| Chicken coccidiosis,the major parasitic disease of poultry,is caused by the apicomplexan protozoan Eimeria.Coccidiosis seriously impairs the growth and feed utilization of infected animals resulting in loss of productivity.Conventional disease controlstrategies rely heavily on chemoprophylaxis or live vaccines.Combined,these factors inflict tremendous economic losses to the world poultry industryin excess of US$3 billion annually.However,the problems of drug residues,drug resistance and the securityand higher cost of live vaccine make us focus on the attractive prospect of new generation vaccine.As coccidiosis is always mixed infection,practical and effective vaccine should be able to resist a variety of coccidia infection.In our previous study,we have screened out common antigen among E.tenella,E.acervulina and E.maxima three specise coccidiosis by the immuno-proteomics.this study we choose one of the common antigens,GAPDH,the homology between the nucleotide sequence of the antigen are more than 91%,to assess the immune protective effect of this common antigen to resistance coccidia infection,develop new multivalent coccidiosis vaccine,we have processed the following studies in particular:Using PCRtechnology,EaGAPDH and EmGAPDH gene were successfully amplified.Then build EaGAPDH and EmGAPDH prokaryotic expression plasmid pET32a-EaGAPDH and pET32a-EmGAPDH by the use of recombinant DN A technology,successfully induced in prokaryotic expression systems(E.coli).Western blot test results showed that the recombinant protein can be naturally recognized by chicken sera infected chicken coccidia.Common antigen GAPDH were respectively connected to eukaryotic expression vector pVAX 1.0 by recombinant DNA technology,to construct eukaryotic recombinant plasmid pVAX-EaGAPDH and pVAX-EmGAPDH,then analyze the immunogenicity of common antigen with a view to eukaryotic expression level(eukaryotic expression plasmid).Two-week-old chicken were intramuscular injected with eukaryotic expression plasmid,non-injected tissues and pVAX plasmid injected tissueswere in the control group.Strengthen immunity was completed a week later,and study the immunogenicity of common antigenrespectively from the cellular immunity and humoral immunity level.Chicken spleen lymphocyte separation was completed respectively 7 days and 14 days post primary immunization,spleen oocytes were dually stained with CD3 +,CD4+,CD8 +monoclonal antibody.The percentages of CD4+/CD3+?CD8+/CD3+ were examined through flow cytometry.Results indicated that the percentages of CD4+/CD3+?CD8+/CD3+of immune group were higher than the control groups,and a significant difference was found in two.This indicated that our recombined plasmiid have effects on T lymphocyte reaction in the body.In addition,the total RNA was extracted by lymphocytes,and cDNA was obtained by reverse transcription.Using Real-Time PCR technology,we analyze the mRNA levels of Thl cytokines(IFN-? and IL-2),Th2 cytokines(IL-4)and other cytokines(TNFSF15,IL-17 and TGF-?4),found that compared with the control group,the experiment groups had a significant difference,and had a higher mRNA level.It's proved that the eukaryotic recomb inant plasmid constructed can promote the secretion of cytokines in the body.Whole blood from chickens was ellected by cardiac puncture at week 1 post-first immunization and week 1 post-second immunization,and the serum was collected.The serum IgG antibody level was measured by indirect ELISA method.The results showed that weekl post-first immunization,the antibody levels had a significant difference between experiment groups and control groups and the former were higher.In addition,the result of weekl post-first immunization was same.In conclusion,the eukaryotic recombinant plasmid we constructedtook participate in the chicken body humoral immunity,and lead to the rise of IgG antibody levels in body fluids.Two-week-old chickens were inoculated with eukaryotic recombinant plasmid pVAX-EaGAPDH and pVAX-EmGAPDH by leg intramuscular injection.A booster immunization was completed 1 week later.Control proup chickens were injected with PBS buffer or pVAX vector at the same injection.Four-week-old chicken were challenged orally with sporulated oocysts of E.tenella,E.maxima,E.acervulina,or all of them.All chickens were euthanized to determine the effects of immunization after 6th day post-challenge.The results of body weight gain,oocyst decrease ratio,lesion score,and anti-coccidial index(ACI)were used to evaluate the efficacy of immunization.The results indicated that theukaryotic recombinant plasmid pVAX-EaGAPDH and pVAX-EmGAPDH could induce better immune protective efficacy. |
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