Protective Effects Of Five DNA Vaccines On Infection Of E.Acervulina Chicken

Avain coccocidiosis is a cytoplastic entozoic parasite disease that causes severe economic loss in the poultry industry.The emergence of drug-resistant parasites and the drugs residues problem restricted the development of poultry industry so that a new approach to prevent avain coccocidiosis should be found.It have proved that stimulating the cell immuno-reaction of the host is an effective approach to prevent avain coccocidiosis.A prominence advantage of DNA vaccine as a new generation of vaccine is that it can induce abroad cell immuno-reaction and form long perdured immunity memory,especially the immune regulative DNA vaccine which was constructed by special antigen together with some cytokine genes has more effect and prospect in the anti- coccocidiosis practice.In this investigation we selected the 3-1E genes of E.acervulina antigen to construct DNA vaccine combined with the chicken cytokineChIFN-γ,ChIL-2,ChIL-17,ChIL- 18to construct DNA vaccine pVAX1.0-3-1E, pVAX1.0-3-1E-IFN-γ,pVAX 1.0-3-1E-IL-2,pVAX 1.0-3-1E-IL-17,pVAX 1.0-3-1E-IL -18 five DNA vaccine.The protective effects of these vaccines were compared in the experiment of chickens against the challenge of E.acervulina.This experiment includes next parts:1 Cloning and sequence analysis of 3-1E from E.acervuinaBased on the published 3-1E nucleotide sequence of E.acervulina,a pair of special primer was designed by using computer software.The gene of 3-1E was amplified by RT-PCR with total RNA extracted from 3-1E oocysts as template,then the gene was cloned into PMD18-T vector.Restriction enzyme digestions,PCR and sequencing confirmed that this amplified product was gene of 3-1E.Comparisons and analysis of the nucleotide acid sequences indicated that the gene of 3-1E included an open reading frame of 513bp.The amplified gene shared 99%homology with that reported.2 The expression of 3-1E and purification of expression protein from E.coliBy the means of DNA recombination,3-1E gene was cloned into PET-28(a) vector,and transformed to E.coli BL21.The recombination protein was expressed by inducing with IPTG.The result of SDS-PAGE indicated that the expression protein was about 22 kD and reached to its peak at about 6 hours after inducing.Most of the recombinant protein existed in thesupematant after ultrasonicated and TritonX-100 wished,but little ininclusion body.3 Constructionofnucleic acid vaccines against E.acervulinaTake advantage of DNA recombination technique,pVAX1.0-3-1E, pVAX1.0-3-1E-IFN-γ,pVAX 1.0-3-1E-IL-2,pVAX1.0-3-1E-IL-17,pVAX 1.0-3-1E-IL -18DNA vectors were constructed.After identification by restriction enzyme digestion,the recombination DNA Plasmids were injected into chest muscleofl 4-day old chicken respectively.The chicken were killed 7days post second immnuization and the muscle tissue where plasmids were injected and no-injected were checked by RT-PCR and Western-blotting,to make sure that the plasmids in the chicken muscle were successfully transcriptied and expressed.4 Protective effects of nucleic acid vaccines on chicken against E. acervulinaChickenweredividedinto nine groups:pVAX1.0-3-1E group,pVAX 1.0-3-1E-IFN-γgroup,pVAX1.0-3-1E-IL-2group,pVAX1.0-3-1E-IL-17group,pVAX1.0-3-1E-IL18 group,unimmunized-unchallenged control group,unimmunized challenged control group and 3-1E recombinant protein.Each group had 30 chickens and was injected with vaccine through chest muscle.The vaccine was inoculated at 14 days and 21 days of age and each chicken was challenged with 1.0×10~5 E.acervulina sporulated oocytes at 28 days.All chickens were killed at 34 days of age and the protective effects were estimated by the index of lesion score of duodenum,OPG,effect of average body weight gain and anticoccidia index(ACI).The results showed that the immuno-regulative DNA vaccine pVAX1.0-3-1E-IFN-γ,pVAX1.0-3-1E-7-IL-2,pVAX1.0-3-1E-IL-18,pVAX1.0-3-1E-I L-17 had good protective effects than DNA vaccine pVAX1.0-3-1E and protein vaccine 3-1E.The ACI of pVAX1.0-3-1E-IFN-γand pVAX1.0-3-1E-7-IL-2 were more than 180.The pVAX1.0-3-1E-IL-18,pVAX1.0-3-1E-IL-17,pVAX1.0-3-1E and protein vaccine 3-1E presented the ACI of these four vaccine were 176.07, 174.7,171.31 and 160.3 respectively.