Cloning Expressing And Preliminary Founctional Studies Of Eimeria Tenella Microneme Protein1

Eimeria tenella is intracellular parasite in the caecum of domesticchicken causing coccidiosis, a severe infection particularly of poultry. Conventionaldisease control strategies rely heavily on chemoprophylaxis and, to a certain extent,live vaccines. Due to the increasing emergency of drug-resistant strains and theconsumers’ needing green food, many novel methods have been explored to controlthe disease. The decisive measure is using the recombinant vaccines and/or DNAvaccines to control the avain coccidiosis. A major hurdle in development of thosevaccines is searching for the protective antigens. Microneme organelles are presentin all apicomplexan protozoa and contain proteins that are critical for parasitemotility and host cell invasion. One apicomplexan-wide family of micronemeproteins has been identified with members that are characterised by the possessionof hrombospondin type I repeats, conserved adhesive motifs which are implicated inbinding to glycosaminoglycan chains. We chose one critical microneme protein(MIC1) to research:Based on the published nucleotide sequences of MIC1genes in GeneBank, theMIC1fragments were amplified by RT-PCR. Then, By the means of geneticengineering technique, the MIC1and MIC1-AD genes encoding sequences weresubcloned into pMAL-c2X vector, respectively, and transformed into E.coliBL21(DE3). The recombinant protein were expressed by inducing with IPTG. Thetarget protein were detected by SDS-PAGE, and the result of the SDS-PAGEindicated that the recombinant protein(MIC1and MIC1-AD) were about116KDaand66KDa, respectively, and both of them were soluble. The two protein werepurified using the MBP-tag recombinant protein purification system.According to the animal experiment program, the recombinant protein（pMAL-c2X-MIC1and pMAL-c2X-MIC1-AD）were injected into breast musclethree times at the7,14and21day-old chicken, respectively. And chicken were orally challenged with live E. tenella (Guangdong strain) oocysts about3×104at7days following the last immunization. In order to evaluate the protective effects ofthe immunization, survival rate, relative weight gain rate, reduction of caecum lesionscores, oocysts amount in caecum content and anticoccidial index(ACI) wereanalysed. The result indicated that the recombinant protein could reduce the oocystamount in caecum content and decrease the caecum lsion scores, moreover, theeffect of the recombinant protein with high-dose were better than the recombinantprotein with low-dose.The serum antibody titer were determined by ELISA, and the serum wasobtained following the first, second, third immunization and7days after challenging.The mRNA expression of IFN-α, IFN-β, IFN-γ, TNF-α, IL-1β, IL-6, IL-12p40,which were from the caecum in chicken, were detected by Real-time QuantitativeRT-PCR. The results indicated that the immunization with high-dose group had ahigher antibody levels, and the mRNA expression levels about seven kinds ofcytokines were also significantly higher than the positive and negative controlgroups.