Identification And Analysis Of Biological Function Of Cryptosporidium Parvum Glycoprotein Gp40 Receptor Protein ENO1

Cryptosporidium is apicomplexan parasite that mainly infects the intestinal epithelium.Although cryptosporidiosis has been reported around the world,children in some developing countries and under-resourced areas bear a heavy burden.Furthermore,Cryptosporidium is the leading cause of diarrhea in children and AIDS patients worldwide.Cryptosporidiosis usually causes stunted growth in children and can develop chronic and fatal diarrhea in immunocompromised people.Of the nearly 44 effective species of Cryptosporidium identified,Cryptosporidium parvum is currently considered to be an important species associated with fecal-oral transmission between humans and domestic animals.Transmission occurs via the fecal–oral route,and sources of Cryptosporidium infection include contaminated water or food or contact with infected people or animals.After entering the intestine,the oocysts release infected sporozoites with the ability to infect the intestinal epithelial cells,and then secrete multiple proteins to participate in the invasion throughout the process.Cryptosporidium parvum gp40 protein is a secreted protein identified in the process of sporozoites invading host cells and plays an important role in the invasion process.So far,the molecular mechanisms of interaction between the Cpgp40 protein and the host cell proteins are unknown.Therefore,the present study is the first to screen the potential interaction possible proteins with Cpgp40 protein by GST-Pull down and LC-MS/MS technology.Then we verified the possible interaction relationship.The functional characteristics of receptor proteins in C.parvum sporozoites adhesion and invasion in vitro were preliminarily explored,which laid a foundation for further study of related invasion mechanism,prevention of Cryptosporidium infection in animal husbandry and development of cryptosporidiosis related drugs.Firstly,C.parvum gp40 gene was constructed into the prokaryotic expression vector p GEX-4T-1.Recombinant plasmid p GEX-4T-1-gp40 was transformed into expressing bacteria and inducing expression at 1 m M IPTG,37°C.And then recombinant GST-gp40 proteins and control GST proteins were successfully obtained,and SDS-PAGE and Western blot detection and identification of recombinant proteins were performed.Finally,the highly purified gp40 protein was obtained by glutathione agarose affinity chromatography,and the rabbit polyclonal antibody of anti-Cpgp40 was successfully prepared,which laid a good foundation for subsequent experiments.After obtaining the purified proteins,we identified a total of 26 proteins by GST-Pull down technology and LC-MS/MS technology.Based on the result of the Venn diagram,a total of 14 proteins with the possibility of interaction with the Cpgp40 protein were screened.Most of them distribute on the cell membrane and cytoskeleton of the HCT-8 cells.We detected the changes of the m RNA levels of several proteins located in the cell membrane and cytoskeleton in LC-MS/MS results at different time points during C.parvum invasion of HCT-8 cells.We found that ENO1 gene m RNA level was significantly downregulated 3h after C.Parvum invasion,which was the adhesion invasion stage.It is worth noting that ENO1 is widely studied in parasite infection and mainly participates in the degradation of extracellular matrix.Therefore,based on the results of the experiment and the bioinformatics analysis of these genes,we selected the ENO1 protein as a follow-up experimental study.Subsequently,we successfully verified the interaction of Cpgp40 protein and ENO1 protein of HCT-8 cells in vitro using bimolecular fluorescence complementarity and immunoprecipitation,laying the foundation for the following experiments.Antibodies and si RNA specific to ENO1 showed the ability to neutralize C.parvum infection in vitro,which indicated ENO1 play an important role in infection of HCT-8 cells.The addition of exogenous proteins and the transfection of overexpressed vectors also indicate that ENO1 proteins has a facilitating effect in C.parvum invasion of HCT-8 cells.In addition,we further demonstrated that ENO1 is involved in the degradation of extra cytoplasmic matrix during the invasion of C.parvum and the energy supply process of C.parvum in the developmental stages within cells.Further studies are needed to fully understand the specific role and functional mechanism of ENO1 in the invasion of the host to determine the feasibility of it as a vaccine candidate for cryptosporidiosis in vivo.In summary,this study preliminarily explored the interaction between Cryptosporidium parvum gp40 protein and host cell protein,laying a foundation for studying the invasion mechanism of Cryptosporidium parvum.