| Avian coccidiosis is one of important parasitic diseases which cause serious economic losses to the poultry industry. Encoded by mic gene, microneme converges at the apical end of the sporozoite and merozoite of Eimeria tenella. It has been suggested that this protein is active during the course of coccidiosis infection. Chicken IL-2 gene is a cytokine which plays an important role in immune regulatory network and it also can stimulate T-lymphocyte proliferation. In this study mic4 gene fragment of E. tenella and chicken interleukin-2 gene were cloned into pET28b(+), pVAX1.0 and pcDNA4.0 vectors respectively. Then in order to evaluate their immune-protective efficiency, the three kinds of DNA vaccines were then used to immunize chicken.Cloning and sequence analysis of mic4 gene of E. tenellaBased on mic4 cDNA sequence published by GenBank, a pair of special primers were designed with computer software. A 804bp DNA fragment was amplified by RT—PCR using total RNA template extracted from E. tenella sporozoites. In order to identify it, the nucleotide was sequenced and compared with existing data published by Tomley in GenBank. The result indicated this mic4 gene fragment encoded 268 amino acids residues, and had a 99.6% homology with mic4 in GenBank, In which 3 nucleotides are differentfrom those reported by Tomley .The mic4 gene fragment encoded a 29.48kDa protein. Expression of mic4 cDNA and purification of recombinant proteinMic4 gene was subcloned into pET-28(b+) vector, then transformed into E. coli (BL21). After induction by IPTG, SDS-PAGE indicated that about 35kDa recombinant protein was expressed . It was found that the target protein expressed to peak on the 5th and 6th hour after induction. To purify the recombinant protein, the inclusion body was isolated and washed by 1%Triton-X100. Then it was renatured by dialyzation in PBS. Finally, the concentration of recombinant protein was 11.569mg/mL which established a good foundation for the following animal experiments.Construction of immune-regulating DNA vaccine of Eimeria tenella Through the DNA recombinant technique, pVAX1.0-mic4 ,pcDNA4.0(b)-mic4, pcDNA4.0(b)-IL-2-mic4 DNA vectors were constructed. After identification by restrictionenzyme digestion, these three kinds of recombinant DNA plasmid were then injected into 14-day old chicken's breast muscle respectively, then injected muscle tissue samples were analyzed by RT-PCR and western blotting to confirm expression of mic4 fragment and IL-2 gene. The result indicated that both mic4 gene fragment and IL-2 gene were successfully expressed in injected muscle tissue.Protective effects of immune-regulating DNA vaccines on chicken challenged withE. tenellapET28b(+)-mic4 , pBV220-IL-2 recombinant protein and pVAX1.0-mic4 , pcDNA4.0(b)-mic4, pcDNA4.0(b)- IL-2-mic4 DNA vaccine were inoculated into chicken breast muscle at the age of 14 and 21 day respectively. Chicken were then challenged with E. tenella sporulated oocysts at 28 days old. In order to evaluate the protective effects of these DNA vaccines against E. tenella, the parameters including weight gain, oocyte production (OPG), the caeca lesion score and anti-coccidial index(ACI) were observed. The Results indicated that these DNA vaccines were effective since they could reduce the relative oocyst production, increase relative weight gain, and significantly reduce the caeca lesion score. The caeca lesion score of pVAX1.0-mic4 , pcDNA4.0(b)-mic4, pcDNA4.0(b)- IL-2-mic4 were respectively 1.45, 1.85, 1.19, and the relative weight gain were respectively 92. 48%, 93. 08%和P95. 53%, and ACI were respectively 167.98, 164.58, 173.63. As reported above, pcDNA4.0(b)- IL-2-mic4 is the best one of the three DNA vaccines, therefore it is promising to protect avian against E. tenella. |
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