The Rescue And Identification Of Recombinant Rabies Virus Containing VP2 Gene Of Canine Parvovirus

Rabies is an ancient zoonosis caused by rabies virus. It cause almost 100% mortality once clinlcal symptom appeared. It is one of the highest mortality of acute infectious disease. Canine parvovirus disease is a highly contagious infectious disease caused by canine parvovirus. It mainly cause hemorrhagic enteritis and non suppurative myocarditis. VP2 is the main component of capsid of canine parvovirus, which can induce the body to produce neutralizing antibodies and has become the main protein of genetic engineering vaccine of canine parvovirus.Firstly, VP2 gene was cloned and sequence analysised from 10 epidemic materials of infected dogs of Hebei and Gansu in this study. The results showed that 80% samples were belonged to CPV-2a and 20% samples were belonged to CPV-2b, which indicated that CPV-2a was the predominant gene type in Hebei and Gansu provinces. VP2 protein was expressed by prokaryotic system then polyclonal antibodies were prepared by immunizing guinea pig with puried protein. Western blotting results showed that the recombinant protein had good immuneoreactivity and immunogenicity.Secondly, based on the reverse genetic system of rabies virus, bivalent genetic engineering vaccine against canine parvovirus and rabies virus was developed in this study.The attenuated strain of rabies virus BNSP was used as skeleton vector to construct the full-length cDNA of rabies virus,. The full-length cDNA and helper plasmids were mixed together to transfect into Vero E6 cells, and immunofluorescence test showed that the recombinant virus rBNSP-CPV-VP2 was rescued successfully. Then the biological characteristics of recombinant virus were identified. The recombinant virus was passaged 8 generation on Vero E6 and virus titers of the first, thirth and fifth passage were checked, the highest titer can reach to 109FFU/mL. VP2 gene of recombinant virus of each generation was detected by RT-PCR and confirmed the stability of VP2 gene; Western blotting showed that VP2 protein was successfully expressed in recombinant virus.Next, the pathogenic and immunological properties of recombinant virus were evaluated by BALB/c mouse model. The results of pathogenicity showed that mice injected the recombinant rabies virus did not find any clinical symptoms.The recombinant virus was inactivated and the vaccine was prepared according to a certain proportion with GEL adjuvant after the safety test, manwhile, the test of immunity was carried out with the antigen group and the PBS control group. The BALB/c mice were immunized at day 0 and 14. Blood was collected at day 7, 14, 21, 28 and 35 respectively. The rabies virus IgG antibody and canine parvovirus IgG antibody were detected by ELISA, meanwhile the rabies virus neutralizing antibody level was measured by RFFIT. The IL-4 and IFN-γ levels were measured using ELISPOT method. The BALB/c mice were challenged with 30 LD50 of CVS by intracerebral inoculation to evaluate the protective effect of vaccine. The results of ELISA and ELISPOT results showed that both group of BNSP-VP2 and BNSP-VP2+GEL can induce humoral and cellular immune response, and GEL adjuvant can enhance immune response. The result of RFFIT indicated that the neutralizing antibody of BNSP-VP2 group can reach to 7.19 IU/ml, meanwhile the level of BNSP-VP2+GEL group can reach to 21.21 IU/ml. The challenged results showed that the survival percentage of BNSP-VP2 and BNSP-VP2+GEL can reach to 66.67% and 100%, respectively.In summary, the epidemic situation of canine parvovirus in some provinces of China was analyzed in this study, manwhile the VP2 protein was expressed by prokaryotic system. In addition, rabies virus reverse genetic technology was used to rescue recombinant rabies virus that contain VP2 gene of cannnine parvovirus. The mice immunized with inactiveted recombinant antigen vaccine can induce high specific antibody against rabies virus and canine parvovirus, which can resist to lethal attack of rabies virus.This study laid a foundation for the development of bivalent genetic engineering vaccine against canine parvovirus and rabies virus.