The Study On Immuno-Regulating DNA Vaccine PEtK2-IL-2 Against Eimeria Tenella

Avian coccidiosis is an important parasitosis, which led to serious economic losses. Eimeria tenella calmodulin-domain protein kinase (EtCDPK), converged at the apical end of the sporozoite was encoded by pEtK2 gene. It had been shown that this protein could stimulate T-lymphocyte proliferation of chicken infected with coccidia. Chicken interleukin-2 (IL-2) plays an important role in immune regulatory network. In this study, pEtK2 gene of Eimeria tenella and chicken IL-2 gene were cloned into pET28a(+), pVAXl.O and pcDNA4.0(c)vectors respectively. The DNA vaccine were then used to immunize chicken in order to evaluate their immuno-protective effect.1. Cloning and sequence analysis of pEtK2 gene of Eimeria tenellaBased on pEtK2 cDNA sequence published by GenBank, a pair of special primers were designed by computer software. A 1464bp DNA fragment was amplified by RT-PCR using total RNA template extracted from Eimeria tenella sporozoites. In order to identify this gene, the nucleotide was sequenced and compared with existing data in the GenBank published by Dunn. The result indicated this pEtK2 gene included an open reading frame (ORF) which encoded 487 amino acids residues, and had a 99% homology with pEtK2 in GenBank, in which 10 nucleotides were different from those reported by Dunn. Seven nucleotides produced sense mutation, while the other three nucleotides resulted in non-sense mutation. The pEtK2 gene encoded a 55kDa calmodulin-domain protein kinases (EtCDPK ) which was thought to play an important role in the sporozoite invasion.2. Expression of pEtK2 cDNA and purification of the recombinant proteinpEtK2 gene was subcloned into pET28a(+), and then transformed into E. coli (BL21). After induced by IPTG, 55kDa recombinant protein was expressed and could be detected by SDS-PAGE. Analysied by TCL-scanning, it was found that the target protein reach its peak at about 5 hour after induction, which covered more than 32 percent of the total proteins. To purify the recombinant proteins, the inclusion bodies were isolated and washed with TritonX-100. After which; the proteins were renatured by dialyzation in PBS and the lymphocyte transformation test was performed. The result shew that pET28a(+)-pEtK2 recombinant protein could stimulate the proliferation of lymphocytes from chicken infected with coccidian.3. Construction of immuno-regulating DNA vaccine against E. tenellapVAXl-pEtK , pcDNA4.0(c)-pEtK2 , pVAXl-pEtK2-IL-2 , pcDNA4.0(c)-pEtK2-IL-2 DNA vectors were constructed by the DNA recombinant technique. After identification by restriction enzyme digestion, the recombinant DNA plasmids were then injected into chest muscle of 14-day old chicken respectively, then muscle tissue samples were analyzed by RT-PCR and western blotting to confirm the expression of pEtK2 gene and IL-2 gene. The result indicated that both pEtK2 gene and IL-2 gene were successfully expressed in chicken chest muscle tissue.4. Protective effect of immuno-regulating DNA vaccine on chicken challenged with E. tenella.pET28a(+)-pEtK2 recombinant protein and pVAXl-pEtK2, pVAXl-pEtK2- IL-2, pcDNA4.0(c)-pEtK2, pcDNA4.0(c)-pEtK2-IL-2 DNA vaccines were injected into chicken chest muscle twice at the 14 and 21 days old respectively. Chicken were then challenged with fresh E. tenella sporulated oocysts at 28 days old.. In order to evaluate the protective effects of these DNA vaccines against E. tenella infection, the following parameters were surveyed such as weight gain, oocytes production (OPG), the caeca lesion score and anti-coccidial index(ACI). The results illustrated that the DNA vaccines were high effective immuno-regulatory vaccines for they could reduce the relative oocyst production , increase relative weight gain ratio. The caeca lesion score was also significantly reduced. The relative oocytes production of the groups inoculated with pVAXl-pEtK2-IL-2 and pcDNA4.0(c)-pEtK2-IL-2 were 29.89% and 23.26% contrasting with the positive control group. The caeca lesion score of the groups inoculated with pVAXl-pEtK2-IL-2 and pcDNA4.0(c)-pEtK2-IL-2 were 0.8...