The Immunogenicity Of EmTFP250 Gene Of E.maxima

The coccocidiosis of chicken is a parasitic protozoa disease.It is harmful to the growth and development of chickens and causese economic loss to the poultry industry. Current methods for coccidiosis control in the poultry industry have many drawbacks,such as drug-resistance and the potential reversion to virulence.Therefore,nucleic acid vaccines containing important parasitic antigens have been suggested as an alternative to the virulent or attenuated vaccines.It has proved that an effective approach to prevent chicken coccocidiosis is to stimulate the cell immune response in the host.Nucleic acid vaccine is a biotechnology vaccines,it can induce abroad cell immune response and form long perdured immunity memory.E.maxima is one of the four common coccidians that induce severe coccocidiosis of chicken.EmTFP250 is antigen in agamocytogeny of E.maxima.It is the member of the TRAP family,and belongs to EtMIC protein.This gene's total length is 7990 base pairs with ORF of 7083bp and encode 246kDa protein.In this report,we divided EmTFP250 gene to eight fragments according to the gene epitope.This eight fragment genes were designated as Em1(231-1187),Em2(1124-1988),Em3(1850-3113),Em4(3071-4214), Em5(4133-4964),Em6(4916-5882),Em7(5783-6341) and Em8(6242-7310) and cloned respectively to pcDNA4.0 plasmid for the detection of their protective effects on the chicken against the challenge of E.maxima.The Em1(231-1187),Em2(1124-1988),Em3(1850-3113),Em4(3071-4214), Em5(4133-4964),Em6(4916-5882),Em7(5783-6341),Em8(6242-7310) gene were cloned by RT-PCR.The result indicated that the Em1,Em2,Em3,Em4,Em5,Em6,Em7,Em8 gene contained 980,881,1297,1149,810,1014,579,1092bp and encoded a peptide of 326,293,432,383,270,338,193,364 amino acid residues to part.Comparison of the Em1, Em2,Em3,Em4,Em5,Em6,Em7,Em8 gene with the previously reported EmTFP250 gene in segmentation,the result indicated that they had 99%above homology in both nucleotide and amino acid sequence.By the means of DNA recombination technique,pcDNA4.0-Em1,pcDNA4.0-Em2, pcDNA4.0-Em3,pcDNA4.0-Em4,pcDNA4.0-Em5,pcDNA4.0-Em6,pcDNA4.0-Em7 and pcDNA4.0-Em8 vectors were constructed.After identification by restriction enzyme digestion,these recombinant DNA plasmids were extracted and injected into the leg muscle of 14 days old chickens.A week later,the injected tissue was sampled respectively to check whether these DNA plasmid expressed or not by RT-PCR.The results indicated that Em1, Em2,Em3,Em4,Em5,Em6,Em7 and Em8 genes could be successfully transcribed in the injected tissues.pcDNA4.0 vector,recombinant plasmid pcDNA4.0-Em1,pcDNA4.0-Em2, pcDNA4.0-Em3,pcDNA4.0-Em4,pcDNA4.0-Em5,pcDNA4.0-Em6,pcDNA4.0-Em7, pcDNA4.0-Em8 were inoculated respectively into the leg muscle of chicken twice at 14 and 21 day-old of age,and the control groups were also set up,then challenged with E.maxima sporulated oocyst at 28 days old.The indexs of weight gain,oocysts per gram feces(OPG),reduction of lesion scores and anti-cocccidial index(ACI) were done respectively to check the protection of these DNA vaccines against E.maxima infection. The results indicated that all of the immune-regulating DNA vaccines pcDNA4.0-Em1, pcDNA4.0-Em2,pcDNA4.0-Em3,pcDNA4.0-Em4,pcDNA4.0-Em5,pcDNA4.0-Em6, pcDNA4.0-Em7 and pcDNA4.0-Em8 could well protect chicken from E.maxima infection. The best recombinant DNA vaccine are pcDNA4.0-Em1,pcDNA4.0-Em3,pcDNA4.0-Em6 and pcDNA4.0-Em8,so they coule be satisfying candidate nucleic acid vaccines against E.maxima.