Preliminary Study On The Peptide Mimics Of Blood Group A Antigen

Part I Screening peptide mimics of blood group A antigenObjective:To screen peptides that can mimic the blood group A antigen.Methods:By using monoclonal antibody against blood group A antigen, a phage display 12-mer peptide library was biopanned and positive clones were selected by ELISA, micropanning. DNA encoding the peptides displayed on the positive phages were sequenced and the corresponding peptides were deduced. Agglutination inhibition test was used to assess the group A antigen-mimicking ability of the phage displayed peptides.Results:1. After three rounds of panning and the corresponding confirm test, seven phage clones can bind to NaM87-1F6 specifically.2. Six phage clones displayed the same peptide EYWYCGMNRTGC (named P5, phage clone C5 represent all the six phage clones in the follows), one displayed peptide QIWYERTLPFTF (named P17).3. The agglutination results showed that C5 and C17 can inhibit the agglutination of group A red blood cell. Phages from the primary library, as negative control, could not inhibit the agglutination.Conclusion:Our results suggest that peptides EYWYCGMNRTGC and QIWYERTLPFTF can mimic the epitope of blood group A angiten. The two peptides can be used to develop new methods to detect and absorb anti-A antibody. Part II The expression of group A mimicking peptides in GST fusion formObjective:To construct the fusion expression vector containing the genes encoding peptide P5 (or P17) and GST protein and determine the blood group A antigen mimicking ability of the fusion protein.Methods:The P5 (or P17) and GST genes were amplified by PCR and ligated as P5-GST (or P17-GST) gene. The two genes were cloned into pET28b plasmid and named pET28b-P5-GST, pET28b-P17-GST respectively. Restrict digest and DNA sequence analysis were used to confirm the constructed plasmids. Then the two plasmids were transformed into E.coli BL21 (DE3) respectively, expression of the fusion proteins were induced by IPTG. The fusion proteins were purified by Ni-NTA resin. The agglutination inhibition test was used to determine the group A-mimicking ability of the fusion proteins.Results:1. The pET28b-P5-GST and pET28b-P17-GST plasmids were constructed successfully.2. The fusion proteins (P5-GST, P17-GST) were expressed efficiently and purified successfully.3. The purified fusion proteins could inhibit the agglutination induced by anti-A antibody in a dose dependent manner.Conclusion:Our results suggest that P5-GST and P17-GST can mimic the blood group A antigen. They are the potential materials to substitute for natural group A antigen. Part III Preliminary study on the ability of the peptide mimics to determine the blood group antibodyObjective:To study the feasibility of the two peptide fusion proteins, P5-GST and P17-GST, to detect the blood group A antibody.Methods:The plasma samples were collected from health blood donors. The tube agglutination test was performed for blood group typing and titering. The ELISA based on P5-GST or P17-GST was constructed to test the blood group antibody. The ability of the ELISA method to test anti-A antibody was compared with that of the classic agglutination test by statistic analysis.Results:1. One hundred and twenty plasma samples were collected, among them ninety samples were anti-A antibody positive and the other were anti-A negative.2. In ELISA based on P5-GST fusion protein, the receiver operating characteristic (ROC) curve showed that area under curve (AUC) were 0.873(P<0.001). The Spearman correlation coefficient between the ELISA value and the antibody titer got from haemagglutination assay were 0.728(P<0.001). As for P5-GST, 0.280 was the best cutoff value for ELISA with 80% sensitivity and 76.7% specificity. ELISA based on P17-GST could not show the ability to detect anti-A antibody.Conclusion:Genetic engineering peptide-GST fusion protein, EYWYCGMNRTGC-GST, can detect anti-A antibody and has the potential to be used to develop method for detecting anti-A antibody.