| Coccidiosis is a wide spread parasites disease happened in the large-scalechicken farm easily, and the pathogens genus is Eimeria spp. Coccidiosis can lead topoultry grow slow and drop feed conversion rate, which indirectly caused the farmingeconomic losses. If more serious, there will be a lot of chickens to dead, resulting insignificant economic losses for farmers. At present, it was reported that coccidiosisinflicted a huge loss of about500million pounds on the poultry industry of the worldeach year. Now, conventional disease control strategies have mainly depend on drugto prevent and control coccidiosis, but the drug resistance of parasites has becomeincreasingly prominent. Meanwhile, the drug residues, the high prices of drug andother problems also prompted people to find new approaches to prevent coccidiosis inchickens. Eimeria is an obligate intracellular parasite belongs to apicomplexan, it hasa complex life cycle, including stages of intracellular and extracellular. The two stageswill invade intestinal epithelial cells of chicken，grow and develop in the intestinalcell. The sporozoite is the first developmental stages of chicken when Eimeria invadeintestinal cells, but how to invade, is not very clear. Study on the differentiallyexpressed genes of sporozoite stage will help us understand the invasion, growth anddevelopment mechanisms of Eimeria. Of course, it would provide us some new ideasfor the development of new vaccines and new drugs. Eimeria tenella is one of themost important species causing avian coccidiosis and is frequently used as a modelspecies to study Eimeria spp. In our previous study, some differentially expressedgenes of sporozoites of E.tenela were identified by using the suppression subtractivehybridization(SSH) and cDNA microarray, and some ESTs were obtained. In thisstudy, one differentially expressed gene of sporozoite was cloned and identifiedaccording one EST sequence(clone number: ZB7-B08, GenBank accession number:ES351380.1).1．Cloning and analysis of a new gene ZB7-B08full-length cDNA of Eimeria tenellaA differentially expressed gene EST of E.tenella sporozoites (clone number:ZB7-B08) was cloned and a full-length cDNA sequence was obtained containing a complete open reading frame of the differentially expressed gene by the rapidamplification of cDNA ends (RACE) technique. In the NCBI website, homology blastfound the gene has68%similarity with the eukaryotic translation initiation factor3subunit7like of Neospora caninum, suggesting that the gene is eukaryotic translationinitiation factor3of E. tenella (EteIF3). In addition, the transmembrane domain,conserved domains, signal peptide, the antigenic sites and hydrophobicity of the genewere predicted by some corresponding bioinformatics softwares. The results showedthat the gene encoded a polypeptide of571amino acids, and theoretical molecularweight is about65KD, which according with the molecular weight size of the dsubunit of eIF3, so the gene was called EteIF3d. Moreover, there were notransmembrane structure, no signal peptide, but had a lot of antigenic sites. Real-timequantitative PCR analysis the gene expression level in four different developmentalstages (unsporulated oocysts, sporulated oocysts, sporozoites and the secondgeneration merozoites) of E. tenella, the results revealed that the gene expressionlevel is relatively high in the sporozoites and the second generation merozoites stage,and the highest expression level is in the stage of second generation merozoites.2．Expression of EteIF3d gene of Eimeria tenella and antigenic validation of theproteinEteIF3d gene was ligated to prokaryotic expression vector pET28b (+), and therecombinant plasmids were transformed into E. coli BL21(DE3), then it was inducedto express recombinant protein. SDS-PAGE showed the molecular weight ofrecombinant proteins was about69KD and reached a expression peak after inducting6hours by adding IPTG. the recombinant protein was inactive and appeared in formsof inclusion bodies. The protein were denatured with urea and purified by Ni-columnaffinity chromatography. Western-blot revealed that the recombinant proteinHis-EteIF3d has excellent antigenicity. In order to get soluble recombinant proteinand do further study function of the protein, EteIF3d gene was ligated to Pichiapastoris expression vector pPIC9k, and the recombinant plasmids were transformedinto yeast cells GS115by electroporation technology. The protein was produced byrecombinant yeast cells which were induced by methanol, and the EteIF3d of E. tenella was expressed in eukaryotic expression system of Pichia pastoris.Western-blot revealed that the protein has excellent antigenicity too.3．The invasion and development analysis on EteIF3d gene of Eimeria tenellaUsing an antibody against recombinant EteIF3d, The localization of EteIF3d insporozoites and during first schizogony in vitro was examined by indirectimmunofluorescence experiments. E.tenella sporozoites were inoculated into chickenembryo fibroblast cell line(DF-1) in vitro first, then anti-EteIF3d polyclonal antibodyimmunized rabbit were used as the primary antibody to analyze the localization andthe expression of the EteIF3d gene in different developmental stages of E.tenella. Theresults showed that expression level of EteIF3d was gradually increasing along withthe time of sporozoites invade in different periods. With the development of thesporozoites, the protein was gathered at the top of the cells, and its expression levelwas also gradually increasing and reached the maximum in the merozoitestage stage.These results suggested that EteIF3d maybe play a role during the developmentprocess of the sporozoite and merozoite. And it maybe also involved in invasion ofintestinal epithelial cells of sporozoite and merozoite. To confirm the protein in theinvasion of E.tenella sporozoites, the inhibition experiment of EteIF3d in vitro wasstudied. Sporozoites were incubated2h at different concentrations with purifiedanti-EteIF3d IgG (50μg/mL,100μg/mL,200μg/mL, and400μg/mL). After that thesporozoites were inoculated into DF-1cells and analysed by flow cytometry. Theresults revealed that the rate of sporozoites invade host cells were gradually decreasedalong with antibody concentration increased. So these results indicated that theprotein indeed played an important role during sporozoites invade the host cells.4．Immunoprotection of chickens against Eimeria tenella by EteIF3d proteinIn order to observe the immune protective effection of EteIF3d against E.tenella,two groups of chicks were immuned with the different dose(50μg and100μg) ofrecombinant protein His-EteIF3d by intramuscular injection.7days after secondaryimmune, the chickens were inoculated with1×104sporulated oocysts of E.tenella.Cecum and spleen of chickens in different periods were collected, The immuneprotective effection of the protein were evaluated by body weight gain, oocysts reduction rates, lesion scores etc., the changes of some cytokites and specific serumIgG were investigated. The results showed that the immune group was better thaninfected control group in weight gain, oocyst reduction rates, lesion scores and otheraspects, and the effect of high-dose immune group were more better; In humoralimmunity, the antibody titers in chickens immuned with His-EteIF3d weresignificantly different from those of the control groups. The specific IgG antibodylevels of chickens immuned recombinant proteins were slight changed in the wholeimmune and infection stages and there were relatively high antibodies levels in thehigh-dose immune group; In Cellular immunity, the expressions of some cytokines(IL-2, IL-4, IL-8, IL-10, IL-18and IFN-γ) in cecum and spleen were analyzed duringdifferent immune stages. The results revealed that the expressions of differentcytokines were different not only in different immune dose but also in differentimmune and infection stages, which have provided the foundation for further studyingabout function of cellular immunity during coccidia invade the chicken body. |
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