Expression And Function Verification Of CDPK4 And CDPK6 Proteins In Cryptosporidium Parvum

Cryptosporidium is a zoonotic parasitic protozoa with diarrhea as its main clinical manifestation.There is no effective treatment in clinical practice.Comparative genomics analysis found that protein kinases are the largest members of the Cryptosporidium protein family,and each Cryptosporidium has about 80 protein kinase family members.Cryptosporidium parvum Calcium-dependent protein kinases（CDPKs）are serine/threonine（Ser/Thr）protein kinases.The principle of action of CDPK is to receive and conduct Ca2+signals,and the change of Ca2+ concentration can regulate the physiological processes such as invasion of cells and movement of the worm.Previous studies showed that CDPK1 specific inhibitors can effectively treat cryptosporidiosis,suggesting that CDPKs may be associated with the invasion of host.In order to further understand the role of the CDPK family of Cryptosporidium parvum,CDPK4（cgd7₄0 gene coding）with unique zinc finger structure and CDPK6（cgd4₃330 gene coding）with typical CDPK structure were selected from 9 CDPKs for gene cloning,plasmid construction,protein expression and potential functional analysis.The protein CDPK4 with a size of about 100 KDa was obtained in the E.coli expression system,but no enzyme activity was detected.The highest expression level of CDPK4 was detected by real-time fluorescence quantification at 12 h after infection with the host,and it was also expressed at 48 h and 72 h.CDPK4 was found in the anterior position of sporozoites by indirect immunofluorescence.Neutralization experiments showed that the polyclonal antibody of CDPK4 inhibited the invasiveness of Cryptosporidium parvum into the host by about 34%.Among the nine inhibitors screened,only the inhibitor No.8 had an inhibition efficiency about 38%.In addition,a soluble protein CDPK6 having a size of about 90 KDa was obtained in the E.coli expression system,but no enzyme activity was detected.The protein function analysis was carried out by the same method.The results showed that CDPK6 had a higher expression level at 2 h after invasion of the host,and the highest expression at 6 h,and it was distributed in the periphery of the sporozoites.The neutralization efficiency of the ploycloned antibody was about 24%.None of the 10 inhibitors screened had any inhibition efficiency.It is speculated that CDPK6 does not play a key role in the invasion process of Cryptosporidium parvum,but may play a role in other stages.The results have laid a foundation for elucidating the bioselection function of CDPKs,which is helpful to further understand the role of CDPK family proteins in the process of invasion,growth and development of Cryptosporidium,so as to provide basis for the development of new therapeutic drugs for cryptosporidiosis...