Immune Protection Of The Antigent NPmz19 Against E.necatrix

Avain coccocidiosis,an enteritis caused by Eimeria infection,is of major economic importance to the poultry industry around the world.The development of poultry industry was restricted by the emergence of drug-resistant parasites and the drug residues problem so that a new approach to prevent avain coccocidiosis should be found.Stimulating the cell immuno-reaction of the host has been approved to be an effective approach to prevent avain coccocidiosis.The DNA vaccine,as the third generation of vaccine,has the obvious advantage to induce abroad cell immuno-reaction and form perdured immune memory. Especially,the immune regulative DNA vaccine,constructed by special antigen together with cytokine gene,has more prospect in the anti-coccocidiosis practice.In this investigation pVAX1.0-NPmzl9,pVAX1.0-NPmz19-IFN-γ,pVAX1.0-NPmzl9-IL-2 were constructed by the gene recombinant technology with the antigen NPmzl9 of Eimeria necatrix and the chicken cytokine IFN-γ,IL-2.The protective effects of the above mentioned eukaryotic recombinant plasmids together with NPmz19 protein were tested in the protective experiment of chickens against the challenge of Eimeria necatrix.Based on NPmzl9 sequence published on GenBank,a pair of special primers were designed with computer software.NPmz19 gene was amplified by RT-PCR with the total RNA extracted from E.necatrix merozoites as template.In order to identify it,the nucleotides were sequenced and compared with the existing data published on GenBank. The result indicated this NPmz19 gene encoded 960 nucleotides,319 amino acids residues, with a whole open reading frame,6 nucleotides lesser than the NPmz19 on Gen_Bank,the sequence has submitted to GenBank,the accession number is EU795423,and it had a 94% nucleotide homology and 88%amino acid homology with NPmz19 on GenBank,51 nucleotides and 24 amino acids are mutated.The NPmz19 gene encoded a 35kDa protein.NPmz19 gene was cloned into pET32a(+) vector,then transformed into E.coIi BL21. After induced by IPTG,SDS-PAGE indicated that about 35kDa recombinant protein was expressed.It was found that the target protein expressed to peak on the 4th and 5th hour after induction.To purify the recombinant protein,the inclusion body was isolated and washed by 1%Triton-X100.Then it was denatured by urea and renatured by dialyzation in PBS.Finally,the concentration of recombinant protein was 4.6mg/mL.With the DNA recombinant technique,the recombinant plasmids pVAX1.0-NPmz19, pVAX1.0-NPmz19-IFN-γ,pVAX1.0-NPmz19-IL-2 were constructed.After the restricted enzyme digestion,these three recombinant plasmids were then injected into the leg muscles of 14-day old chicken respectively,the injected muscles were analyzed by RT-PCR and western blot to confirm the expression of the antigen NPmz19 and cytokine gene.The result indicated that they can successfully expresse in chicken's muscles.pET32a(+)-NPmz19 recombinant protein and pVAX1.0-NPmz19,pVAX1.0-NPmz19 -IFN-γ,pVAX1.0-NPmz19-IL-2 eukaryotic recombinant plasmids were injected into chicken's leg muscles at the age of 14 and 21 day respectively.Chicken were then challenged with E.necatrix sporulated oocysts at 28 days.In order to evaluate the protective effects of these eukaryotic recombinant plasmids against E.necatrix,the parameters including weight gain,the oocysts per gram of caecum of caecum,the small intestine lesion score and anti-coccidial index(ACI) were observed.The Results indicated that these recombinate protein and eukaryotic recombinant plasmids were effective since they could reduce the relative oocyst production increase relative weight gain,and significantly reduce the small intestine lesion score.The small intestine lesion score of pVAX1.0NPmz19,pVAX1.0NPmz19-IFN-γ,pVAX1.0NPmz19-IL-2 and pET32a(+) -NPmzl9 were respectively 1.03,0.97,0.69,1.53,1.04×10~5,the counting number of caecum were respectively 1.04×10~5,0.93×10~5,0.65×10~5and 1.56×10~5 and the relative weight gain were respectively 91.18%,93.78%,96.61%and 85.56%,and ACI were respectively170.88,182.98,188.71,160.26.As reported above,pVAX1.0NPmz19-IL-2 is the best one of the three eukaryotic expression vectors,therefore it is promising to protect avian against E.necatrix.