Expression And Preliminary Functional Identification Of L-lectin And ADF Proteins Of Cryptosporidium Parvum

Cryptosporidium is a highly pathogenic,water and food-borne zoonotic parasitic protozoa that can cause severe diarrhea in humans and various animals.Treatment for cryptosporidiosis is limited,due to the lack of effective drugs and vaccines.The main reason for this would be that the molecular mechanism of the interaction between Cryptosporidium and host cells and the immune regulation mechanism of the host are not well understood.Using the previous proteomics data of the research group,it was found that the expression of Cryptosporidium parvum(C.parvum)proteins containing lectin＿leg-like and ADF-H domains was up-regulated during gliding and invasion.In Apicomplexa,animals and plants,the lectin＿leg-like domain protein was reported to be mainly involved in protein transport and secretion,innate immunity,UPR signaling pathway,etc.,and the ADF-H domain protein was reported to be mainly involved in the invasion of hosts and self-growth,etc.Based on the above studies,it was speculated that lectin＿leg-like and ADF-H domain proteins might play a certain role in the process of C.parvum infecting the host,but the specific functions were unclear.Therefore,this study was the first to analyze the expression and function of L-lectin(containing lectin＿leg-like domain)and ADF(containing ADF-H domain)proteins in C.parvum.1.Bioinformatics analysis,prokaryotic expression and polyclonal antibody preparation of L-lectin and ADF proteinIn this study,we first analyzed the biological information of L-lectin and ADF proteins.L-lectin protein contained a lectin＿leg-like domain,which was a type I transmembrane protein,and the carbohydrate binding sites(E-121 and G-261)were conserved,but there were certain differences in the metal binding site(S-163).The ADF protein contained an ADF-H domain,which was an actin depolymerization factor,and the binding sites of G-actin(S-4 and G-5)and F-actin(K-93 and D-124)were conserved,but there were certain differences in the binding sites of F-actin(T-77,K-79 and E-121).Then the L-lectin and ADF genes were amplified and connected to the p GEX-4T-1 expression vector,induced and expressed in E.coli,purified by affinity chromatography,and then immunized with New Zealand big-eared rabbits to prepare polyclonal antibodies that the potency is 128 k.2.Preliminary identification on the function of L-lectin proteinThe prepared L-lectin protein polyclonal antibody was used to identify the natural L-lectin protein of C.parvum.The expression of L-lectin gene was detected by real-time fluorescent quantitative PCR,and it was found that the expression of L-lectin gene peaked at 12 and 24 hours post-infection.The indirect immunofluorescence test showed that L-lectin was localized in the wall of oocyst,the membrane surface of the middle and front end of the sporozoite and the cytoplasm of merozoites.The antibody blocking test showed that the blocking effect of the antibody was most obvious when the antibody was diluted at 1:50(inhibition rate=58.1%).Glycosyl binding experiments showed that D-mannose and mannan had a certain binding ability to L-lectin protein.After D-mannose and mannan were co-incubated with oocysts,it was found that they had a significant inhibitory effect on the invasion of oocysts.The binding ability of L-lectin protein to HCT-8 host cells was detected by ELISA and IFA.It was found that L-lectin protein could bind to HCT-8 host cells in a dose-dependent and saturated manner.After the recombinant L-lectin protein was co-incubated with HCT-8 cells,it was found that the recombinant protein had the most obvious effect of inhibiting C.parvum invasion at 200 n M(inhibition rate=42.5%).A total of 40 proteins that might interact with the L-lectin protein of C.parvum were screened out by GST-Pull Down combined with mass spectrometry identification.Through subcellular localization,glycosylation and functional analysis,two glycoproteins(Immunoglobulin heavy constant alpha 1,Metatropic glutamate receptor 1)located on the cell membrane were successfully screened.Whether the two glycoproteins are interacting with L-lectin to mediate the invasion of C.parvum requires further identification.3.Preliminary identification on the function of ADF proteinThe prepared ADF protein polyclonal antibody was used to identify the natural ADF protein of C.parvum.The expression of ADF gene was detected by real-time fluorescent quantitative PCR,and it was found that the expression of ADF gene peaked at 12 hours post-infection.The indirect immunofluorescence test showed that ADF was localized in the cytoplasm of oocysts,the middle region of sporozoites and the cytoplasm of merozoites.The antibody blocking test showed that the blocking effect of the antibody was most obvious when the antibody was diluted at 1:50 (inhibition rate was 58.1%).Actin sedimentation assay showed that ADF protein depolymerized but did not cosedimentation with actin filaments and its ability of F-actin depolymerization was p H independent.Molecular docking assay showed the model of ADF and F-actin is highly reliable,forming the multiple interactions,such as hydrogen bonds,salt bridges,etc.These results will lay a foundation for understanding the biological functions and molecular mechanisms of the lectin＿leg-like and ADF-H domain protein families of C.parvum,and provide a theoretical basis for studying the pathogenicity of C.parvum,host immune regulation and screening of drugs and vaccines for cryptosporidiosis.