| Chicken coccidiosis is a kind of parasitic disease caused by a variety of Eimeria,of which Eimeria tenella has the strongest pathogenicity,causing huge economic losses to the chicken industry every year.The most effective way to prevent and control coccidia is to block their invasion.Therefore,screening and identifying the key proteins of coccidiosis invasion is the first task to elucidate the pathogenic mechanism of coccidia to carry out effective prevention and control.The coccidiosis invasion process is complicated,and many proteins such as rod-shaped protein,microfilament protein and surface antigen protein have been proved to be related to coccidiosis invasion.Studies have shown that secreted proteins may play an important role in the process of coccidiosis invasion.Research on the invasion related secreted proteins will help to screen and identify key proteins for coccidiosis invasion and deepen the coccidiosis invasion mechanism and coccidia-host interaction understand the mechanism and provide more important reference data for the preparation of more effective anticoccidial drugs or vaccines.In this study,TMT-labeled quantitative proteomics technology was used to screen and identify the secreted proteins related to E.tenella's invasion of DF1 cells in vitro,and the functional analysis of two E.tenella invasion-related proteins SAG14 and EF1? was performed.This study is mainly divided into the following four parts: 1.Proteomic analysis of secretion of Eimeria tenella invading DF1 cells in vitroIn order to study the invasion mechanism of coccidia and the interaction mechanism between coccidia and host cells,it is important to screen and identify the invasion related proteins.In this study,the sporozoite secreted protein of uninfected cells was used as a control.Quantitative proteomic analysis was performed by TMT-labeled sporozoite secreted proteins invading DF1 cells at three time points of 0.5 h,1 h and 2 h.The results showed that a total of 208 effective proteins were quantified,including 153 differential proteins,including 82 up-regulated proteins and 71 down-regulated proteins.Statistical analysis of the differential proteins revealed that 15 co-expressed up-regulated proteins and 9 co-expressed down-regulated proteins were identified.The GO secondary annotation and subcellular structure localization classification analysis of the differential proteins showed that the differential proteins were mainly concentrated in the biological processes such as cellular processes and metabolic processes,and were mainly involved in the composition of cells such as cells,organelles and macromolecular complexes.It mainly plays the role of molecular functions such as catalytic activity,adhesion connection,structural molecular activity,antioxidant activity and transportation activities,and is mainly located in the extracellular and cytoplasmic parts.2.qPCR and Western blot to verify the results of omicsThis study verified the reliability of omics results by combining qPCR and Western blot.Firstly,12 differential proteins were selected to design qPCR primers for verification.The results showed that 10 of the 12 differential proteins had consistency at the mRNA transcription level and protein expression level,and the coincidence rate was 83.33%,which initially verified the reliability of omics.After that,surface antigen 14(SAG14),elongation factor 1?(EF1?)and serine protease inhibitor(SERPIN1)were selected as Western blot verification proteins in omics results,mainly for gene cloning,protein expression and purification,and polyclonal antibody preparation.Western blot verification using ACTIN antibody as the internal control antibody showed that SAG14,EF1? and SERPIN1 were secreted proteins,and the coincidence rate was 100%,which further verified the reliability of omics results.3.Preliminary study on the function of invasion-related protein SAG14Some studies have shown that surface antigen proteins may have an important role in the coccidiosis invasion.In this study,SAG14 secreted proteins from omics results were selected for preliminary functional investigation,including bioinformatics analysis,sporozoite localization,cell adhesion,and in vitro invasion.Evaluation of blocking effect and animal immune protection.The results of bioinformatics analysis showed that SAG14 had a signal peptide and a transmembrane region;sporozoite localization results showed that SAG14 was localized on the surface of sporozoites;SGA14 was displayed on the surface of yeast to adhere to the cells,and the results showed that the yeast with SAG14 displayed on the surface The control group(16.22%±2.84%)had a more significant adhesion effect on cells,with an adhesion rate of 41.89%±2.38%;the in vitro invasion blocking test results showed that SAG14 antibody can significantly reduce the number of sporozoites of invading cells,The highest inhibition rate is 48.40%±3.98%.The immune protection of SAG14 was evaluated through animal experiments.The results showed that after immunization of SAG14 recombinant protein,the chicks can significantly reduce the weight loss of the chicks caused by E.tenella infection,reduce the cecum disease of the chicks and reduce the amount of oocysts.The anticoccidial index was 142.20,and the anticoccidial effect was moderately effective.ELISA analysis showed that chicks infected with E.tenella could stimulate the chicks to produce high titer SGA14 antibodies to resist coccidiosis invasion,showing that SAG14 has a partial immune protection effect on chicks.4.Preliminary study on the function of invasion-related protein EF1?The EF1? secreted protein in the omics results was selected for preliminary functional exploration,including bioinformatics analysis,sporozoite localization,cell adhesion,in vitro invasion blocking effect and animal immune protection evaluation.The results of bioinformatics analysis showed that EF1? has a PTZ00141 conserved domain with the function of temporary elongation factor 1?;sporozoite localization indicates that EF1? can be localized on the surface of sporozoites;EF1? is displayed on the surface of yeast to adhere to the cells,and the results show that EF1? is displayed on the surface Compared with the control group(16.22%±2.84%),the yeast had a more significant cell adhesion effect,the adhesion rate was 35.00%±4.32%;in vitro invasion blocking test results showed that EF1? antibody can significantly reduce the sporozoites of invading cells In terms of quantity,the highest rate of intrusion suppression is 42.07%±4.46%.The immune protection of EF1? was evaluated through animal experiments.After the chicks were immunized with EF1? recombinant protein,the results showed that the weight loss of the chicks caused by E.tenella infection could be significantly reduced,the cecal lesions of the infected chicks and the oocyst output were reduced.The EF1? anticoccidial index was 165.53,and the anticoccidial effect was good.ELISA analysis showed that chicks infected with E.tenella could induce chicks to produce high titer EF1? antibodies to resist coccidiosis invasion,showing that EF1? has partial immunoprotective effect on chicks.In this study,TMT labeled quantitative proteomics technology was used to systematically analyze the secreted proteins of E.tenella invading DF1 cells in vitro.A total of 153 proteins with significant differences were identified,including 82 up-regulated proteins and 71 down-regulated proteins.It was verified that SGA14,EF1? and SERPIN1 were secreted proteins.Preliminary functional investigations of the secreted proteins SGA14 and EF1? revealed that they were all related to sporozoite adhesion and invasion,and had partial immune protection for chicks. |
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