Preparation And Immunogenicity Of Human Cytomegalovirus Clinical Strain Strain UL148 Gene Encoding Protein

Objective:To preparation the UL148Gene antibody of Human Cytomegalovirus in clinical strains andanalysis the Immunogenicity.Methods:1. On the based of the amino acid sequence of the gene D2UL148HCMV clinical strainsby the Bioinformatics, The secondary structure were analyzed by the SOPMA、GOR、HNN Programs. In combination with transmembrane, hydrophilicity, accessibility,flexibility and antigenicity analysis to selected the potential candidates of linear B-cellepitopes.2.3antigenic peptides were synthesized by the chemical synthesis which had the higherscore. The antigenic peptides were used to immune the rabbits (via the initialimmunization and strengthen immunization) to get the immunized rabbit serum. TheSaturated ammonium sulfate was used to purify the rabbit serum antibodies, and thetiters of3antigenic peptides were identified at different times and theimmunogenicity could be identified by the Elisa.3. The HCMV virus were inoculated in the human foreskin fibroblasts (HFF), and thenthe protein of cells that were infected was extracted. The HCMV UL148genecorresponding sensitivity and specificity of the antibody response was identified by theWestern Blot.Results:1. Comprehensive variety of forecasting methods, the265-275,221-229,18-25amino acidsequence regions of the HCMV UL148gene are main random coil or β-turn model inthe secondary structure and have the higher antigenic index. The P1(HCMVUL148265-275)、P2（HCMV UL14818-25）、P3（HCMV UL148221-229）are determinedfor the HCMV UL148gene candidates for B-cell epitopes. 2. The three peptides are immunized to the rabbits in1w,3w,5w, after the initialimmunization (1w) and strengthen immunization(3w,5w) can induce the titer ofimmune serum antibody increase; Three peptides can stimulate the body to produceantibodies, the titer of antibody increased the number of immune and rise up to1:12800or more by detected of the Elisa.3. Identified by the Western Blot, human foreskin fibroblasts(HFF)which infected by theHCMV, appear positive with the P3peptide (HCMV UL148221-229)antibody, however,the P1, P2peptide antibody are negative.Conclusion:1. Successfully to screen three HCMV UL148gene candidate B cell epitope which hadgood repeatability and high antigenic index by the Bioinformatics.2. Three peptides were synthesized by the chemical method, which could immunized therabbits to produce antibodies, along with the immunization, the titer of antibodyenhanced.3. The P3peptide (HCMV UL148221-229)antibody could be combined with HCMV UL148gene, which played an important foundation on the study of functional andimmunogenic of the HCMV UL148protein.