思路。
[Abstract]:The screening of the first part of the mimic peptide of blood type A antigen
Objective:
Screening mimic peptides of blood type A antigen.
Method:
The phage random 12 peptide library by antibody of A type NaM87-1F6 three rounds of screening, 32 phage clones were randomly selected, the phage enzyme linked immunosorbent assay (ELISA), the binding force of phage screening and identification of phage antibody and micro - Determination of specific phage DNA sequence and its deduced polypeptide sequence show. Agglutination inhibition test detection of phage display the simulation effect of polypeptide on blood group A antigen.
Result:
1. after three rounds of screening and corresponding identification, 7 specific phage clones of anti A antibody NaM87-1F6 were obtained.
2., of which 6 phage (C5,22,23,26,27,31) displayed polypeptide sequence was EYWYCGMNRTGC (the polypeptide sequence was named P5, the phage displayed the polypeptide represented by C5), and another phage clone C17 showed the polypeptide sequence was QIWYERTLPFTF (named the peptide sequence P17).
3. agglutination inhibition test showed that the phage displayed these two peptides could inhibit the agglutination of A type red blood cells. The negative control phage peptide library could not inhibit this agglutination.
Conclusion:
The results suggest that polypeptide EYWYCGMNRTGC and QIWYERTLPFTF can mimic the epitope of blood group A antigen. These two kinds of peptides have potential to establish new diagnostic and affinity filtration methods for anti A antibody.
GST fusion expression of second partial blood type A antigen mimic peptides
Objective:
To construct a fusion expression vector expressing the mimic peptide of blood type A antigen, and to identify the ability of the fusion protein to mimic the blood group A antigen.
Method:
The amplification of PCR blood group A antigen mimic peptide (P5, P17) and glutathione S- transferase (GST) gene encoding, and connected into P5-GST and P17-GST into prokaryotic expression recombinant plasmid pET28b digested plasmid pET28b-P5-GST and pET28b-P17-GST. by enzyme digestion, sequencing of recombinant BL21 (DE3). For the expression of bacteria, the expression of P5-GST and P17-GST fusion protein induced by IPTG, purified protein Ni-NTA column. Identification of ELISA fusion protein show competition simulation effect on the activity of phage peptide antigen, hemagglutination inhibition test analysis of blood group A antigen fusion protein simulation ability.
Result:
1. successfully constructed the prokaryotic expression plasmid pET28b-P5-GST and pET28b-P17-GST. of the fusion of P5, P17 and GST genes
2.P5-GST and P17-GST genes are highly expressed and purified in host bacteria.
3.P5-GST and P17-GST proteins inhibit the agglutination of A type red cells in a concentration dependent manner.
Conclusion:
The fusion protein P5-GST and P17-GST expressed in prokaryotic expression can simulate A antigen of natural blood group. It has potential to replace natural A antigen for developing new anti A antibody detection methods and filtering materials.
A preliminary study on the detection of blood group antibodies by third parts of simulated polypeptide fusion protein
Objective:
To explore the application value of polypeptide fusion protein EYWYCGMNRTGC-GST and QIWYERTLPFTF-GST in the detection of blood group A antibody.
Method:
Collect more health blood donors, blood type antibody ABO titer setting, agglutination test; with the expression of GST fusion peptide EYWYCGMNRTGC (P5-GST), based on QIWYERTLPFTF (P17-GST) ELISA blood group antibody detection method; statistical analysis and comparison of ELISA method and classical agglutination test for detection of antibody potency.
Result:
1. the blood plasma of 120 healthy blood donors was collected, of which 90 were anti A antibody positive and 30 anti A antibodies were negative.
The area of 2. ELISA based on the detection of plasma P5-GST anti A antibody test receiver operating characteristic curve under the curve was 0.873 (P0.001), ELISA results A value and the Spearman correlation coefficient between the agglutination antibody titers were 0.728 (P0.001). Based on the P5-GST ELISA in the threshold of 0.280 is its sensitivity, specificity respectively 80% and 76.7%. P17-GST ELISA could not show the ability to detect A antibodies based on efficiency.
Conclusion:
The gene engineering polypeptide EYWYCGMNRTGC-GST fusion protein has a certain ability to detect the blood type A antibody, which provides a way of thinking for the development of a new blood type antibody detection method.

【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R392

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