| Canine distemper, caused by canine distemper virus, is one of the most acute and highly contagious disease in canine farming, fur cultivation and wildlife conservation. Traditional attenuated vaccine has intrinsic shortcomings despite its important role in controlling the occurrence of CD: it can cause immunosuppression and lesion of CNS, it may be lethal some susceptible animal and possess a wild host spectrum. Moreover, mutation of CDV can also cause epidemic of this disease. To develop a new kind of safe and efficient CDV genetically engineered vaccine, we constructed two genetic recombination eukaryotic expression plasmid pcDNA-F and pcDNA-H that express two envelope glycoprotein genes F and H, with the Chinese isolates of CDV on the basis of initiative exploring of the molecular property of this two glycoproteins, which is tightly related to the pathogenicity and immunity of CDV and subsequently detected their protection immunogenicity in mice and dogs. These above works can be divided into 4 parts:1) Cloning and expression of envelope glycoprotein gene ofCDV OP strainVirus was isolated from the CDV OP strain infected Vero cells to extract its RNA as template of RT-PCR, and then we amplified two fragments of the fusion (F) protein gene (including the fusion domain, transmembrane domain and partial attachment domain, and the length is 769bp, 832bprespectively.) and the 5' and 3' region of haemagglutinin (H) protein gene (945bp and 904bp respectively). According to the direction of the correct ORF, PCR products were inserted into downstream of GST in the pGEX-6p-l vector. After transformation of BL21 host bacterium, the recombinants expressed efficiently with the induction of 1.0mM IPTG at 37 ℃. Expression products of the envelope glycoprotein gene were identified by SDS-PAGE to be 54kD, 56kD and 59kD, 58kD respectively. Results of indirect fluorescence assay (IFA) showed that the serum of mice immunized with the expression products can response to virus-infected cells specifically, and the titer of the neutralizing antibody is 1:16. These data suggests that the expressed envelope glycoprotein gene of CDV in vitro reserves partial immunogenicity of the natural protein, and it's promising to produce sub-unit vaccine based on these experiments. Meanwhile, the antibody can be used to diagnose in related research.2) Sequence analyses of envelope glycoprotein genes ofChinese isolatesUsing RNA extracted from the Chinese isolates (CDV-YZ0101, CDV-YZ0102) as template, we amplified the 1989bp F protein gene and the 1824bp H protein gene with RT-PCR. The PCR products were cloned into pGEM-Teasy vector routinely, and the positive recombinants were identified by blue-white screening and endonuclease digestion. F gene and H gene we got were then sequenced and analyzed. For H gene, nucleotide and predicted amino acid of the two Chinese isolates is 98% identical to each other. Comparison from ChangChun isolates and other field isolated from American and Japan showed the 96% and 93-95% identity respectively, but only 90-91% identity with the vaccine strains (Ondersteroop strain, Convac strain). Amino acid alignment shows that there are 8-9 potential asparagines glycosylation site in the protein of the Chinese isolates andother isolates, while there are only four in OP-CDV. Phylogenesis study suggests that our isolate and ChangChun isolate can be clarified to the same lineage, which is different from American and Japanese isolates, and there are evident difference between field isolates and vaccine strains. For F gene, the two Chinese isolates showed 99% identity. Comparison from the Germany isolate, phocine distemper virus type n (PDV-2) and vaccine strain , the major difference is the long signal peptide domain while the mature protein (F0) exhibit high identity, and all of the 13 serine residues, four potential asparagine glycosylation sites and two hydrophobic regions supposed to affect the fusion function are completely identical. These data supports the view that F protein is more conser...
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