Expression And Functional Verification Of The Insulinase Family Member INS-5 And INS-1 Of Cryptosporidium Parvum

Cryptosporidium spp.are zoonotic parasites and important diarrhea-causing pathogens.Compared with other apicomplexan protozoa,Cryptosporidium spp.have quite compact genome,expressing as only one copy of most genes,but have a large number of insulinase（INS）family members.More than twenty INSs were found in divergent Cryptosporidium spp..It is speculated that the insulinase family members may have important functions during the invasion and development of Cryptosporidum spp..In this study,we expressed INS-5（encoded by cgd2₂760 gene）and INS-1（encoded by cgdl₁680 gene）and analyzed their preliminary function.The recombinant plasmids were constructed,transformed into the Escherichia coli system and expressed the target protein.After immunization with rabbits,polyclonal antibodies were produced and used for immunofluorescence analyses.The expression level of the target protein after invasion of C.parvum in vitro was analyzed by real-time quantitative PCR.The location of the target proteins in C.parvum developmental stages were determined by indirect immunofluorescence analysis.The blocking effect of antisera on the invasion of C.parvum was assessed by neutralization assay.Results showed that the expression of INS-5 was the highest at 2 h postinfection,INS-5 was localized at the anterior,middle and posterior regions of sporozoites,and the inhibition rate of the anti-INS-5 antisera against the C.parvum invasion was 39%.The expression of INS-1 was the highest at 48 h and 72 h post-infection,INS-1 was localized at the anterior region of sporozoites,and the inhibition rate of the anti-INS-1 antisera is 32%.It is speculated that INS-5 may be involved in the invasion process of sporozoites;although INS-1 is associated with sporozoite invasion,it is more likely to play a role in the development of pathogens in host cells.According to the different expression patterns of INS-5 and INS-1,it is speculated that the expression of the insulinase family members may be regulated by the development of Cryptosporidium spp.,and thus play different roles during different life stages.These results laid the foundation for understanding the biological function of insulinase in Cryptosporidium spp.,which may help to further elucidate the molecular mechanism of Cryptosporidium invasion and development,so as to provide a theoretical support for formulating new immunotherapy strategies.