Expression And Functional Verification Of Virulence Factor CaM And ATG8 Proteins Of Cryptosporidium Parvum

Cryptosporidium parvum is a parasitic protozoa belonging to Cryptosporidium of Cryptosporididae.It is parasitic in humans and a variety of mammals,and can cause severe diarrhea and even life-threatening for hosts with low immunity and incomplete immune function.At present,there is no effective therapeutic drug for cryptosporidiosis.Virulence factors play an important role in the adhesion,colonization,invasion and escape from host immunity of Cryptosporidium,which may be one of the important causes of Cryptosporidium infection.Among the many virulence factors of apicomplexan parasites,calmodulin is considered to be a calcium dependent enzyme activity regulatory protein,which plays an important role in the growth and development of apicomplexan parasites.Especially in the process of parasites invasion into host cells,Ca M can assist parasites invasion by regulating Ca²⁺signal.Autophagy related protein 8 is the key protein causing autophagy,which is very important for the survival of apicomplexan parasites.When parasite is under stress conditions such as nutritional deficiency and drug stimulation,it can maintain its own nutritional needs by starting autophagy.At present,there are few reports on the role of Ca M and ATG8 in the host mechanism of C.parvum infection.Based on this,on the basis of C.parvum transcriptome sequencing and protein qualitative analysis,the recombinant proteins of C.parvum Ca M and ATG8 were expressed in prokaryotic,and their functions were preliminarily studied.The following results were obtained:（1）C.parvum transcriptome sequencing and protein qualitative analysis:The subtype of Cryptosporidium oocysts passed from calves in this study was identified as C.parvum IId A19G1.Transcriptome sequencing of the oocyst showed that the oocyst of Cryptosporidium contained 3805 genes which encoding 3805 proteins.A total of 4431 signal pathways were enriched by GO analysis,including protein phosphorylation and DNA binding;A total of 207 signal pathways were enriched by KEGG analysis,including dopaminergic synapse and neurotrophin signal pathway.The qualitative analysis of protein showed that the Cryptosporidium oocysts contained 675 proteins.A total of 1136 signal pathways were enriched by GO analysis,including peptide biosynthesis process and structural molecular activity;A total of 56 signal pathways were enriched by KEGG analysis,including RNA transport and alanine metabolism disorder.There are 248 proteins shared by transcriptome sequencing and protein qualitative analysis.These proteins exist in signal pathways such as pyruvate metabolism and RNA degradation.（2）Sequence analysis,prokaryotic expression and functional study of Ca M:Sequence analysis of Ca M protein gene shared by C.parvum oocyst transcriptome and proteome showed that the protein has no signal peptide and transmembrane region,and there are two chiral domains in its tertiary structure.After prokaryotic expression of the Ca M protein,it was found that the recombinant Ca M was expressed in the supernatant,with a molecular weight of 26 k Da.The recombinant Ca M was purified by nickel column,and the purified protein was immunized with rabbits to prepare Ca M neutralizing antibody,the serum titer reached 1:100000.q PCR showed that the expression level of C.parvum Ca M was significantly lower than that of the oocyst at 6 h,8 h and 12 h after the oocyst infected HCT-8 cells,but began to rise at 24 h,reached the peak at 36 h and was significantly higher than that of the oocyst,and then began to decline.At 48 h,its expression level was significantly lower than that of the oocyst.Indirect immunofluorescence test showed that Ca M was mainly located in the middle of the sporozoite,and mainly distributed around the nucleus of the parasites after C.parvum infected the cells.The neutralization test of polyclonal antiserum showed that the maximum neutralization efficiency of Ca M polyclonal antiserum against C.parvum invasion reached 30.69%at 1:100 dilution,suggesting that Ca M may participate in the process of C.parvum invasion into host cells.（3）Sequence analysis,prokaryotic expression and functional study of ATG8:Sequence analysis of ATG8 protein gene,which only exists in the transcriptome of C.parvum oocyst,found that the protein has no signal peptide and transmembrane region,and its tertiary structure showed that it was a loose globular protein.After prokaryotic expression of the ATG8 protein,it was found that the recombinant ATG8 was expressed in the sediment with a molecular weight of 14 k Da.The recombinant ATG8 was purified by nickel column,and the purified protein was immunized with rabbits to prepare ATG8 neutralizing antibody.The serum titer reached 1:100000.q PCR showed that the expression level of C.parvum ATG8increased gradually after the oocyst infected HCT-8 cells,reached the peak at 12 h and was significantly higher than that of the oocyst,and then began to decline.The expression level of C.parvum ATG8 was significantly lower than that of the oocyst at 24 h,36 h and 48 h.Indirect immunofluorescence test showed that ATG8 was distributed in the whole cytoplasm of sporozoites and mainly around the nucleus of parasites after C.parvum infected the cells.The neutralization test of polyclonal antiserum found that the maximum neutralization efficiency of ATG8 Ca M polyclonal antiserum to C.parvum invasion at 1:200 dilution was8.24%.In conclusion,this study conducted transcriptome sequencing and protein qualitative analysis of C.parvum oocysts.On this basis,Ca M and ATG8 were selected for prokaryotic expression,and the protein expression,localization and neutralization efficiency of C.parvum invasion were studied.It was found that the expression level of Ca M decreased in the early stage and increased in the late stage after C.parvum oocyst infection,while the expression level of ATG8 increased in the early stage and decreased in the late stage after infection;Ca M was mainly distributed in the middle of the sporozoite,while ATG8 was distributed in the whole cytoplasm of the sporozoite.After C.parvum infected the cells,Ca M and ATG8 were mainly distributed around the nucleus of the parasites;The maximum neutralization efficiency of Ca M and ATG8 polyclonal antiserum against C.parvum invasion was 30.69%and 8.24%,respectively.The results deepen the understanding of the effects of calmodulin and autophagy related proteins on C.parvum infection,lay a theoretical foundation for further clarifying the invasion mechanism and pathogenesis of C.parvum,and also provide new ideas for cryptosporidiosis vaccine design and drug research and development.