| Recent studies showed that CpG DNA, such as bacterial DNA, plasmid DNA and oligodeoxynucleotides (ODN) which all contain unmethylated CpG motifs, could be used for novel vaccine adjuvant to enhance the humoral and cellular immune responses. It was reported that CpG DNA can activate plasmacytoid dendritic cells (pDCs) and B cells directly, which express TLR9, and secondarily activate monocytes^nacrophages, NK cells, T cells and some non-immune cells, improve the ability of neutrophils to provide effective host defense, and produce Thl-like proinflammatory cytokines, interferons, and chemokines, to activate host defense mechanisms leading to innate and acquired immune responses. Therefore ,CpG DNA is expected to be a novel adjuvant for prophylactic and therapeutic vaccination for cancer, allergy and infectious diseases. However, one certain CpG motif is not always optimal to all host, so it is very important to screen CpG ODN with strong immunostimulatory activity on canine in vitro.Canine distemper (CD), a highly contagious disease with worldwide distribution, could cause severe economic losses to the canine fanning, far cultivation and wildlife conservation. The causative agent of the disease, canine distemper virus (CDV), is a member of Paramyxoviridae whose genome is a non-segmented single-strand negtive-sense RNA. The CDV NJ-15 strain is a virulent CDV isolated from dogs in Nanjing. Traditional attenuated vaccine against CDV has intrinsic shortcomings despite its important role in controlling the occurrence of CD. It can cause immunosuppression and lesion of CNS, and it may be lethal some susceptible animal and possess a wild hot spectrum. Moreover, mutation of CDV may also cause epidemic of CD. In order to develop a new kind of safe, efficient and convenient CDV genetically engineered vaccine, we constracted one genetic recombinant eukaryotic expression plasmid pcDNA-H and another recombinant prokaryotic expression plasmid pET-H, which express the H gene or its fragments, with the Nanjing isolate of CDV on the initiative exploring of the molecular property of H protein that is tightly related to the pathogenicity and immunity of CDV andsubsequently detected their protection immunogenicity in mice and dogs. These above works can be divided into 4 parts:1 T-A Cloning and Sequence analyses of hemagglutinin protein gene of Nanjing isolate Virus was isolated from the CDV NJ-15 strain infected Vero cells to extract its RNA as template of RT-PCR, and then we amplified the full-length 1815bp hemagglutinin protein (H) gene . The PCR products were inserted into pMD 18-T vector routinely, and the positive recombinants were identified by blue-white screening and endonuclease digestion. H gene we got were then sequenced and analyzed, the nucleotide and predicted amino acid of Nanjing isolate is 99% identical to the vaccine strain-Onderstepoort stain of CDV. Comparison from other isolates shows the 90~96% identity respectively. Amino acid alignment shows that there are only 4 potential asparagines glycosylation site in the protein,while there are 8-9 in other isolates, such as Changchun isolate, American isolat and Japan isolate.etc.2 Cloning and Expression of the Fragment of Canine Distemper Virus Haemagglutinin(H) Protein GeneThe 1058bp fragment of H gene of CDV NJ-15 was gained by digesting the plasmid pMD-H with EcoR I and 5a/ I, then it was cloned into the expressing plasmid vector pET-28b(+).The recombinant was transformed into the host strain Rosetta?and induced to express by l.OmM BPTG at 37C.The expression product was identified by SDS-PAGE and found to be 38KDa as expected one and confirmed by Western blot with CDV Onderstepoort strain antiserum. The results revealed that in vitro expression of the fragment of H gene had the critical antigenitic epitopes of CDV.3 Cloning and Transient Expression of the Full-length Gene Encoding the Nanjing strain of Canine Distemper Virus Haemagglutinin(H) protein in Mammalian CellsThe H protein gene was gained by digesting the plasmid pMD-H w... |
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