The Development And Preliminary Application Of Multiple Antigenic Peptide Microarray Plateform

AIM: Since Human Genome Project has been put in practice, proteomics has already become the research hotspot in life science following genomics. Facing the tremendous bioinformation, researchers all over the world developed nucleic acid microarray and protein microarray for high throughput and parallel analysis of the bioinformation, which broke through the traditional methods revolutionarily. The microarray analysis has become one of the most important technologies in the screening of biological markers, the analysis of interaction between biological molecules, the functional research of biological molecules and diagnosis and prognostic evaluation of diseases. Multiple Antigenic Peptide (MAP) microarray is another technical innovation after nucleic acid microarray and protein microarray. It combines the advantages of parallelism, high throughput, automation and low cost, aiming at the active sequences of target protein molecules and taking nowaday technologies of proteomics research into a deeper level. This study aims to establish a fast, high-throughput, automated and low-cost microarray platformfor identification, typing, and screening of microbial pathogen and assistant development of peptide vaccine basing on the design and synthesis of multiple antigenic peptide.METHODS: According to the works we had done previously, we chose Human cytomegalovirus and Helicobacter pylori as our subjects and analyzed the secondary structure, hydrophilicity, surface accessibility and flexibility of Human cytomegalovirus glycoprotein B, phosphoprotein pp150 and Helicobacter Pylori urease beta subunit. On the basis of the indexes mentioned above and the information pressed before about the linear epitopes of the two microbial pathogens, we selected several specific epitopes and ensured that they had not cross epitopes with other unrelated microorganisms through BLAST tool. The multiple antigenic peptides containing selected sequence were synthesized by Fmoc solid phase chemistry and purified through High Performance Liquid Chromatography, the molecular weights of MAPs were identified by MALDI-MS subsequently. After the multiple antigenic peptides were printed on nitrocellulose membrane by computer-controlled high-speed robotics, the nitrocellulose membrane was blocked with 2 % bovine serum albumin solution. The multiple antigenic peptide microarray was finished by assembling the membrane with macromolecular bibulous materials and plastic outer shell. Some microarrays were selected at random for quality control and the others were used for clinical trial. The results were compared with those obtained by ELISA method.RESULTS: Assisted by computer analysis and the information pressed before about the linear epitopes of the two microbial pathogens, we designed 4 multiple antigenic peptides, 2 for Helicobacter Pylori urease beta subunit(DKSIKEDVQF and SVEVGKVADL) and 2 respectively for Human cytomegalovirus glycoprotein B(TTVTSGSTKD) and phosphoprotein pp150 (NSPWAPTAPL). Through BLSAT analysis, they had not cross sequences with other unrelated microorganisms. After synthesized by Fmoc solid phase chemistry, the peptides were purified by High Performance Liquid Chromatography. The results of identification by MALDI-MS (Matrix Assisted Laser Desorption Ionization-Mass Spectrometry) is consistent with theoretical molecular weights. The multiple antigenic peptides were also identified for its antigenicity by ELISA with control sera, and then used for preparation of multiple antigenic peptides microarray. In the clinical trial of 120 random sera, the micrarray performed almost equally with the ELISA using recombinant antigen and microbial lysate antigen. Compared with the ELISA, the microarray had more than 90 % sensitivity and specificity as anti-Ure IgG and anti-PP150 IgG were detected. The CV were lower than 7 % between microarrays, which showed a good repeatability. CONCLUSION: Starting from Helicobacter pylori urease beta subunit, Human cytomegalovirus glycoprotein B and phosphoprotein pp150, we primarily established the multiple antigenic peptide microarray analytical platform. According to the results of clinical trial and comparison between the microarray and ELISA method, the microarray has important potential to be applied to clinical diagnosis and identification of epitope for the two microbial pathogens. As the result of high specificity of multiple antigenic peptide containing single epitope, the detective background was at a very low level, no nonspecific interaction was observed, and there was clear difference between positive and negative results, showing that multiple antigenic peptide microarray had higher specificity than early microarray based on recombinant...