The Construction Of Recombinant Plasmid PSMART2b-Rhomboid3 And Its Preliminary Application Against Eimeria Tenella In Vivo

Coccidiosis in Chicken is caused by Eimeria spp which parasite in the chicken intestines.E.tenellabecomes the focus of prevention and treatment among all species of Eimeria spp for the highest pathogenicity.The coccidiosis caused by Eimeria tenellaseriously affects the growth and development of chicken,egg production rate,etc.so it is very harmful to the poultry industry.According to relevant statistics,the annual economic losses caused by the Eimeria tenella was more than 30 billion dollars all over the world.However,at present,the prevention and treatment of this disease mainly depends on chemical drugs and live coccidiosis vaccines.But long-term use of drugs to prevent coccidiosis can lead to drug-resistant strains and drug residues,and the live vaccine may cause the diffusion of virus and virulence reversion.Therefore,it is necessary to develop a safe vaccine with good immune effect for the prevention and control of coccidiosis.In this study,Rhomboid3 protein of E.tenella was used as antigen molecule,and the eukaryotic expression vector pSMART2 b was used to transfer and express the antigen molecule.The recombinant plasmid was transducted into 293 cells for expression,then immunized chickens with recombinant plasmid.The cellular immunity and humoral immunity of chickens were measured to evaluate the protection function of recombinant plasmid pSMARAT2bRhomboid3 on the Eimeria tenellaas well as to evaluatethe potential of recomninant plasmid as coccidiosis vaccine.The construction of recombinant plasmid pSMARAT2b-Rhomboid3.The total RNA of E.tenella was extracted and reverse transcribed into c DNA.The Rhomboid3 gene was amplified by PCR and ligated with p MD-18 T and pSMARAT2 b,then cloned into DH5?.And the Rhomboid3 gene was 774 bp.pSMARAT2b-Rhomboid3 was identified by sequencing and enzymed digestion.Then the recombinant plasmid pSMARAT2b-Rhomboid3 was transducted into 293 cells with LipofectamineTM2000 and expressed,then detected by western blotting with positive serum against E.tenella,the resultshowed that Rhomboid3 protein was immunogenic.Immune response induced by recombinant plasmid pSMARAT2b-Rhomboid3 in chicken.The recombinant plasmid pSMARAT2b-Rhomboid3 were immunized by intramuscular injection and the immune doses were 100?g per and 50?g per chicken.The chickens were immunized for 3 times at intervals of one week.Then the cellular immunity level,cytokines levels(IL-2?IL-4?IL-10 and IFN-?),proliferation of T lymphocyte and cytotoxic T lymphocyte activity were tested.The results showed that cytokines levels(IL-2 ? IFN-?),proliferation of T lymphocyte and cytotoxic T lymphocyte activity increased significantlycompared with the control group,(P<0.01).The protection efficacy induced by the recombinant plasmid pSMARAT2bRhomboid3 against E.tenella challege.The chickensimmunized by pSMARAT2b-Rhomboid3 were challenged with E.tenella oocysts.The results showed that mmunized doses of 50?g and 100?g compared with control group,decreased the number of oocysts,weight gain and cecal lesion reducedsignificantly.The ACIs were 181.25 in group 50?g and 184.22 in group 100?g,respectively.This result show the potentialof coccidiosis vaccines.