| Adenovirus(Ad)-based gene delivery systems have been widely used as vehicles for gene therapy and as vaccine vectors.The nonhuman Ad that are currently used as vaccine vectors for their natural host species, with an excellent safety record. A recombinant canine adenovirus type 2 expressing the rabies virus attenuated strain SRV9 glocoprotein has been constructed in our lab previously. The immunogenic effectiveness of rabies was shown effective, but it was still lower than immunization with live or inactive rabies virus. The immunogenic effectiveness of recombinant vaccine may be effected by many factors, and greatly compromised by the levels of the transgene product and the levels of preexisting immunity. In this study, we attempt to take advantage of the adenovirus capsid-hexon protein for presenting peptide epitopes to construct the CAV-2 gene delivery vectors with effective and stability.The capsid of each Ad virion is composed of three major proteins: hexon, fiber, and penton base. Hexon, the largest and most abundant of the Ad capsid proteins, with 720 copies/virion. In the mature virus, hexon exists as homotrimeric capsomeres which make up the facets of the icosahedral virion. The crystal structures of HAV2 and HAV5 hexons have been solved, revealing a complex molecular architecture. The base of each monomeric subunit consists of twoβ-barrel motifs, and three long loops extend out from the base structure to form the tower region of each molecule. Sequences within these loop domains protrude to the surface of the capsid to form the exterior of the virion. Alignments from different adenovirus serotypes show that the sequences located on the capsid exterior are poorly conserved in both length and amino acid sequence, termed hypervariable regions (HVRs). Futhermore, based on early sequence alignments, seven HVRs were identified throughout the human adenovirus hexon molecule. However, recent work has suggested that HVR7 is composed of three separate poorly conserved regions, bringing the number of HVRs to nine. Because the HVRs are poorly conserved between serotypes and do not appear to be involved in maintaining the structural integrity of hexon, it was hypothesized that small changes could be made to these domains without affecting the viability of the virus. In addition, many work has verified that the HVRs of the human adenovirus hexon can accommodate modifications without affecting viral formation and stability.In order to study the possibility of modification of CAV-2 hexon protein, and establish the antigen epitope displayed technology based on CAV-2 capsid, the structure of CAV-2 was analysised by bioinformatics, and then we genetically incorporated rabies virus glycoprotein epitope into different HVRs of CAV-2 hexon to test whether the normal biology of recombinant CAV-2 was affected by the mutation.In this study, the hexon protein full-length of two canine adenovirus type 2 attenuated stains, YCA-18 and CC0710, were obtained by PCR, cloned and sequenced. Then, a comprehensive comparison of hexon protein sequences of 15 different mastadenovirus serotypes was performed for mapping of conserved and variable regions, and the secondary and tertiary structure of hexon protein were predicted by homology modeling in order to deduce the location of nine HVRs in loop 1 and 2. The Glycoprotein antigen epitope(WSPIDI) of rabies virus and flanking Lys-Gly-Ser spacers were genetically incorporated into HVRs 2, 3, 5 and 7 of CAV-2 hexon gene by overlapping PCR. To rescue viruses, these modified plasmids were transfected into the MDCK cells with LipofectamineTM 2000, but no evidence of virus growth was detected even after mang times of transfection. We performed SDS-PAGE and Western blotting analysis of MDCK cells after transfection to examine whether the recombinant plasmids could replicate, transcribe and translate. The experimental data indicated that the modified plasmids were indeed expressed well in cells. The result suggested that the HVRs which were deduced and mutated may be the important to the encasement and/or stability of recombinant virus, and may not accommodate with foreign peptides. The structure model of the modified CAV-2 hexon were further analysised. The structure of loop1 become to loose or tighten by the mutation of HVR2, HVR3 and HVR5. A additionalα-helix was formed after the linear epitope incorporated into HVR2, while theβ-pleated sheet is substitute by a random coil in HVR3 and HVR5. HVR7 appeared to present on surface loop2, the random coil become toβ-pleated sheet after mutation. Furthermore, the partial spatial conformation of hexon may be affected by the incorrect interaction between hexons or with other capsid components. The loops(amino acids 157 to 160) is involve in interaction between hexon monomer, and may be affected by replacement. To reconstruct the CAV-2 hexon modification gene transfer vector with the experience of HAV-2 and HAV-5, could not get the recombinant CAV-2. The modification may affect the partial spatial conformation of hexon or the interaction between capsid proteins, and hamper the encasement of virion. Finally, the recombinant viruses were not successfully rescued in MDCK cells. Our study provided valuable information for studies involving CAV-2 displayed gene transfer vectors. |
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