C.parvum And C.andersoni Oocyst Wall Proteomic Analysis And Identification Of Diagnostic Marker Protein

Cryptosporidium is a common parasitic protozoan that can cause water-borne and food-borne diarrhea outbreaks worldwide.Global intestinal center study identifies cryptosporidiosis as one of the leading causes of moderate to severe diarrhea in children under 2 years of age,second only to rotavirus Slowness,cognitive impairment,and even death.At present,effective drug treatments and vaccines for Cryptosporidium are not yet available,so their research has important public health significance.Based on this,it is important to establish an early diagnosis method for human and animal cryptosporidiosis.There have been many ELISA detection methods for the direct detection of Cryptosporidium oocysts and indirect detection of worms,but the use of Cryptosporidium oocyst wall as an immunogen ELISA detection method has not been studied.Due to in vitro detection technology,sporozoite antigens cannot be detected.Therefore,using the oocyst wall as an immunogen may be more sensitive and efficient.In this study,C.parvum and C.andersoni oocysts were used for in vitro excystation to collect and purify the oocyst wall.Qualitative omics analysis of oocyte sac wall protein components,and the use of software and previous research data to predict the protein localized on the oocyst wall surface for recombinant expression,in order to screen the surface of the oocyst wall of C.parvum and C.andersoni.It can be used as a diagnostic marker protein for detection,thereby achieving rapid in vitro detection of Cryptosporidium.1.Isolation and purification of the oocyst wall of C.parvum and C.andersoni.In this study,C.parvum and C.andersoni oocysts kept in the laboratory were used to stimulate the decapsulation of Cryptosporidium by bile salt,decapsulating solution,and 37℃ water bath heating.The rate was over 80%,and the activity was good.Subsequently,the gradient of Percoll cell separation solution was used to isolate and purify the oocyst wall of Cryptosporidium.Among them,in accordance with the requirements of subsequent proteomics experiments,In this study,through the improvement of the method of Cryptosporidium oocyst wall separation,a large number of high-purity Cryptosporidium oocyst wall can be quickly obtained,laying a foundation for further research on the structure and molecular composition of Cryptosporidium oocyst wall.2.Qualitative proteomics identification of Cryptosporidium parvum and Cryptosporidium andersoni oocyst wallIn this study,non-calibration,high performance liquid chromatography fractionation,and qualitative proteomics based on mass spectrometry were used to perform proteomics identification of the oocyst wall of C.parvum and C.andersoni,respectively.It showed that 798 proteins were identified at the stage of C.parvum oocyst wall,and 1 032 proteins were identified at the stage of C.andersoni oocyst wall,accounting for 20%（798/3876）and 26%（1032/3876）.Based on proteomics data,we performed a systematic bioinformatics analysis on all identified proteins,including protein function annotation,subcellular localization,COG/KOG function classification,and protein function enrichment,and performed on all differentially expressed proteins Function classification and function enrichment.We found a large number of ribosomes and proteasome-related proteins in both the oocyst wall component of C.parvum and the oocyst wall component of C.andersoni,indicating that ribosomes and proteasomes play an important role in the process of Cryptosporidium excystation.In addition,in this study,a series of classic oocyst wall proteins（COWP1-9）were identified in the oocyst wall extracts of C.parvum and C.andersoni,thereby validating the purification and extraction methods used in this experiment.3.Cloning,expression and localization of related proteins of C.parvum and C.andersoniBased on bioinformatics predictions and previous research data,this study screened C.parvum encoded by the cgd7₅140 gene with a cysteine-rich surface protein and C.andersoni by the cand₀20680 gene.The protein may be located on the surface of the oocyst wall of Cryptosporidium and may be used as a detection target protein.In this study,specific primers were designed based on the gene sequence,and the Cpcgd7₅140 and Cacand₀20680 genes were amplified by PCR,and the recombinant cloned plasmid and expression plasmid were constructed for prokaryotic expression.The recombinant protein was expressed in the form of inclusion bodies.The target protein was purified by a combination of high concentration urea and a desalting column.Finally,the high concentration protein Cpcgd7₅140-GST protein（1.98 mg/mL）and Cacand₀20680-GST protein（2.02 mg/mL）).Can be used for subsequent tests.Anti-Cacand 020680（1.56 mg/mL）and anti-Cpcgd7₅140（1.50 mg/mL）antibody titers were>512K when the purified recombinant protein was used as an antigen to immunize New Zealand white rabbits.In order to further verify the localization of the target protein expressed in vitro on C.parvum microcysts and C.andersoni,the indirect immunofluorescence method was used to locate the target protein.Cacand₀20680 recombinant protein was localized at On the surface of Cryptosporidium oocysts,specific red fluorescence was observed around the oocysts;Cpcgd7₅140 recombinant protein was located in the entire oocysts,and specific red fluorescence was observed in the entire oocysts.It is speculated that the Cacand₀20680 protein and Cpcgd7₅140 protein can be used as targets for in vitro detection of C.andersoni and C.parvum,respectively.