Screening And Application Of Epitope In Glycoprotein M Of Human Cytomegalovirus

Human cytomegalovirus is a betaherpesvirus that infects a large proportion of the human population. Infection is usually asymptomatic in immunocompetent individuals but may be life threatening in immunocompromised hosts. As yet, the accuration of detecting HCMV by serological methods in clinical need to be improved in future, and there is no effectious vaccine to prevent its infection.Through subtractive strategy by normal goat IgG and targeted on the polyclonal antibody against HCMV, an epitope in glycoprotein M(gM) of HCMV, named CMVMME, was screened from phage random peptide display library. The X-XXXX motif was highly homologous with sequence of gM from compute analysis. After immunizing Balb/c mouses, the antibody against CMVMME binded specially not only the HCMV particles identified by capturing- ELISA, but also the recombinant protein gM confirmed by Western Blot. So CMVMME was a linear epitope of gM.To deeply understand the difference of CMVMME after single residues mutation, some conservative residues were mutated into glycine residue(G) respectively. Once Glutamine(Q) residue was mutated, the activity of CMVMME to bind the antibody against HCMV was reduced obviously through ELISA and Western Blot. Otherwise other residues had not the same characteristics. The result suggested that the bioactivity of CMVMME to bind antibodies can be effected by the conformation of Glutamine residue.To verify the neutralizing activity of the antibody against CMVMME, 50TCID₅₀ virus particles mixed with 50μg B65275G IgG, 50μg IgG against CMVMME, 50μg normal goat IgG and 50μg normal mouse IgG respectively were cultured for an hour in incubator at 37℃. Then the mixtures were used to infect the human embryonic lung(HEL) cell. It is finded that the antibody against CMVMME inhibited the virus from entrying into the HEL obviously confirmed by counting the numbers of the immediately early proteins of 68 kD tagged by FITC and expressed in HEL cell after virus infection. On the contrary, the normal mouse and goat IgG didn't. As the antibody against CMVMME could neutralize the activity of HCMV to bind their permissible cells, CMVMME was proved to be a neutralizing eiptope. After cloned into the bacterial random peptide library and expressed, the recombinant CMVMME binded the antibody B65275G specially, and the sera from immunized Balb/c mouses also binded gMaa30-78.To identify CMVMME to bind the human sera from HCMV infection, at first, 8 in 40 samples were detected HCMV IgG positive by two different ELISA kit. The recombinant peptide AD1 of gB binded 5 samples, the recombinant AD2 6 ones, and CMVMME 3 ones. But 7 in 8 samples binded the mixture of AD1/AD2/CMVMME.In summry, CMVMME not only can used to detect the virus,but also may be used as candidate to prevent the HCMV. All these results will be of great help for study on HCMV in the future.