Expression And Function Of Cryptosporidium Parvum P60 Protein

Cryptosporidium spp.are zoonotic apicomplexan protozoa,which mainly cause moderate to severe diarrhea or even death to the host.Cryptosporidium spp.contains nearly40 species,in which Cryptosporidium parvum is the most important zoonotic species and composed of different subtype families.Comparative genomics analyses indicate that there exists some highly polymorphic regions among different subtype families in C.parvum,including cgd6＿60 gene,which encodes protease P60,and high level of genetic recombination is also found in the region surrounding cgd6＿60 gene.Thus,protease P60 probably played an important role in Cryptosporidium life cycle.To understand the biological function of protease P60,the follwing works were conducted in the present study:1)Bioinformatics analysis of P60.Comparative genomics analyses indicate that cgd6＿60 gene was highly polymorphic,and bioanalytical methods were used to predict that P60 contained multiple functional domains.Secondary structure analyses indicate that P60 contains 4 strutures including α-helix/β-strand /β-turn/randomcoil,whereas P60 C-terminal protein mainly had a randomcoil structure which was located at the disorder region of the full-length protein.In addition,the C-terminal protein contains all functional domains of the full-length protein and is the functional center of the P60 protein.2)Expression of the full-length P60.A recombinant plasmid was constructed using p ET-28 a plasmid and full-length cgd6＿60 gene,then transferred into E.coli BL21 Condon Plus(DE3)-RIPL(BCPD)expression strain to induce the expression of P60 protein.However,due to the high molecular weight(160 k Da)and complicated structure of the full-length P60 protein,the amount of P60 protein was too low to obtain the purified protein.Therefore,we choosed the C-ternimal region with concentrated function domains in P60 protein for function analysis.3)Expression and functional verification of P60-C-terminal protein.The same methods were used to induce expression of P60-C-terminal protein and the polyclonal antibody was obtained using the purified protein to immunize rabbits..The real-time fluorescence quantitative PCR(RT-q PCR)was used to detect the relative expression levels of cgd6＿60 gene at different stages of invasion.The indirect immunofluorescence experiments were conducted to localize P60 protein in oocysts,sporozoites,and intracellular stages.In vitro serum neutralization was applied to evaluate the inhibition of polyclonal antiP60-C-terminal protein antiserum against the invasion of C.parvum.The results indicated that the expressed products for P60-C-terminal protein contained the expected peptide of 37 k Da and one fragment of 55 k Da,which may be the dimer of the former.The expression of cgd6＿60 gene was highest at 36 h post-infection.The P60 was expressed at the sporozoites of the oocysts,the anterior and middle parts of the sporozoites,and the merozoites in the intracellular stages.In addition,anti-P60-C-terminal protein antibodies reduced the invasion of C.parvum sporozoites by over 31%.Therefore,the above results indicated that P60 played some roles in the invasion,and also involved in the growth and development of intracellular stage for C.parvum.The results of this study will facilitate the understanding of the molecular mechanism of invasion and development,and lay the foundation for the screening of drug targets for Cryptosporidium spp.