Effect Of Different Domains Of Apical Membrane Antigen 1 On Invasion Of Emieria Sporozoites And Screening Of Interacting Proteins

Eimeria tenella is the most harmful protozoa to chickens.Apical membrane antigen-1(AMA1)is an important component during the formation of motor-linked loops before entry to host cells.A preliminary study on the characteristics of Eimeria tenella AMA1(EtAMA1)was conducted in our lab and found that the mRNA and protein translation levels of Et AMA1 in sporozoites were higher than those of the second generation merozoites.Both the sporozoite membrane proteins and cytosolic proteins are expressed EtAMA1,while are mainly expressed in the second-generation merozoites cell membrane proteins.The EtAMA1 antibodies can significantly inhibit the invasion of sporozoites into host cells.The present study was conducted to explore the effects of different domains of EtAMA1 on the Eimeria tenella invasion of host cells.1.Establishment of primary chicken embryo fibroblasts cells culture model forEimeria tenella9-day-old SPF chick embryos were used.The heads,wings,viscera and limbs of the embryos were removed from the aseptic environment.The chicken embryo tissue pieces were rinsed with sterile PBS to prepare a braided tissue,which was digested,centrifuged,filtered,and the like.Generation cells were cultured,tested,and passaged to establish a chicken embryo fibroblast culture system.Eimeria tenella sporozoites were inoculated into primary cells of chick embryo fibroblasts to observe the growth and development of the parasites in cells for up to 76 h.The sporozoites began to invade cells at 5 h after inoculation,and most of the worms had developed within the cells at 24 h,lumps of schizonts appeared at 72 h,and the merozoites began to overflow from cells at 76 h.Judging from the life cycle of Eimeria tenella,these experimental results were similar to those observed in chicken caecum test,indicating that the in vitro culture model of chicken embryo fibroblast primary cells was successfully established which lay the foundation to study the important role of EtAMA1 in Eimeria tenella invasion 2.Effect of different domains of EtAMA1 on Eimeria tenella sporozoites invasioncellsThe primers were designed according to Et AMA1 domainⅠ(EtAMA1-DⅠ)sequence.Et AMA1-D Ⅰ gene was cloned,prokaryotic recombinant expression plasmid pGEX-4T-2-EtAMA1-DⅠwas constructed,and transferred into E.coli BL21 competent cells to induce the expression of proteins.After purification of the recombinant protein using Ni column affinity chromatography,New Zealand white rabbits were immunized to prepare polyclonal serum antibodies.Western blot showed that the recombinant protein had good immunogenicity and reactogenicity.Incubation of sporozoites with synthetic EtAMA1-D Ⅲ and signal peptide and prepared rEtAMA1-DⅠantibody had certain inhibitory effects on parasite invasion.Gliding and adhesion experiments showed that EtAMA1-DⅢ had a partial effect on the pre-invasion movement.The results showed that the DⅠ,DⅢ,and signal peptides in EtAMA1 all played a role in during process of Eimeria invasion.3.Study on the interaction between Et AMA1-DⅠand rhoptry neck protein 2Using the cDNA of Eimeria tenella sporozoite as a template,the E.tenella rhoptry neck protein 2(EtRON2)and EtAMA1-DⅠgenes were amplified by PCR and the recombinant eukaryotic expression plasmid pBiFC-VN155-EtAMA1-DⅠand pBiFC-VC155-EtRON2 were constructed.Both recombinant eukaryotic expression plasmids were transfected into BHK cells for expression and their expression were identified by indirect immunofluorescence(IFA).As a result,specific green fluorescence was detected,indicating that the constructed recombinant plasmids pBiFC-VN155-EtAMA1-D Ⅰ and pBiFC-VC155-EtRON2 were expressed in eukaryotic cells BHK.The interaction between EtRON2 and EtAMA1-DⅠ was validated by the BiFC technique.The BiFC results showed that the co-transfected pBiFC-VN155-EtAMA1-DⅠand pBiFC-VC155-EtRON2 group,the positive control group of p BiFC-bJunVN173 and p BiFC-bFosVC155 showed specific green fluorescence in transfected BHK cells,while the negative control group did not stimulate the green fluorescence,indicating that there is an interaction between EtAMA1-DⅠand Et RON2.Therefore,it is speculated that the interacting between EtAMA1-DⅠand EtRON2 would be play a key role in Eimeria tenella cell invasion.4.Screening of host cell proteins interacting with EtAMA1Previous studies had shown that AMA1 can interact with some parasite proteins,which was an important step to parasite invading cells succefully.But to date,no cells proteins interacting with AMA1 has been reported.In the present experiment,host cell(DF-1 cell)proteins interacting with the extracellular domain of Et AMA1 were selected for screening.The recombinant prokaryotic expression product GSTEtAMA1 was expressed and purified.The purified GST-rEtAMA1 was verified by Western blot to have a single target band at approximately 72 kDa,confirming that the obtained protein is GST-Et AMA1 fusion protein.The GST-EtAMA1 protein,GST protein and PBS were incubated with glutathione agarose beads and incubated with DF-1 cell protein.The eluent was subjected to SDS-PAGE.The results showed that the bands among them were significantly different.The differential bands were excised and analyzed by mass spectrometry.The obtained protein peptides were blast-matched to the Uniprot chickens protein database,and 14 potential proteins that interacted with DF-1 cells in the extracellular domain of EtAMA1 were initially obtained.Including actin,tubulin,guanine nucleotide binding protein,ribosomal protein and some conserved proteins.The results laid the foundation for further elucidating the molecular mechanism of the extracellular domain of EtAMA1 that plays an important role in the coccidia invasion of host cells.