| Avian coccidiosis is an intestinal disease caused by infection with any of several species of the protozoan Eimeria, and was distributed globally. Its prevalence in intensive farms was up to 50% to 70% and the mortality rate was 20% to 30%, up to 80% in severe infection. The annual economic loss caused by coccidiosis in the global poultry industry was about ￡ 500 million. Among the recognized seven Eimeria species from chicken, E. tenella was is the most virulent parasite and was one of the most widely distributed species. In our previous works, yeast two-hybrid system was used to screen the sporozoites proteins interacting with the invasion-related protein—apical membrance antigen-1(AMA1) and fourteen potentially interacting proteins were obtained. The numbered 56-2 EST sequence was selected and was cloned and characterized in the present study. In addition, its interacting relationship with EtAMA1 protein was verified and potential as a novel DNA vaccine candidate molecule was also evaluated. 1. Cloning and bioinformatic analysis of Eimeria tenella serine / threonine protein kinase(EtSTK)The full-length cDNA sequence was cloned by RACE using these primers designed according to the obtained 56-2 EST sequence. The obtained sequence was analyzed by bioinformatics tool and found that it was 100% identity with ZB1-A08 gene differentially expressed between sporozoites and unsporulated oocyst. This protein contains a serine / threonine protein kinase domain and was named as Eimeria tenella Serine / threoni ne protein kinase(EtSTK). The full-length cDNA sequence of EtSTK was 1799 bp and the open reading frame(ORF) was 1524 bp, which encodes 507 amino acids with a molecular weight of 54 kDa and an isoelectric point of 8.36. The EtSTK protein has no signal peptide and may contain 24 antigenic sites. Real-time PCR showed that the EtSTK transcription level in sporozoite was significantly higher than the unsporulated oocysts, sporulated oocysts and second generation merozoites(P<0.05), which showed that the level of transcription was developmentally regulated. 2. Prokaryotic expression and molecular characterization of EtSTKThe pCold I-EtSTK prokaryotic expression plasmid was constructed and was then transfected into BL21(DE3) E. coli competent cells. The soluble recombinant EtSTK protein was expressed successfully and was purified by using Ni affinity chromatography. The antiserum against EtSTK was successfully obtained after immunizing rabbits with the purified recombinant protein. Western blot showed that the recombinant protein had good immunogenicity. IFA showed EtSTK was mainly located on the surface of apex of sporozoite and was transferred to the top of sporozoites after invasion of DF-1 cells. During the subsequent development, the EtSTK protein mainly concentrated on the surface of trophozoites and immature schizonts. When the parasites develop to mature schizont, the protein was mainly in the anterior region of the first generation merozoites. Invasion inhibition assay in vitro showed that anti-EtSTK polyclonal antibody IgG-treated sporozoite inhibition rate was significantly increased, as compared with the normal rabbit IgG treated-group, and the inhibition rate was positively correlated with the concentration of antibodies. The inhibition rate was up to 70% when the antibody concentration was 400 μg/mL, suggesting that the EtSTK protein plays an important role in processes which sporozoites invade the host cell. 3. Verification of the interacting relationship between EtSTK and EtAMA1In order to construct eukaryotic recombinant expression plasmids of Eimeria tenella Serine/threonine protein kinase(EtSTK)gene and express it in DF-1 cells, the EtSTK gene was amplified by PCR with the cDNAs of the sporozoite of E. tenella as template. The PCR products were subcloned to pcDNA3.1-flag vector. The recombinant plasmids were transfected into DF-1 cells. The expression of recombinant EtSTK protein in DF-1 cells was detected with Western blot and indirect immunofluorescene(IFA). A protein about 54 kDa which was in accordance with molecular weight of target protein was recognized by recombinant EtSTK antiserum in Western blot. Green fluorescence was observed by IFA in the transferred cells. These results indicated that the recombinant plasmids of pcDNA3.1-flag-EtSTK were successfully constructed and expressed in DF-1 cells. Then on this basis, Co-IP and GST Pull-Down technology were used to verify the interacting relationship between EtSTK and EtAMA1. Co-IP showed that anti-EtSTK polyclonal antibody can precipitate the EtSTK-EtAMA1 complex and anti-EtAMA1 monoclonal antibody also can precipitate the EtAMA1-EtSTK complex. GST Pull-Down showed that EtSTK protein can be captured successfully by the EtAMA1 bait protein. Both Co-IP and GST Pull-Down results showed that EtSTK protein may interact with EtAMA1 protein, verifying the previous results obtained by yeast two-hybrid system. 4.Protective efficacy of eukaryotic expression plasmid pcDNA3.1-flag-EtSTK against E. tenellaDoses of 100 μg/bird and 200 μg/bird of eukaryotic recombinant plasmid pcDNA3.1-flag-EtSTK or empty plasmid pcDNA3.1-flag were immunized 7-day-old chickens with intramuscular way, and the non-immunized challenged group and non-immunized non-challenged group were set. The second immunization conducted at 14-day-old with the same immunization dose and immunizing way as the first immunization. At 21 days old, all chickens except the unchallenged control group were inoculated orally with 1×104 sporulated oocysts of E. tenella Shanghai strain. The weight gains(WGs), cecal lesion scores and oocyst production were used as evaluate protective efficacy. Results showed that WGs of all immunized groups during immunization were similar to those of non-immunized group(P>0.05). The WGs of immunized groups during challenge were more than those non-immunized challenged group, but the difference was not significant(P>0.05). The WGs of non-immunized challenged group was less than those of non-immunized non-challenged group, but the difference was not significant(P> 0.05). The oocyst production of eukaryotic recombinant plasmid group was significantly lower than that of non-immunized challenged group(P<0.05), the oocysts reduced rate was 74.07%-78.13%. The oocyst production between the empty plasmid group and the non-immunized challenged group was not significant(P>0.05), the oocysts reduced rate was 40.39%-46.03%. The lesion scores of recombinant plasmid groups and empty plasmid groups were similar to that of non-immunized challenged group(P> 0.05), were significantly lower than that of the non-immunized challenged group(P<0.01). These results showed that the eukaryotic recombinant plasmid pcDNA3.1-flag-EtSTK have partly protective effect against E. tenella infection. 5. Screening the host cell proteins interacting with EtSTK proteinThe recombinant eukaryotic expression plasmid pcDNA3.1-flag-EtSTK was transfected into DF-1 cells. The cells protein was collected and was used as conduct Co-IP. SDS-PAGE results showed that electrophoretic bands between the anti-EtSTK antibody treated- and the untreated DF-1 protein were significantly different. These samples were analyzed by shotgun mass spectrometry and the obtained peptides were BLAST with Uniprot chicken(Gallus_gallus) protein databases. A total of 28 proteins were found in the anti-EtSTK antibody treated-DF-1 protein and 345 were found in the untreated-DF-1 protein. Only 12 proteins were found in both treated and untreated DF-1 protein, including chicken myosin, vimentin, shock protein 70, glucose-regulated protein. These protein peptides obtained in this study lay the foundation to further study the molecular mechanism that EtSTK play an important role in the host cells invasion. |
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