Rabies Virus Glycoprotein / Fusion Gene Dna Vaccine

Rabies is severe disease to animal and human being caused by rabies virus(RV) with a characteristic of almost 100% death rate. Immunization prevention is a available and effective method to control the rabies. Glycoprotein and Neucleoprotein are the main protein which can induce protective immunizing reaction in vivo. The experiment were conducted to construct a plasmid containing the G/N melt gene of rabies virus in order to develop the new kind of rabies vaccine characterized higher effectiveness and safety. The protein expression and protein immunity were measured by infecting pETGN into E.coli BL21, pIRESGN infecting into cell strains of BHK21. At the same time, the plasmid was injected into the healthy, no immunization of rabies dogs. The results indicated that plasmid containing G/N melt gene caused immunity reaction and provided some evidences for the further clinical utilization1. two special primers were designed according the nuclide sequence of G and N gene of rabies virus SAD-B19 strain published by NCBI, and the primers of G gene contained the Confine digest enzyme of BamHI, PstI, PstI and NotI were added into primers of N gene according to the experiment protocol. The G and N gene of rabies virus SAD-B19 strain were amplified by PCR, the target genes were collected and digested by Confine digestion enzyme of PstI. After collecting the G and N gene, T4DNA ligase was used to link the two genes. The same operation was conducted to linger the melt gene with pMD-18T vector under T4 DNA ligase function. After nuclide sequence determination, there was no significant difference between the target gene and published gene in website of NCBI. After Jameson-Wolf method of epitope advantage analysis, there was no new epitope in the new melt gene.2. The clone plasmid containing G and N melted gene of rabies virus was incided by BamHI and NotI, the target gene were linked with pET32(a) which were treated at the same enzyme under the T4 DNA Ligase, after identification with BamHI, PstI or NotI, the plasmid was translated into E. coli BL21, the target protein was translated under being induced by IPTG, SDS-PAGE was introduced to analyzed the protein, after Western Blot, the protein was reaction with rabbit anti rabies virus IgG and goat anti rabbit IgG. This result showed that the protein had character of immunogenicity.3. In order to constructing the eucaryon clone plasmid, also, two pairs of primer were designed according the published nuclide sequence of rabies virus SAD-B19strain in NCBI, and the NotI, PstI and BamHI were contained into primers based on the next experiment design, respectively. After being amplified by PCR, the G gene and N gene were incided by PstI, then to melt under condation of T4 DNA ligase as tool enzyme, finally, the melt gene linked with pMD-18T vector then clone plasmid of pMDGN was constructed. The sequence determination showed that there was no significant difference of epitope between the plasmid and published sequence.4. Clone plasmid of pMGGN and pIRESneo were incided by NotI and BamHI to constructed the eukaryotic expression plasmid, the target gene were got back to link together under function of T4DNA ligase. The plasmid of pIRESGN was constructed after identification of enzyme inciding, then, translated into E. coli DH5α, after large scale preparation of plasmid, the pIRESGN was transinfected into BHK-21cell. The total mRNA was extracted by TRIzol(R) method, the melt gene of G and N was amplified by RT-PCR, after that, FITC lane goat anti rabbit IgG was introduce into the method of immunofluorescence. The results indicated that the melt gene of G and N encoding the protein was identified and also showed immunogenicity.5. The healthy, adult and no immunization dog were injected the naked DNA with dose of 100ug/kg to determine the immunogenicity of pIRESGN, interval of immunization was 10days, and the blood of dogs were collected to lab. Analysis. ELISA method was introduced to determine the antibody level while the rabies virus was introduced to cover the plate. When blood was diluted into 100×solution, the OD₄₉₀ had a extend that increasing of antibody were found following the immunized times. However, there was no significant difference among the dogs. CD₄⁺, CD₈⁺ and CD₄⁺/CD₈⁺ in blood also had some regular when the content of these item were determined. The amount of CD₄⁺, CD₈⁺ increased with the immunized time delayed, the CD₄⁺/ CD₈⁺ was decreased inversely. Perhaps, the result showed that cell immunity played the more important role than humoral immunity while the DNA vaccine of rabies virus immunized into body. The denatured rabies virus and ConA were introduced to stimulating the leukomonocyte. The result indicated that OD₅₉₀ of leukomonocyte were increased following the immunized time increased.After amplification of G and N gene of rabies virus SAD-B19 strain, melting target genes and construction of pETGN, pIRESGN, the target plasmid which were transifected into E. coli BL21, BHK-21 cell and dog to determine the immunogenicity of melt gene of G and N extracted from rabies virus SAD-B19 strain had enough immunogenicity, and support the utilization of rabies virus DNA vaccine with scientific evidences.