Preliminary Study On The Characteristics Of Cryptosporidium Parvum Enolase And Eukaryotic And Prokaryotic Expression Of Cryptosporidium Parvum Calmodulin-like Protein Gene

Cryptosporidium parvum is an intestinal apicomplexan parasite, which is one of main pathogens that cause diarrhea disease of human and animal in the world. C. parvum is also considered a pathogen causing lots of water-borne diarrhoeal disease outbreaks. At present, there is still no specific treatment for cryptosporidiosis. Enolase is not only a key enzyme of glycolysis in the apicomplexan parasites, but mediated interaction between host and pathogen. Calmodulin-like protein (CML) has a similar structure with calmodulin, which may be an important intracellular Ca2+signal sensor and has important biological function.C. parvum Enolase gene was amplified by PCR with cDNA of C. parvum oocysts. The amplified products were cloned into pMD18-T and the DNA of recombinant pMD-Enolase plasmid were extracted. The plasmids were digested with double enzymes and the objective fragments were connected with pET28a (+) which had been digested with same enzymes. After identifying by double restrict enzymes digestion and gene sequence analysis, prokaryotic recombinant plasmid pET28a-Enolase was transformed into BL21(DE3) competent cells for expressing recombinant proteins. The recombinant proteins were purified by Ni-NTA His Bind Resin affinity chromatography. Antigenicity of fusion proteins was analysed using Western blot technology. Two New Zealand rabbits were immunized with the fusion proteins to obtain the corresponding antibody. The titer of the polyclonal antibody was detected by ELISA assay and the reactivity of polyclonal antibody was identified by Western blot. The results showed that C. parvum Enolase gene was successfully amplified and its nucleotide sequence similarities compared with the GenBank published sequence was99.1%. SDS-PAGE analysis showed that recombinant fusion protein Enolase (rEnolase) was successfully expressed in an abundantly soluble form, with molecular weight approximately55kDa. Western blot analysis showed that the purified fusion protein can be recognized by positive serum from rabbit and His-Tag mouse mAb which indicated that purifed fusion protein was successfully expressed and had good antigenicity. ELISA detection showed that the titer of polyclonal antibody was1:51200, but not reacted with the rabbit muscle Enolase at the same coated amount, which indicated that the polyclonal antibody has good specificity.Enzyme activity of rEnolase and location of Enolase on C. parvum oocyst were detected. The enzyme activity of rEnolase measured by positive reaction of2-D-PGA and PEP transformation was33.5U/mg. The best suitable pH value was7.0-7.5. Little effect on the activity of rEnoalse was found at the temperature from4℃to46℃. The Michaelis constant of rEnoalse was0.7383mmol/L and the maximum reaction rate was0.0579mmol/L· min. Enolases were found on the surface of C. parvum oocysts by indirect immunofluorescence detection.C. parvum CMLe gene was amplified by PCR with cDNA of C. parvum oocysts. The amplified products were cloned into pMD18-T and the DNA of recombinant pMD-CMLe plasmid were extracted. The plasmids were digested with double enzymes and the objective fragments were connected with pVAX-1which had been digested with same enzymes. After identifying by double restrict enzymes digestion and gene sequence analysis, Hela cells were transfected with the recombinant plasmids by FuGENE(?)HD transfection reagent method. The expression of CML gene in Hela cells was measured by Western blot and indirect immunofluorescence. The results revealed that the eukaryotic expression plasmid pVAX-CML was construction successfully. Indirect immunofluorescence and Western blot showed that the recombinant CML proteins were expressed successfully in Hela cells and the proteins with antigenicity.C. parvum CML gene was amplified by PCR with cDNA of C. parvum oocysts. The amplified products were cloned into pMD18-T and the DNA of recombinant pMD-CML plasmid was extracted. The plasmids were digested with double enzymes and the objective fragments were connected with pGEX-6P-1which had been digested with same enzymes. After identifying by double restrict enzymes digestion and gene sequence analysis, prokaryotic recombinant plasmid pGEX-CML was transformed into BL21(DE3) competent cells for expressing recombinant proteins. Recombinant proteins were purified by High-Affinity GST Resin affinity chromatography. The antigenicity of fusion proteins was analysed using Western blot technology. The results showed that CML gene was successfully amplified and its nucleotide sequence similarities compared with the GenBank published sequence was100%. SDS-PAGE analysis showed that recombinant fusion protein CML (rCML) was successfully expressed in soluble and inclusion-body form with molecular weight approximately51kDa. Western blot analysis showed that the purified fusion protein can be recognized by positive serum from rabbit, which indicated that the fusion protein has good antigenicity.C. parvum Enolase gene was successfully expressed in E. coli and the Enzyme activity of rEnolase and location of Enolase on C. parvum oocyst were detected. Eukaryotic and prokaryotic expression plasmids of C. parvum CML gene were constructed and rCML were successfully expressed. These works provided the basis for further researches of characteristics and functions of the Enolase and CML and seeking for novel prevention and control methods of cryptosporidiosis.