Construction, Expression And Immunogenicity Of Gene Vaccines And Recombinant Adenovirus Of CCV

In order to clarify the novel molecular biological characteristics of canine coronavirus strain DXMV (CCV DXMV) isolated from the liver of a died giant panda, three pairs of specific primers were designed for amplification of spike protein gene, membrane protein gene and nucleoprotein gene by RT-PCR, respectively, according to the genomic sequence of CCV strain Insavc-1 published in GenBank. The PCR products were cloned into pGEM-T and sequenced. Then the nucleotide sequences of the three structural protein genes were analysed by DNAMAN and DNAstar software, respectively. The full length of spike protein gene of strain DXMV was 4362bp,which encoded 1453 amino acids. The initiative 18 amino acids were signal peptide. The membrane protein gene was 789bp, encoding of polypeptide of 263 amino acids which had a 17 amino acids constituting signal peptide. The overall length of nucleoprotein gene was 1146bp, which encoded 382 amino acids. The homologies of nucleic acid and deduced amino acid sequence of spike protein, membrane protein and nucleoprotein among the different isolates of CCV strain accessed by GenBank were 22.7-99.7% and 38.3-99.7%, 77.7-99.5% and 81.7-98.9%, 91.4-99.8% and 92.1-99.7%, respectively. The variation parts of spike gene mainly existed in front half of nucleotide sequence, especially the region 350-370nt, 439-478nt and1718-1818nt, while the region 1060-1700 showed highly conservative bases. Phylogenetic analysis based on the complete spike and membrane gene, indicated that there were two different genotypes of CCV, genotype I and genotype II. The strain DXMV, together with most classical isolates such as K378, C54, V6, V1 and Insavc-1 belonged to genotype II, whereas E1mo02 and 2303 were included into genotype I. There were high homologies among the strains of genotype II, while the low identities between the genotype I and genotype II. There are 34 potential N-glycosylation sites in deduced S protein for strain DXMV and V54, but 33 for strain Insavc-1. The N-glycosylation site 566-568 was possessed for most virulent strains but not for vaccinal strain Insavc-1. Moreover, analysis of hydrophobicity,antigenic index and surface probability plot of spike and membrane proteins, indicated that there were some differences between strain DXMV and Insavc-1. Jameson-Wolf antigenic index of spike and membrane proteins of strain were significantly higher than that of the vaccinal strain Insavc-1, which revealed the possibly reasons that strain DXMV had better immunogenicity than that of Insavc-1 in molecular aspects. Three eukaryotic expression vectors, pVAXS1, pVAXM and pVAXN, used as gene vaccines of canine coronavirus were successfully constructed, and expressed in MDCK cell line. The S1 gene fragment of strain DXMV, which encoded major antigenic region A, B, C and D of S protein, was amplified from pTS by PCR with a pair of specific primers and cloned into pVAX1 vector. The recombinant pTM and pTN were digested by KpnI/NotI and interesting gene fragments were recovered and further subcloned into pVAX1, respectively. The three constructed expression vectors, namely pVAXS1, pVAXM and pVAXN were used for transfection in MDCK cell. Transcriptions of interesting genes were detected by RT-PCR. The interesting proteins were detected by indirect immunofluorescent technique and indirect ELISA method. The results showed that transcription of interesting genes could be detected at 36 h post-transformation, and expressed proteins could be detected by the two methods mentioned-above after 48-96 hours of transformation. In order to construct a recombinant canine adenovirus type 2 (CAV-2) to express the spike glycoprotein of canine coronavirus (CCV), complete expression cassette harboring S1 gene of CCV was subcloned into the shuttle vector pVAXE3, then further cloned into the backbone vector pPoly2-CAV2 containing complete genome of CAV-2. To gain the recombinant canine adenovirus, the recombinant plasmid pCAV-2-CCV-S1 was linearized by ClaI/AscI to release recombinant genome, and then transfected into MDCK cell. The recombinant virus CAV-2-S1 was gained through 4 times of passaging in MDCK, and the classical cytopathic effects (CPE) of CAV-2 could be observed. The recombinant S1 protein expressed by recombinant virus, which was identified by western blot analysis, could be specifically recognized by polyclonal serum against CCV. Eight groups of mice were immunized by intramuscular injection with the different combinations as following: sole expression plasmid, two expression plasmids or three expression plasmids. Indirect ELISA, serum neutralization test and lymphocyte...