The Role Of Specific Peptides Targeting Emeria Tenella Calcium Binding Protein Inhibit Sporozoites Invasion Into Host Cells

Chicken coccidiosis causes huge economic losses to the poultry industry every year.Currently,the control of chicken coccidiosis mainly depends on anti-coccidiosis drugs and live oocysts vaccination.However,there have been severe potential problems such as drug residues,drug resistance and virulence reversion of live vaccines for a long time.The development of new vaccines or drugs aiming at controling chicken coccidiosis is a research hotspot in recent years.The calcium binding protein of Eimeria tenella calcium binding（EtCab）protein is located on the surface of E.tenella sporozoites and plays an important role during invasion of parasites into host cells.In this study,the recombinant EtCab protein was used as the target to screen the binding affinity peptides from the phage 12-peptide library,and the role of the screened affinity peptides inhibiting the invasion of parasites into host cells was systematically detected in vitro and in vivo.Firstly,the recombinant protein EtCab was expressed and purified in prokaryotic system,and the protein was then immunized to New Zealand rabbits.The titer of polyclonal antibody was determined by ELISA method,and the specific binding of polyclonal antibody to natural EtCab protein in E.tenella sporozoites was detected by Western blot and indirect immunofluorescence.SDS-PAGE analysis showed that the recombinant EtCab protein was expressed in soluble form and its molecular weight was about 39 k Da.The rabbit anti-EtCab polyclonal antibody showed a titer of 2¹⁶ and effectively recognized the natural EtCab protein in E.tenella sporozoites.With the aim to screen the twelve peptides that can specifically recognize the recombinant EtCab protein,the recombinant EtCab protein was used as target for four rounds of biological screening using phage display technology,and 30 bacteriophages with affinity were selected.Sequence analysis of 30 affinity phages showed that 13 of 30dodecapeptides were Y peptides,12 were G peptides,3 were V peptides,and the other 2 were different sequences.Then three polypeptides Y,G,V with high repeatability were selected to perform the following research work.The affinity of target bacteriophages was further identified by enzyme linked immunosorbent assay（ELISA）,indirect ELISA and competitive ELISA.The results showed that the three affinity bacteriophages could specifically bind to recombinant or natural EtCab protein and compete with rabbit anti-EtCab protein polyclonal antibody.Based on the above results,three target affinity peptides were then synthesized.In order to construct an in vitro method to objectively calculate the invasion rate of sporozoites into cells,sporozoites were labeled by fluorescence proble,and the labeling efficiency was quantified by flow cytometry analysis.Fluorescence signals of intracellular sporozoites were detected.The results showed that 97.4%of the sporozoites were labeled with fluorescence,and the fluorescence signal of MDBK cells invading by fluorescence-labeled sporozoites could be detected.The results showed that an effective in vitro model for accurately calculating sporozoites invasion rate based on flow cytometry analysis was successfully established.Then the maximum non-toxic concentration of the three affinity peptides to MDBK cells was detected by CCK-8 method,and the concentration was all 125μg/m L.The inhibition rates of three target affinity peptides on sporozoites invasion into host cells were evaluated by flow cytometry analysis.The results showed that the selected three peptides Y G,and V peptide could effectively inhibit sporozoites invasion into cells to some extent,among which both Y peptide and G peptide displayed significantly higher inhibitory effects than that of V peptide（p<0.001）.To ensure that the corresponding phages of the three peptides can effectively exert anti-coccidial effects in the cecum of chicken after oral administration,the release of target phages encapsulated by sodium alginate microspheres was evaluated in vitro in simulated gastric fluid（SGF）and simulated intestinal fluid（SIF）.The results showed that sodium alginate encapsulation could protect the bacteriophage from degradation by gastric acid and maintain a high titer after incubating with simulated gastric fluid with low p H condition.When passing through the simulated intestinal fluid,bacteriophages in sodium alginate microspheres began to gradually release,and almost all bacteriophages were released after 120 min.For in vivo experiment,21-day-old SPF chickens were orally gavaged with encapsulated and unencapsulated phages,respectively,and the distribution and variation with time of affinity phages in the intestinal tract of chickens were detected.The results showed that 1 hour after oral administration of bacteriophage,the phages were mainly distributed in the first half of digestive tract,2 hours after oral administration of target phages,the titer of phages in cecum increased significantly,and 4 hours after oral administration of bacteriophage.A certain number of bacteriophages can still be detected in the cecum of chickens.For purpose of evaluating the anti-coccidial protective effects of bacteriophages corresponding to three affinity peptides,90 SPF chickens with 21-day-old were randomly divided into 9 groups,including TBS group,sodium alginate microsphere encapsulated phage Y group（phage Y+Alg）,sodium alginate microsphere encapsulated phage G group（phage G+Alg）,sodium alginate microsphere encapsulated phage group（phage V+Alg）,phage Y group（phage Y）,phage G group（phage G）,phage V group（phage V）,sodium alginate hollow microsphere group（Alg）and E.tenella infection control group.Except for in TBS group,all chickens were treated with 1.0×10⁵ sporulated oocysts and orally administered 1.0×10¹⁰ PFU corresponding sodium alginate microspheres encapsulated or unencapsulated phages,and then continued oral administration of the same dose of phages for two days.Seven days after infection,the protective effects against coccidiosis were evaluated by average weight gain,oocyst excretion rate,cecal lesion score,anti-coccidiosis index,cecal gross lesion and pathological changes.The results showed that phage Y+Alg group,phage G+Alg group,phage V+Alg group,phage Y group,phage G group and phage V group could provide to some extent anti-coccidial protective effects,and sodium alginate microsphere encapsulated phage group could offer better protective effects,and the anti-coccidial index of phage Y+Alg group,phage G+Alg group,phage V+Alg group,phage Y group,phage G group and phage V group was 170.9,160.33,120.07,145.20,129.44 and 107.62,respectively.The gross and histological pathological changes in cecum of chickens in E.tenella infection control group and Alg group were significantly obvious than those in TBS control group,while those in other groups orally gavaged with phages were significantly alleviated,among which phage Y+Alg group showed the best protective effects.In order to analyze the mechanism of anti-coccidiosis effects of specific peptides targeting EtCab protein from the point of view of bioinformatics,the spatial binding of EtCab to three affinity peptides was analyzed by molecular docking method.Firstly,the 3D structure of EtCab protein was predicted by I-Tasser server,and the homology of three affinity peptides was modeled by modeller software.And then,the above model is optimized and molecular docking is carried out by using Clus Pro online website.The date showed that the EtCab protein model with reasonable structure was successfully constructed.Y-peptide,G-peptide and V-peptide could bind to EtCab protein through hydrogen bond,and the binding effect of Y-peptide was the best,followed by G-peptide and V-peptide.Based on the above analysis,the relationship between conservative amino acid residues and affinity peptide binding sites in Cab protein sequences of five species of Eimeria was further compared.It was found that Y-peptide and G-peptide could bind to three amino acid residues in Cab protein conserved in five Eimeria species,while V-peptide could only bind to one conservative amino acid residue.In summary,the EtCab affinity peptides（Y peptide,G peptide and V peptide）screened in this experiment can effectively inhibit the invasion of E.tenella sporozoite into host cells in vivo and in vitro,and have a certain protective effects against coccidiosis.This study provides a reference for the development of small molecule drugs or vaccines against coccidiosis.