| It is well known that avian coccidiosis, especially caused by Eimeria tenella is an important parasitosis which endanger poultry seriously. At present, there are many problems existing in preventing and curing it with conventional methods. With its polyclonal antibody, the gene of SO7 was found by Profous-Juckelka from A.gtllcDNA expressing library of partly sporulated oocysts, and it had been showed that its protein could yield protective immunity in some degree after administered chicken. Chicken IL2 is a cytokine with biological activities which plays an important role in immune adjust network, It works as intensifier on immunity. In this experiment, the S07 gene of E. tenella and chicken IL2 were amplified and cloned into pVAXl.O and pcDNA4.0(c) plasmid vectors respectively or in series to construct immune regulating nucleic acid vaccines. These vaccines were used to immune chicken. In order to check their effects on inducing special immune reactions and preventing chicken from E. tenella challenge. This experiment includes next parts: Cloning and sequence analysis of SO7 from E. tenellaBased on the published SO7 nucleotide sequence of E. tenella, a pair of special primer was designed by using computer software. The gene of SO7 was amplified by RT-PCR with total RNA extracted from E. tenella sporozoite 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 SO7. Comparisons and analysis of the nucleotide acid sequences indicated that the gene of SO7 included an open reading frame of 651bp and encoded 216 amino acid residues, only one nucleotide in 288nt different from the reported sequence, and was nonsense mutation. The amplified gene shared 99.8% homology with that reported, and encoded 22.7KD protein. The expression of SO7 and purification of expression protein from E. ColiBy the means of DNA recombination, SO7 gene was cloned into pET32a(+) vector, and transformed to E. coli BL21.The recombinant protein was expressed by inducing with IPTG. The result of SDS-PAGE indicated that the expression protein was about 40KD and reached to its peak at about 5 hours after inducing. Most of the recombinant protein existed in the inclusion body after ultrasonicated and TritonX-100 wished, but little in supernatant. Construction of immuno-regulating nucleic acid vaccines of E. tenellaTake advantage of DNA recombination technique, pVAX1 .0-SO7 pcDNA4.0(c)-SO7 pVAX1.0-SO7-IL2 DNA vectors were constructed. After identification by restriction enzyme digestion, the recombinant DNA plasmids were injected into chest muscle of 14-day old chicken respectively. The chicken were killed 7 days post second immunization and the muscle tissue where plasmids were injected or no-injected and blood were checked by RT-PCR and Western-blotting, to make sure that the plasmids in the chicken muscle were successfully transcriptied and expressed. The results were positive. Protective effects of immuno-regulating nucleic acid vaccines on chicken against E. tenellaSO7 IL2 recombinant protein and pVAX1.0-SO7 pcDNA4.0(c)-SO7 pVAX1.0-SO7-IL2 nucleic acid vaccines were injected into chicken muscle twice at the 14 and 21 days old respectively. Chicken were challenged with fresh E. tenella sporulated oocysts at 28 days old. In order to evaluate the protective effects of these nucleic acid vaccines against E. tenella infection, weight gain, oocysts production (OPG), reduction of lesion scores, and anti-coccidial index (ACI) were surveyed. The results illustrated that the nucleic acid vaccines, especially the immuno-regulating nucleic acid vaccine were effective. The pVAX1.0-SO7-IL2 recombinant nucleic acid vaccine could reduce the relative oocyst production, increase relative weight gain ratio, and the caeca lesion score was also significantly decreased. The relative oocytes production of the groups inoculated with pVAX1.0-SO7-IL2 was reduced 22.8% comparing with the positive control group. The caeca lesion score of groups inoculated with pVAX1.0-SO7-IL... |
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