The Screening Of Affinity Peptides From Apical Antigen AMA1 Protein In E.tenella And The Study Of Its Anti-coccodia Effect

Chicken coccidiosis is one of the most deadly pathogens,which caused huge economic losses in poultry industry.Although the way of prevention and control of drug feed additive of coccidiosis has played an important role in safeguarding,but with a series of problems,for example,coccidiosis drug resistance and drug residues has produced,there is an urgent need to find new drug target molecules to exploit new drugs.In the study of parasites such as Plasmodium and Toxoplasma gondii has been shown that the apical membrane antigen 1（AMA1）is a promising drug target.Apical membrane antigen 1（AMA1）apicomplexan plays an important role during invasion of parasites into host cells.However,report about the immune function of E.tenella sporozoites AMA1（Et AMAl）was few.In this study,we investigated the inhibitory effect of Et AMA1 extracellular domain gene fragment（Ectodomain,Ecto AMA1）recombinant protein affinity ligand on the infection of Eimeria infection.1. Random 12-mer Phage Display Peptide Library was used to panning the expressed recombinant Ecto AMA1 protein for four rounds.Thirty monoclonal phages were selected randomly and subjected to sequence comparison.The results show that five different peptides were screened by panning on the Ecto AMA1 recombinant protein.Two peptides that have good homology with the RON2 binding region ang the highest binding activity to the Ecto AMA1 were synthesized.Their sequences are LPCYPGYYQITI（L）named L and CWTISLFGGVTQ（C）named C,respectively.Indirect ELISA results showed that the two polypeptides were able to specifically bind to the recombinant Ecto AMA1 protein and Et AMA1 protein.Competitive ELISA results showed that anti-Ecto AMA1 recombinant protein polyclonal antibody inhibited the binding of affinity phage to recombinant Ecto AMA1.2.The ability of L-peptide and C-peptide to inhibit sporozoite invasion in vitro was evaluated.First,MTT method was used to detect the maximum non-toxic concentration of L and C peptides to MDBK cells,and determined the maximum non-toxic concentration was 125μg/m L.E.tenella sporozoites were purified by pecoll gradient centrifugation to obtain the more pure E.tenella spores.The purified sporozoites were labeled with CFDA-SE fluorescent probes,and labeled sporozoites（3×10⁵）were used invasion MDBK（1×10⁵）cells for 10 hours,flow cytometry detected the ability to inhibit sporozoite invasion of peptide L,C were at a concentration of 25μg/m L,50μg/m L,75μg/m L,100μg/m L,125μg/m L.The results showed that L peptide and C peptide had a certain inhibitory effect on sporozoite invasion of MDBK cells.L peptides were superior to C peptides at different concentration gradients.3.The anti-coccidiosis ability of L-peptide and C-peptide was evaluated in vivo.Fourty chicks were randomly divided into 4 groups（n=10）,PBS group,L peptide treatment group,C peptide treatment group and Eimeria tenella infection control group.At the age of 21 days,group L,group C and Eimeria tenella infection control group were infected with 1×10⁴ E.tenella oocysts in oral.At 0-2 days after infection（subspore breeding stage）,L group was orally treated with L peptide corresponding to the positive phage,1×10¹² pfu per day;C group was orally treated with C peptide corresponding phage,1×10¹² pfu per day.The chickens were sacrificed on the 8th day after the challenge,and the weight gain,the cecal lesion score,the cecal eye lesion and the pathological lesion,OPG and ACI were measured.The results showed that the indexes of group L and group C were significantly higher than those of Eimeria tenella infection control group.Compared with group C,the indexes of group L were slightly better than those of group C.The OPG of PBS group,L group,C group and Eimeria tenella infection control group were 200,164.45,153.34 and119.45.4.Swiss-Model software was used to build three-dimensional model of Et AMA1.Autodock4.2 software was used to analysis the L peptide with Et AMA1,C peptide with Et AMA1 docking situation.The binding of Et AMA1 to L peptide and the binding of Et AMA1 to C peptide were studied.The results showed that the homology modeling score was more than 90 points,and the model results were reasonable and reliable.Between L peptide amino acid and Et AMA1 active center amino acid,C peptide amino acid and Et AMA1 active center amino have both intermolecular hydrogen bond and hydrophobic force.L peptide and C peptide in space can combinate well with Ecto AMA1.In summary,the results of this study show that the two Ecto AMA1 recombinant protein affinity ligands can bind to Et AMA1 well.,which can inhibit the invasion of host cells by E.tenella spores and play a protective role in anticoccidial infection.The study provides a reference for chicken vaccine development based on Et AMA1.