Interaction Of Eimeria Tenella Microneme Protein And Host Epidermal Growth Factor Receptor

Eimeria spp. cause coccidiosis, which is economically one of the most severediseases in modern poultry industry. Among Eimeria spp., E. tenella is the mostsevere one. The relationship between the parasite and the host is the foundation ofdisease’s generation and development, however, the Eimeria invasion process remainsobscure. Thus, a deep research on the interaction between parasite and host at theearly stage will have a significance in the mechanism of parasite invading into hostcells.Microneme is the smallest secretory organelle of the Apicomplexan. Themicroneme proteins play a vital role in the invasion of parasites. There are five E.tenella microneme gene sequences registered in GeneBank: EtMIC1, EtMIC2,EtMIC3, EtMIC4and EtMIC5. EtMIC4has31tandem repeat EGF-like domains, andEGF can bind epidermal growth factor receptor. Epidermal growth factor receptor(EGFR), which is widely distributed on the cell membranes of human and animals, ismulti-functional glycoprotein and belongs to RTKs superfamily. EGFR is activatedvia binding with its ligand, such as EGF, and then recruits downstream signalmolecules to transduct the extracellular signal into the intracellular. After activation,EGFR will regulate cell propagation, apoptosis, migration, etc. Whether EtMICEGF-like domains bind CER is still unknown, and the single transduction betweenE.tenella and host cells still needs analysis. The research is based on yeast two hybridsystem identifying whether there is an interaction between EGF-like and hostepidermal growth factor receptor and then the co-immunoprecipitation and GST pulldown assays are employed to further verify this interaction.Identification of interaction of EGF-like and CER EGF-like and CER fragment were generated via RT-PCR. The recombinant bait vector pGBKT7-EGF-like andprey vector pGADT7-CER were co-transformed yeast AH109in order to identify theinteraction of EGF-like and CER. The results showed that the EGF-like can beexpressed in AH109, and recombinant bait vector has no transcriptional activationeffect. At last, the interaction of EGF-like and CER was identified by GAL4yeast twohybrid system.Verification of interaction of EGF-like and CER The recombinant plasmidspcDNA3.1-myc/his-CER and pcDNA3.1-HA-EGF-like were constructed thenco-transfected293T cell lines. The lysates of transfected cell were used to doco-immunoprecipitation. The prokaryotic expression recombinant EGF-like proteinwas applied to GST-pull down assay to verify the interaction of EGF-like and CER.Results showed Co-immunoprecipitation verified the interaction of the two proteins.GST pull down assay verified the interaction. The above results laid a foundation ofelucidation of E. tenella invasion mechanism and the discovery of vaccine candidates,besides, played an important role in coccidosis prevention.