Expression And Functional Verification Of Insulin-degrading Enzymes INS21 And INS4 Of Cryptosporidium Parvum

Cryptosporidium spp.infects host intestinal epithelial cells and are one of the important pathogens of human and animal diarrhea.The pathogen can be transmitted by water,food,animal-animal,animal-human and so on.Comparative genomics analysis found that the genome of Cryptosporidium is highly compact,most of the genes are single copies,but the insulinase（INS）family of the M16 family of metalloproteinases contains about 20 genes,means that the family may play a role in the invasion and the development of Cryptosporidium.Previous work indicates that INS-20-19 may play an important role in the invasion and early development of Cryptosporidium,but it is not known about the function of other family members.In this study,INS21（encoding by cgd7₂080 gene,56.2 kDa）with a smaller molecular weight and INS4（encoding by cgd2₉30 gene.116.2 kDa）with intact domain were selected as subjects to study their roles in the invasion and growth of Cryptosporidium.The recombinant plasmid was constructed by ligating the gene of interest to the plasmid of PET-28a,and the recombinant plasmid was transformed into E.coli BL21 for prokaryotic expression and purification to obtain a recombinant protein.A polyclonal antibody was prepared by immunizing rabbits with the purified protein as an antigen.Real-time quantitative PCR was used to determine the expression of the target gene in C.parvum culture in vitro.The indirect immunofluorescence method was used to locate the expression of the target protein in the life cycle of C.parvum,and the neutralization experiment detected whether the protein of interest plays a role in the invasion of C.parvum.The results showed that INS21 was expressed in inclusion bodies,and a protein of 56 kDa was obtained.The antibody titer was 1:32000.In the natural state,a band corresponding to the expected size was obtained in the sporozoites,the expression level of INS21 reached the highest peak at 2 h after infection,and distributed at the front and middle ends of sporozoites.The neutralizing efficiency of polyclonal antibody was about 31%.INS4 was expressed in the supernatant,and a protein of 116 kDa was obtained.The antibody titer was 1:6400.In the natural state,a band slightly smaller than the expected size was obtained in the sporozoites,The expression level of INS4 reached the highest peak at 2 h after infection and distributed at the mid-end of sporozoites.The neutralizing efficiency of polyclonal antibody was about 36%.The above results indicate that INS21 and INS4 may be involved in the invasion or early development of C.parvum.The findings help to further understand the biological functions of the insulin protease family.