Specific Peptides Binding Emeria Tenella Microneme Protein 3 Inhibit Parasites Invasion Into Host Cells

Emeria tenella microneme protein 3?Et MIC3?is an important protein that is secreted by the organelle of E.tenella during sporozoitesinvasion into host cells.It can bind to specific receptor molecules on the surface of chicken cecal epithelial cells,thus mediating the subsequent invasion process,and cause coccidiosis which caused huge economic losses to poultry industry.With the emergence of the disadvantages presenting by conventional main treatment measures?anti-coccidial drug?and prevention methods?live attenuated vaccines?,for example drug-resistance Eimeria srains and high cost etc,new methods were urgently needed to explored to treat coccidiosis based on theparasites invasion mechanism.S ome studies have been found that Et MIC3 can be used as a candidate vaccine to prevent coccidiosis.Et MIC3 is consists of seven tandem repeat domains?A,B,C1,C2,C3,C4,D?,among which contained three incomplete repeat domains at both ends?A,B,D?and four complete repeat domains in the middle?C1,C2,C3,C4?.With the analysis of conservatism and antigenicity,the function of B and C1 fragments?Et MIC3-BC1?in the process of invasion was mainly explored in the present study.Phage display techniques is a new technology,which was used to study antigen and antibody structure,analyze antigen epitopes,and screen antigen andantibody mimic peptide.Phage display techniques plays an important role in parasitic disease research and prevention.In this study,phage display technique was used to screen affinity peptide binding Et MIC3-BC1 protein and to investigate its inhibitory effects against parasites invasion into host cells.1.Prokaryotic expression of Et MIC3-BC1 recombinant proteins and preparation of polyclonal antibodies.In this experiment the p ET-30a-Et MIC3-BC1 recombinant expression plasmid was firstly constructed by using enzyme digestion sites Bam H?and Xho?,and prokaryotic protein expression was carried out in E.coli?BL21?by using inducer IPTG.The target protein was purified and recovered after SDS-PAGE verification.Then the renatured protein was used as antigen to immunize New Zealand White Rabbits to prepare polyclonal antibody,and the bioactivity of the prepared antibody was detected by Western blot and ELISA methods.The results showed that the p ET-30a-Et MIC3-BC1 recombinant expression plasmid was successfully constructed,the corrosponding protein was expressed,and the prepared polyclonal antibody against the recombinant protein had biological activity.2.Screening of the affinity peptide binding Et MIC3-BC1 recombinant protein.According to the phage display kit instructions,Random 12-mer Phage Display Peptide Library was screened with purified Et MIC3-BC1 protein for four rounds.After the fourth round of biopainning,30independent plaques were randomly selected for amplification,gene extraction,sequencing and derivation of amino acid sequence.The results showed that 8 kinds of affinity peptides sequences were obtained from 30 independent phage clones.Among these sequences,peptides A?AGRLLTPTMSLV?,D?DYHDPSLPTLGK?and W?WKDVHKAWLLEP?that displayed higher repitions and affinity were used for further study.The ELISA and indirect ELISA methods showed that the three peptides could specifically bind Et MIC3-BC1 and sporozoites proteins.The competitive ELISA showed that the prepared polyantibody could competitively inhibit the binding of positive phages and recombinant proteins.The role of affinity peptides A,D and W on inhibiting sporozoites invasion into MDBK cell were detected in vitro.The maximum nontoxic concentration of three affinity peptides to MDBK cells was firstly determined by MTT method and the concentration of 125?g/m L was optimized.The sporozoites?1.0×10⁵/well?,purified by percoll density gradient centrifugation and labeled with CFDA-SE fluorescent probe,were used to invade MDBK cells?1.0×10⁵/well?.The flow cytometry was use to detect the efficiency of the three peptides on inhibiting sporozoites invading into MDBK cells at concentration of 25?g/m L,50?g/m L,75?g/m L,100?g/m L,125?g/m L after incubating for 10 h.The results showed that three peptides had a certain degree of inhibitory effect on inhibiting invasion of sporozoites into cells.And the inhibition effects of peptides A and D were significantly higher than that of peptides W?P<0.01?,and that of peptides A was significantly higher than peptides D?P<0.01?.4.Detection of anti-coccidial ability for three phages corresponding to three affinity peptides in vivo.The purchased 75 chickens with 21-day-old were randomly divided into five groups?n=15?,including PBS group,A peptide treatment group,D peptide treatment group,W peptide treatment group and Eimeria tenella chanllenge control group.In addition to group of PBS?orally inoclated with PBS?,all chickens in each group were orally fed with 1.0×10⁴ E.tenella sporulated oocysts.On days 0?2 post infection?PI?,chickens in PBS group and the Eimeria tenella challenge control group were orally fed with PBS,A peptide group was orally treated with 1.0×10¹² pfu A peptide corresponding to the positive phageper day;D group was orally treated with 1.0×10¹² pfu D peptide corresponding to the positive phageper day;W group was orally treated with1.0×10¹² W peptide corresponding to the positive phage,pfu per day.On day 7 PI,the feces in each group were collected to observe oocysts shedding and calculate oocysts decrease rate.On day 8 PI,all chickens in each group were euthanasized and the changes of weight gain ratio,cecal lesion scores,OPG and ACI were detected,and the gross lesions and histopathological lesions in cecumwere recorded.The results showed that groups treated with A?D?W peptides showed a certain ability to resist coccidiosis,among which the effects in the group treated with A and D peptides were significantly different from those of the challenge control group?P<0.01?and the group treated with W peptide was different from that in challenge control group?P<0.05?.The anti-coccidiosis indexes of the groups of treated with PBS,peptide A,D,W and the challenge control were respectively 200,168.74,161.27,137.36,118.21.5.Atodock molecular docking software was used to evaluate the binding mode of Et MIC3-BC1 proteins and three target peptides.The three-dimensional?3D?model of Et MIC3-BC1 protein was established by Swiss-model,and the spatial binding capacities of A,D,W peptides and Et MIC3-BC1 proteins were analyzed by Autodock.The results showed that there were hydrophobic forces and hydrogen bonds between A peptides,D peptides,W peptides and Et MIC3-BC1 proteins,indicating that all three peptides could bin d to Et MIC3-BC1 proteins in space,and the binding effect of A peptides was the best,followed by D peptides and weak W peptides.In summary,the screened affinity peptides?A,D and W?using Et MIC3-BC1 protein as ligand in this study could effectively inhibit the invasion of E.tenella sporozoites into host cells in vitro and provided protective effects against coccidiosis in vivo.This study provides a reference for developing polypeptide vaccines based on Et MIC3 protein.