Immunoprotection Of Recombinant Eukaryotic Expression Plasmids Of Eimeria Tenella Chimeric Antigen

Eimeriawas a kind of intracellular parasite of Apicomplexa, endangeredseriously the intensification poultry production.At present, prevention and cure ofcoccidiosis depend largely on two ways: drug and vaccine.It was urgent to find betterways because both have existed insurmountable obstacles.This experiment with the strongest morbidity of Apicomplexa as the researchobject, selected four key invasion related protein molecules, and analysis their keyfunction structure domain by bioinformatics, connected their encoding gene of thedomain using splicing by overlap extension PCR (SOE PCR) technology to a newchimeric antigen marked as CA, constructed CA eukaryotic expression plasmid,evaluated the immune protective effect of the plasmid.First, we annotated and spliced the gene sequence of E.tenella invasion relatedprotein (EtAMA1, EtMA1, EtMA2and EtRON2) using bioinformatics andcomparative genomics technology, designed primer on the basis of the sequenceprimers, seleced total RNA of E.tenella (Guangdong strain) second generationmerozites as the template, cloned the four genes open reading frame (ORF) sequenceby PCR, then cloned the encoding gene of extracellular region domainⅡof the fourprotein, connected these genes using SOE PCR. Through the online software, weanalyzed the genes by bioinformatics.Gene sequence length of CA was1452bp,which encoding a protein of484amino acids with a predicted molecular weight of50.84kDa.Second, the eukaryotic expression plasmid pVAXⅠ-CA was constructed.pVAXⅠ-CA was transient transfected into239-T cell and injected into chicken leftleg muscle for expression in vitro and vivo to detection the transcription of CA by realtime PCR. It was indicated that the recombinant plasmid in eukaryotic cells andchicken were transcribed.Third, used the eukaryotic plasmid pVAXⅠ-CA as DNA vaccine to chicken andevaluated the immune protection effect. Chicken were injected with the same dosewhen they are3days-old and10days-old, orally challenged with live E. tenella(Guangdong strain) oocysts about5×104at10days following the last immunization.Which were analyzed that survival rate, relative weight gain rate, reduction of caecumlesion scores, oocysts amount in the ceca content and anticoccidial index(ACI) at7days after challenged.The transcription profiles of IL-12, IL-2and IFN-γ in the ceca were evaluated by quantitative real-time reverse transcription PCR (qRT-PCR) usingSYBR Premix Ex TaqTM, to analyze the cellar immunity induced by eukaryoticplasmid.The results showed that CA have played an important role in anticoccidialinfection with higher ACI value of immune groups than control groups. As a whole,the expression quantity of IL-2、IL-12and IFN-γwas very low in3-days chicken, andimmune group were higher than the control group after immune. It was suggested thatthe expression quantity of cytokines could be induced by CA.Combined with ACI, wefound that the high dose group can induce more lasting cytokines and give a betterprotection effect.In the present study, gene sequence of extracellular region domainⅡof the fourE.tenella invasion related proteins were connected to express in the first time. It wasshown that the CA has immune protection to E.tenella, and could inducecorresponding immunological factors. This test provided a theoretical foundation anda new idea to the research of coccidiosis vaccines.