| Canine distemper virus(CDV)and canine parvovirus(CPV)can cause severe infectious diseases in animals such as canines,martes and pandas which can cause 80% of animal mortality.These virus have brought serious economic losses to the pet breeding industry,fur animal husbandry and wildlife conservation.Nowadays,attenuated vaccines were used to prevent these diseases.However,attenuated vaccines also have some deficiencies,such as high production costs,and limitations in the face of different virus variants.This study aimed to construct a new vaccine.Therefore we inserted the antigen genes of CDV and CPV into the genome of adeno associated virus(AAV)to construct an recombinant adeno-associated virus vaccine.In this study,hemagglutinin gene and fusion gene of CDV and VP2 gene of CPV were selected as antigen genes and inerted into the genome of AAV.The recombinant adeno-associated virus(rAAV)was obtained by using the three plasmids packaging system.After the detection of virus titer and antigen proteins expression,the rAAV vaccine was mixed and immunized 6-week-old Balb/c mice,while the Nobivac Puppy DP vaccine was used as the positive control and the rAAV-GFP virus was the negative control.In the first immunization experiment,the mice were immunized three times according to the immunization protocol of the Nobivac vaccine.Each immunization interval was 14 days.Neutralizing antibody was detected every 14 days after immunization.Then we got good experimental results.In order to verify the effects of the rAAV vaccine,we carried out one immunization test using the same immune pathway and dose as the three immunization experiments,and then continued to monitor the neutralizing antibody level.The results of three times immunization showed that the rAAV vaccine could produce high levels of neutralizing antibody in mice.The titer of neutralizing antibody against CDV produced by rAAV vaccine was higher than that of positive control at all time after immunization.The titer of neutralizing antibody against CDV could reach to1: 500 at the highest level,which was 4 times of that of the positive control.However,thetiter of neutralizing antibody against CPV produced by rAAV vaccine was close to that of the positive control,and the neutralizing antibody titer was between 1: 100 and 1: 200.It is generally believed that when the neutralizing antibody titer is greater than 1: 100,the body can be protected from the attack of virulent strain.The results of once immunization showed that the rAAV vaccine still produced high levels of neutralizing antibody in mice.The titer of neutralizing antibody against CDV exceeded 1: 100 at the 6th week after immunization by rAAV vaccine,but the neutralizing antibody against CDV produced by positive control was always less than 1: 100.And at the 8th week after immunization,the titer of neutralizing antibody against CPV produced by rAAV vaccine and positive control were all over 1: 100.The results showed that rAAV vaccine can achieve effective immunization with only once injection.The results of this study indicate that the rAAV vaccine can stimulate the immune system of mice and produce a high level of neutralizing antibody.And the animal can get effective immunization after only once injection.But the immune effects of CDV and CPV are uneven.The immune effect of CDV is stronger than that of CPV.This phenomenon may be caused by the selection of antigen or the proportion and titer of the rAAV virus.In the following work,we will continue to explore the matching conditions to better balance the immune effects of the two viruses.To sum up,the recombinant adeno-associated virus vaccine carrying antigens of CDV and CPV have the advantages of safe and high efficiency,short immune period and low cost,which is expected to be a new vaccine for the prevention and control of canine distemper and canine parvovirus disease. |
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