| Avian leukosis virus (ALV) is an alpha retrovirus.At present, the infection viruses can be divided into A, B, C, D, E and J subtypes,and the classic exogenous ALV subtypes are A, B, C, and D. E subgroup ia an endogeous ALV,exsisting in the form of provirus.In the early1990s, subgroup J of ALV was mainly identified as a new subgroup(Payne et al,1991).Both A and B subgroups of ALV are more common in chicken flocks,while C and D subgroups of ALV are less common. As a result of taking purification measures by strict cutt off vertical transmission and isolating uninfected offsprings,developed countries have announced that exogenous ALV has been purified since1987. However,the United States recently reported that ALV-A was isolated from Marek’s disease vaccine, while China has been long-term using imported grandparent chickens and vaccines,and never taken purification measures.Therefore,our country’s chicken flocks may already exist ALV-A.In recent years,the ALV-infected chickens serological investigation proved the universality of ALV infection. Furthermore,the isolated ALV-A in China flocks confirmed its widespread.So,in order to get a rapid specific diagnostic reagent and a protective effect vaccine for subgroup A ALV detection and prevention,we do the experiment as follows.1Identification and expression of gp85gene of subgroup A Avian Leukosis VirusThe SDAU09E1strain from Avian Leukosis Virus Subgroup A(ALV-A) infected DF1cells, an ALV-A-gp85DNA fragment of1023bp was amplified from DNA of the infected cells and was inserted into PET-32a(+) plasmid by restriction endonucleases BamH Ⅰ and Not Ⅰ sites. The recombinant plasmid PET-SDAU09E1-gp85was transformed into E coli.BL21(Rosetta) for gp85gene expression.The expressional products were identified by SDS-PAGE, the results showed that molecular weight is52.8kDa.2. Mono-specific serum preparation and specificity of gp85gene of subgroup A Avian Leukosis VirusThe host strain of expression fusion protein was cleaved,purified, dialyzed, refolded and quntatied by spectrophotometric.Then,the fusion protein and the Freund’s adjuvant were mixed by1:1,arid this mixture was used to immunize six weeks old Kunming white mice, and the mice anti-serums were collected from the immunized mice and identified by the indirect immunofluorescence (IFA) method and using ALV-An ALV-B and ALV-J. The recombinant ALV-A gp85protein demonstrated a good antigenecity, mice vaccinated with produced mon-specific serum reactive with subgroups A and B ALV(ALV-A and ALV-B but not subgroup J ALV).This was the first time to demonstrate a mono-specific antiseru specific to ALV-A and ALV-B, it could be used for differencial diagnosis of exogenous ALV infections in DF1cell cultures when in complement with ALV-J specific monoclonal antibodies.3. Vaccine preparation and efficacy of gp85gene of subgroup A Avian Leukosis VirusVaccine of this mixture was prepared through step2method,which was used to immunize4months old imported hens. By detection of antibody level once a week, it is found that the immunization program could induce to maintain the eight weeks specific ALV-A and ALV-B antibody. During the antibody maintaince of SPF hens, we collected the eggs and detected the maternal antibodies in the yolk that contained the anti-ALV-A and ALV-B antibody.However, when we use the fertilized eggs with maternal antibodies to hatch, chicken blood was collected on1,3,5,7,9,14day and antibody was detected by ELISA. It is found that maternal antibodies in the yolk had not been passed to the hatched chickens. The experiment illustrates the preparation of vaccines against the subgroup A ALV has been initially successful, which are able to induce the body to produce antibodies of anti-ALV-A and anti-ALV-B and can be passed to the yolk,while the maternal antibodies are not passed to the hatched chickens, and the experiments still need further research and improvement. |
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