| The coccocidiosis of chicken is a cytoplastic entozoic parasite disease and may cause severe economic loss to the industry of poultry . It have proved that an effective approach to prevent chicken coccocidiosis is to stimulate the cell immunoreaction in the host. A prominent priority of DNA vaccine lies in that it can induce abroad cell immunoreaction and form long perdured immunity memory, especially the immune regulative DNA vaccine which was constructed with some cytokine genes has more prospect in the practice. In this report, we constructed immune regulative DNA vaccine by the combination of MZ5-7 gene of Eimeria tenella with chicken IL-2 and IL-17 and tested their protective effects on the chickens against the challenge of Eimeria tenella.MZ5-7 gene was cloned with RT-PCR from the second generation merozoites of E. tenella that detached from chicken caecum 5 days post infection and its sequence was analyzed on epitope and open reading frame (ORF), then expressing it in prokaryotic cell . The result indicated that the length of MZ5-7 ORF was 945 bp and it had 99.9 % homology with MZ5-7 gene that GenBank has been issued(Access number: L08257) in both nucleotide and amino acid sequence. The prediction of MZ5-7 epitope with DNAStar software showed that its B cell epitope located at the segments of 80-300 and T cell epitop in 123-190 and 220-300. Another pair of primer was designed and then 885bp fragment of the gene without signal-peptide was amplified by PCR and it was inserted into pET28b vector to construct recombinant plasmid pET28b-MZ. After induced with IPTG, a 36.5 kD fused protein was expressed and it was hybridized with serums immunized with the second generation merozoites and sporozoites respectively. The result demonstrated that innate MZ protein was expressed both in the stages of merozoites and sporozoites.IL-17 cDNA of chicken was successfully amplified by RT-PCR in this study from the total RNA isolated from chicken spleen lymphocytes stimulated by ConA for 12 h, 24 h, and 48 h respectively and that isolated from the lymphocytes of caeca tonsil and intraepithelial lymphocytes (IELs) from chicken that had been infected 2 times withEimeria tenella sporulated oocysts ( about 3.6x104/per chicken ) The PCR products were then cloned into pMD18-T vector for sequencing. The gene ORF was 507 bp, and it matched with the predicted size. We Compared it with the published sequence in GenBank (AJ493595) , the homology of nucleic acids and amino acids were 99.4 % and 82.4 % respectively. The gene was later sub-cloned into pET32a vector, designed as pET32a-IL-17. After induced by IPTG , the IL-17 gene was expressed successfully and the fused protein was about 35.3 kD.The open reading frame of MZ5-7 gene, an antigen of Eimeria tenella second generation merozoites, was cloned into plasmid pcDNA4.0 to construct DNA vaccine pcDNA4.0-MZ. With the same methods, the mature interlukin -17 gene and interlukin- 2 of chicken was cloned into downstream of the MZ5-7 gene to construct immune regulatory DNA vaccine pcDNA4.0-MZ-IL-17 and pcDNA4.0-MZ-IL-2 that co-expressive of MZ5-7 antigen with IL-17 or IL-2. After purified, 50ug of these three DNA vaccines were used to immunize one-week-old chickens by breast muscle injection respectively. The transcriptions and expressions of the target genes were checked by RT-PCR and Western-blot respectively on the day 3, 5, 7, 15 and 20 post immunization. The results showed that the transcripted products of MZ5-7 gene can be detected from all injection site muscles immunized with these three DNA vaccines, respectively, on day 5, 7 and 15 post immunization by RT-PCR. The expressing proteins of the plasmids were respectively identified by Western-blot from all injection sites on day 5 and 7 post immunization. The molecular weight of expressive protein of pcDNA4.0-MZ was 36.5 kDa that coincident with the predicted protein of the MZ5-7 gene. While the molecular weight of expressive product of pcDNA4.0-MZ-IL-17 and pcDNA4.0-MZ-IL-2 was about 64 kDa, slightly larger than that of predicted product of... |
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