Expression And Functional Verification Of The MEDLE Family Member Protein Of Cryptosporidium Parvum

Cryptosporidium spp.which can cause zoonotic cryptosporidiosis,is an important cause of diarrhea in humans,ruminants,and other mammals.Comparative genomic analysis of C.parvum（infected people,ruminants and rodents）and C.hominis（just infected people）,which species of genetic similarity and different range of hosts,has shown that these two species have different copies of telomeric genes encoding for secretory insulin hydrolase and the MEDLE family.It was suggested that they could contribute to differences in host specificity among Cryptosporidium spp.However,the current protein familys in the invasion of cryptosporidium function are unknown,especially the MEDLE family which was not found in creatures other than Cryptosporidium.To understand the role of the MEDLE family during the invasion of cryptosporidium,the cgd5₄590 gene,a member of the MEDLE-specific family of C.parvum,was selected for prokaryotic expression.According to the cgd5₄590 gene sequence in C.parvum gene database,a 567 bp gene fragment without signal peptide was obtained by PCR amplification.The target gene was recovered and ligated to pET-28a vector,then transformed into E.coli DH5a for cloning.The monoclonal colonies were identified and sequenced by PCR.The recombinant plasmid was transformed into E.coli BL21 for prokaryotic expression.The recombinant protein（21 kDa）was detected by SDS-PAGE and Western Blot was matched to the predicted size（23 kDa,which contains a signal peptide sequence）.It was further confirmed by Mass Spectrometry.The induced amount was expanded and the expressed product was purified by affinity chromatography to get the purified protein which concentration is 500 μg/ml.The recombinant protein was used to immunize rabbits to prepare antiserum and purified polyclonal antibody.The target protein was screened by indirect immunofluorescence to explore its expression position on C.parvum.At the same time,the expression period of the target gene was analyzed by qPCR technique.Neutralization experiments of polyclonal antibodies was further conducted,C.parvum which incubated by the polyclonal antibody infected the host cells to observe whether the infection rate changes.The results show that the protein expresses on the surface of the sporozoites.The expression of target protein was accompanied by a host invasion process,and the expression reached the maximum after 48 hours of invasion into the host cells.Experimental study of polyclonal antibody neutralizing MEDLE family protein suggested that infection rate of C.parvum to host cells has decreased by 50%.Based on the above results,we hypothesized that the MEDLE-2 protein encoded by the cgd5₄590 gene is involved in the host invasion and is likely to play an important role in this process.