Human cytomegalovirus M epitope and application thereof to medical research field
A human cytomegalovirus and antigenic epitope technology, applied in antiviral agents, pharmaceutical formulations, antibody medical components, etc., can solve the problems of poor immunogenicity of antigenic epitope peptides, and the application research of antigenic epitope peptides is in the primary stage.
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Embodiment 1
[0022] Example 1. Screening of Goat Anti-HCMV Antigen Epitopes
[0023] First, 100 μl goat anti-HCMV polyclonal antibody IgG was used to coat the enzyme-linked plate, and placed at 4°C overnight. After blocking with 3% BSA for 1 h, add a phage display random dodecapeptide library (NEB product), incubate at room temperature for 1 h, and wash non-specifically bound phage with TBST (50 mmol / L Tris-HCl, 0.1% TWEEN20, pH 7.5) , and then use 0.2mmol / L glycine-HCl pH2.2 to elute the specifically bound phage, and the eluate is neutralized with 1mol / L Tris-HCl pH9.0. According to the method provided by the product kit, the titer of the phage in the eluate was determined, and the titer of the eluted phage of normal goat serum IgG was used as a control to determine the P / N ratio. At the same time, the eluted phages combined with TRAP antibody IgG were amplified and their titers were determined for the next round of screening. After 4 rounds of screening, the measured P / N ratio was si...
Embodiment 2
[0024] Example 2. The screened antigenic epitope sequence is compared with the M protein sequence
[0025] Analysis of the screening antigen epitope sequence and the original sequence of the M protein revealed that the antigen epitope sequence was highly homologous to the 32-38 amino acid sequence of the M protein:
[0026] Epitope sequence × L x LSQ x P
[0027] M protein primary sequence 31 H L V LSN f P 38
Embodiment 3
[0028] Example 3. Epitope polyantibody competition inhibits goat anti-HCMV polyantibody binding to virions
[0029] The M antigen epitope was coupled with keyhole limpet hemocyanin (KLH) through covalent bonds, and Balb / C mice were immunized to obtain M antigen epitope polyantiserum. After protein A purification, the M antigen epitope polyclonal antibody with higher purity was obtained.
[0030]According to the method of the kit, the HCMV virus was coated on the wells of the ELISA plate, and after being blocked with 3% BSA for 1 hour, the epitope polyclonal antibody and goat anti-HCMV polyclonal antibody of different concentrations were added, incubated at room temperature for 1 hour, and washed with PBST (20mmol / L PBS, 0.5% Tween-20, pH7.5) wash, then add HRP-labeled rabbit anti-mouse secondary antibody, and use ECL solution to show the OD value of antigen mimotope antibody binding to virus. The experimental results found that with the increase of the amount of goat anti...
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