| Chicken infectious anemia is an immunosuppressive disease caused by the circoviridae genus chicken anemia virus.It mainly affects chicks up to 20 days of age.Atrophy of the lymphoid tissue,aplastic anemia,and other viral,bacterial,fungal infections often cause huge economic losses to the poultry industry.There is no specific drug to treat the disease at present.Hence,vaccine prevention and control of early infection is the key to controlling CAV.The virus titer of CAV in chicken embryo and MSB1 cell is low,making the production cost of CAV inactivated vaccine very high,so it is difficult to be widely used in chicken breeding industry.There is a risk of vertical and horizontal transmission of live vaccines using CAV,which cannot be promoted.Therefore,the timely investigation of CAV molecular epidemiology,mastering the genome characteristics of epidemic strains,and preparation of novel genetic engineering vaccines are currently the main directions of research.1.In this study,56 suspected infectious anemia virus infections were collected from Guangdong in 2016~2017.After grinding and repeated freezethawing treatment,12 strains were identified as chicken infectious anemia virus by PCR.After sequencing,it was found that 12 strains of Guangdong CAV isolates were in the I branch and the evolutionary relationship with the strength strain KF224931 isolated in our laboratory was close.2.VP1 is the main immunogenic protein of CAV and it is also the only capsid protein.The neutralizing antibody produced by the body expressing VP1 protein alone is weak.Only with the help of VP2 protein can form the correct epitope,and induce the body to produce immunity.Neutralizing antibodies for protection.Therefore,coexpression of both VP1 and VP2 genes with fowlpox virus can provide betimmune protection.In this study,the VP1 and VP2 genes of a chicken infectious anemia virus epidemic strain isolated in the laboratory were cloned downstream of the fowlpox virus transfer vector pSY681 promoter to obtain the pSY681-VP1-VP2transfer vector.This plasmid was transfected into chicken poxvirus-infected cells using the Lipofectamine 2000 Transfection Kit.Homologous recombination occurred between the plasmid containing the homology arm of fowlpox virus genome and the virus,and the recombinant fowlpox virus was screened using blue and white spots.Recombinant fowlpox virus DNA was extracted,and VP1 and VP2 genes were successfully inserted into the fowlpox virus genome by PCR.Indirect immunofluorescence and western blot experiments demonstrated that VP1 and VP2genes were successfully expressed in the recombinant virus.3.In this study,10-day-old SPF chickens were randomly divided into 4 groups to be immunized with pSY681-VP1-VP2 recombinant fowlpox virus vector vaccine,chicken infectious anemia attenuated vaccine,and poxvirus vaccine.Peripheral blood was collected on days 7 d,14 d,and 28 d after immunization,and antibody titer of the test chickens was detected using IDEXX CAV antibody detection kit.The test results showed that the antibody level of the rFPV-VP1-VP2 recombinant vaccine group was significantly higher than the blank control group,but it was always lower than the CA V attenuated vaccine group.The dynamic changes of CD3~+,CD4~+,and CD8~+T lymphocyte subsets in the peripheral blood on the seventh day after immunization were detected.The results showed that the content of CD3~+,CD4~+,and CD8~+T lymphocy in the peripheral blood of the rFPV-VP1-VP2 vaccine group was significantly higher than the blank.The levels of CD3~+and CD4~+T lymphocytes in peripheral blood of rFPV-VP1-VP2 vaccine group were significantly higher than those in rFPV group,and there was no significant difference in CD8~+T lymphocyte content between the two groups.The above results showed that SPF chickens elicited an increase inCD4~+and CD8~+T lymphocytes after immunization with rFPV-VP1-VP2and rFPV vaccines,and rFPV-VP1-VP2 vaccines elicited more activation of CD4~+T lymphocytes. |
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