Screening And Identification Of Eimeria Tenella SERPIN1 Host Interaction Protein And Preliminary Study On Its Function

Coccidia can invade the different intestines of chickens.Among them,Eimeria tenella is the most virulent,and it is characterized by disrupting the intestinal nutrient uptake mechanism and leading to weight loss and even death.As a result,coccidia have caused a heavy burden on the poultry industry.Currently,understanding the invasion mechanism of coccidiosis is the most effective preventive method.SERPIN1 protein is a membrane protein with a difference of more than 2.0 times,which was screened by proteomics after E.tenella sporozoites invaded DF1 cells.SERPIN1 is located on the surface of the coccidia and may be a membrane antigen.Serine protease inhibitors play an irreplaceable role in the process of chicken coccidia invasion and immune evasion.However,there are no reports on the host protein that interacts with it.In order to further understand the biological function of SERPIN1 protein.This article screened out the host cecal membrane protein that interacts with SERPIN1 protein,and explored whether SERPIN1 protein has tissue tropism and membrane receptor functions.Provide direction for further research and development of regulation and invasion mechanism.This research is mainly divided into the following three parts:1.SERPIN1 gene amplification,protein expression,and verification of polyclonal antibodiesIn this study,the c DNA of E.tenella was first extracted and used as a template to amplify the SERPIN1 gene fragment.The SERPIN1 gene fragment was connected with the digested empty vector p EGFP-C1 to construct a p EGFP-SERPIN1 recombinant plasmid.The plasmid was transfected into DF-1 cells(at a density of 70%)for eukaryotic expression of the fusion protein.Polyclonal antibodies are provided by our laboratory.Use the whole sporozoite protein to verify polyclonal antibodies.The results showed that the fusion protein was correctly expressed,and the polyclonal antibody could incubate a single band the size of SERPIN1.2.Screening and identifying host proteins that interact with EtSERPIN1 and further exploring the function of MAPK1In this study,the host protein interacting with SERPIN1 protein was identified by the GFP pull-down method and analyzed by mass spectrometry.The analysis results show that there are 66 proteins that may interact with SERPIN1 protein.52 unmatched protein sizes.2proteins with unqualified matching rate.The size of the 12 proteins is consistent with that(6proteins are located in the cytoplasm and 6 proteins are located in the cell membrane).In order to verify the mass spectrometry results,5(4 membrane proteins and 1 pathway protein)proteins were screened: ANXA1,ANXA2,ANXA5,ANXA13,MAPK1.Use yeast double-hybrid system and GST pull-down technology for verification.The results show that SERPIN1 protein interacts with the cecal proteins ANXA2 and MAPK1.When the EtSERPIN1 protein was overexpressed in DF-1 cells,the MAPK1 transcription level of DF-1cells was detected by qPCR to decrease.After Eimeria tenella infected chicks,the MAPK1 transcription level of the chicken cecum increased.3.The role of ANXA2 in coccidia invasion and tissue tropismIn order to further explore the role of ANXA2 in coccidian invasion and tissue tropism,this study designed antibody blocking test,immunohistochemical test,and relative fluorescence quantitative qPCR research methods.The results of in vitro antibody blocking test showed that one-way blocking of anti-EtSERPIN1 antibody can reduce the number of sporozoites that invade DF-1 cells,with a maximum inhibition rate of 37%±1.41%.One-way blocking of anti-ANXA2 antibody can reduce the invasion of DF-The number of sporozoites in 1 cell,the highest inhibition rate is 37.15% ± 1.06%,the two-way blocking of anti-EtSERPIN1 antibody and anti-ANXA2 antibody can reduce the number of sporozoites invading DF-1 cells,the highest inhibition rate is 46.5% ± 2.12%.The results of immunohistochemistry showed that EtSERPIN1 protein has weak binding force to the duodenum,jejunum,and ileum,and strong binding force to the cecum.The average fluorescence intensity ratio after binding to each intestinal segment is 0.62%,2.56%,12.17%,17.46%.The cecum was blocked with anti-ANXA2 antibody,and it was found that the ability of EtSERPIN1 protein to bind to the cecum decreased further indicating that ANXA2 may be the membrane receptor of EtSERPIN1.Relative fluorescence quantitative qPCR showed that the transcription level of ANXA2 of Eimeria tenella at 12 h and 24 h after infection of the cecum was significantly higher than that of other intestinal segments,which inferred that it was in the invasion stage at 12 h and 24 h and played an important role in tissue tropism.This study screened and verified the interaction between EtSERPIN1 protein and ANXA2 and MAPK1.ANXA2 is an interacting host membrane receptor,which has partial immune protection to chicks,and plays an important role in coccidian invasion and tissue tropism.