The Research Of Amino Acids Modification And Immunological Properties Of Human Adenovirus Type3Hexon Hypervariable Region1

Objective To investigate the malleability and immunological properties of humanadenovirus type3hexon hypervariable region1.Methods Modified hexon fragments were amplified by overlapping PCR usingbackbone plasmids pAdΔE3GFP as templates and cloned into pBR322L/R vector toform shuttle plasmids pBR-flag, pBR-his6flag, pBR-his6lgsflag or pBR-AdV4HVR5,after identified by sequencing, co-eletrotransformed into E.coli BJ5183cells toconstructe homologous recombinans plasmids with backbone plasmids pAdΔE3GFP.The resultant homologous recombinants plasmids were screened and identified byPCR, restriction enzyme digestion and sequencing, linearized with AsisI digestionand transfected into AD293cells to rescue modified viruses. Thermostability andgrowth kinetics characteristics of recombinant adenovirus were identified throughthermostability experiments and the growth kinetics respectively; the expression ofexogenous epitopes was identified by Western blot; the binding activity ofexogenous epitopes with Monoclonal antibody and antiserum were detected withELISA assay.Results By PCR, DNA sequencing, the epitope peptides flag, his6flag orhis6lgsflag were successfully incorporated into HAdV-3HVR1region except forepitope AdV4HVR5. Thermostability and growth kinetics characteristics ofrecombinant adenovirus were favorable. These epitope peptides were expressed inmodified hexon proteins and exposed on viron surfaces as assessed by western blotting assay and ELISA assay. Multiple vaccinations with the capsid-modified Ad3induced a humoral response against the epitope insertion in HVR1. Antiserum raisedagainst the rAd3/HVR1-his6flag showed reactivity with the motif “HHHDYK”expressed as GST-2fusion proteins in indirect ELASA. Antiserum raised against therAd3/HVR1-his6lgsflag showed reactivity with the motif “HHHLGSDYK”expressed as GST-1fusion proteins, motif “HHHHHHLGS” expressed as GST-3fusion proteins and motif “LGSDYKDDD” expressed as GST-4fusion proteins inindirect ELASA. In any case, motif “HHHHHHLGSDYKDDDDK” expressed asGST-his6lgsflag fusion proteins exhibited the highest reactivity to serum antibodyagainst rAd3/HVR1-his6lgsflag.Conclusion HAdV-3HVR1can be incorporated and displayed foreign linearepitopes with the length up to17amino acids and induced a humoral response inmice against the epitopes. These results indicate that when multiple epitopes weresimultaneously incorporated into single HVR, a new epitope have been generated,which induces a specifc epitope but a polyvalent humoral immune response.Therefore this study provides valuable information for the generation of multivalentvaccine vectors.