Recombinant Expression Of Antigen Epitope Of Human Cytomegalovirus And Immunogenicity Detection Of Recombinant Protein

Human cytomegalovirus(HCMV)is common in nature,and belongs to double-stranded DNA virus of the herpes virus family.It is studied that HCMV participates in the occurrence of various diseases.It especially causes the infection of immunocompromised populations and fetus.Because the severe diseases caused by HCMV infection have been increasing,scholars have began to study treatment and prevention for HCMV.Because the drug treatment of HCMV can produce extreme side effects,the preparation of HCMV vaccine is the most effective way to prevent and treat HCMV.Glycoprotein B(gB)is one of the most studied target proteins of HCMV,which can induce strong humoral immune.And phosphoprotein65(pp65)is one of the most studied target proteins of HCMV,which can induce strong cellular immune response.According to literature and bioinformatics analysis,490-508 amino acid sequence of pp65 protein and 607-621 amino acid sequence of gB protein have good immunogenicity.So they were immunodominant epitopes for HCMV.The encoding genes of 490-508 aa in pp65 protein and 607-621 aa in gB protein were selected as gene segments.These encoding genes were synthesized artificially as a template for amplification.The genes were cloned into expression vector pET32a(+)after amplified by PCR.After recombinant plasmid was transformed into E.coli BL21(DE3)plys,fusion protein was expressed.The protein was purified by nickel ion affinity column which yield was 30.5%.Then Balb/c mice were immunized with purified protein.After separating serum of mice,the specificity of the antibodies was measured by Western blot.The result showed that the antibody for HCMV has specificity.The titers reach to 1 : 1 0 2 4 0 0 detected by indirected ELISA.And indirect ELISA method of HCMV was developed.The result showed that the recombinant protein can induce significant humoral immunity in mice.The number of CD4+ T cells and CD8+ T cells in peripheral blood were 48.65% and 16% respectively when detected by flow cytometry.The number of CD4+ T cells and CD8+ T cells were significantly higher than control group(38.05% and 13.15%)(P<0.01).The quantities of IFN-?,IL-2,IL-12 were 2.39 pg/L,87.07 ng/L,188.02 ng/L respectively when detected by sandwich ELISA.The quantities of IFN-?,IL-2,IL-12 were significantly higher than control group(1.85 pg/L,52.95 ng/L and 88.51 ng/L)(P<0.01).So the recombinant protein can induce significant cellular immunity in mice.In conclusion,the fusion protein with dominant antigen epitopes of pp65 and gB can induce significant humoral immunity and cellular immunity in mice.It can be used as HCMV subunit vaccine that can prevent and treat HCMV infection effectively.And it will lay the foundation for relevant vaccine research and HCMV relevant diseases prevention and treatment in the future.