Preparation And Identification Of Anti-HCMV GBn1 Antibody

Background:Human cytomegalovirus(HCMV) infection is common in the world. Its clinical symptoms are mainly seen in people with hypoplasia of the immune system or immunosuppression. Envelope glycoprotein B(gB) of HCMV plays an important role in HCMV infection and induction of immune response. In addition,gBnl is a major pathogenic genotype,and holds an obvious quantitative advantage in HCMV infected Chinese.Objective:gBn1 which was synthetic peptide,was used to prepare anti-HCMV gBn1 antibody. The antibody would help clinical testing of HCMV gBn1 and preparation of HCMV vaccine in the future.Method:Based on the GenBank sequence (M60929) , gBn1 antigen peptide amino acid sequence was decided by use of computer-aided design. The peptide was synthetized and conjugated with Keyhole Limpet Hemocyanin (KLH) to heighten immunogenicity of gBn1 antigen. After the peptide gBn1 conjugated KLH immunized rabbits (including the initial vaccination and four times of enhanced immunization), rabbit immune serum were obtained, then purified by affinity purification. Direct ELISA assay was used to detect anti-gBn1 antibodies and identify the level of antibody. Immune cells staining was to identify the antibody and antigenic cross-reaction. Western-blot and immunoprecipitation methods were to identify the specificity and sensitivity of antibody.Result:Through the ELISA method and antigenic cross-reaction, it was showed that anti-gBn1 antibody could specificly bind to strains of HCMV Towne, titer could reach 1:64000, and the antibody could not response to the AD169 strain and gBn3 strain. Immunocytochemical staining showed that it was coincide with PCR typing gBn1 genotype, and there was no specific cross-reactions with the two other types (gBn2 and gBn3). The lysate of HCMV Towne strain, HCMV AD169 strain, HCMV gBn3 strain and normal cell lines were as antigen,which reacted to the rabbit antiserum in Western blot analysis. It showed that the serum and Towne strain appeared a clear band in molecular weight of about 110KD,which was the same as the molecular weight of HCMV gB; no other bands appeared. We considered the antibody could identify the same synthetic peptide fragments of HCMV, that was, gBn1-HCMV, but could not identify the other type strains of the virus,showing good specificity of the anti-gBn1 antibody.Conclusion:By ELISA method, immune cells staining and Western-blot, the anti-gBn1 antibody was able to sensitively and specificly identify HCMV gBn1 virus strains. It indicated that anti-gBn1 antibody was successfully prepared in this study . The antibody can be used in a wide range of applications,such as the diagnosis and treatment of HCMV infection and disease, research on the structure and function of the HCMV,and antibody analysis.