Preparation Of Specific McAb-PE40 Chimeric Toxin And Its Kill Action To Cryptosporidium Parvum

Four hybirdoma (1D5, 2E9, 3D6 and 4G4) producing McAbs against sporozoites oocyst antigens of C.parvum were developed by fusion of SP2/0 mouse myeloma cells and spleen cells from BALB/C mice immunized with sonicated oocysts.The antibody isotypes of the IDS, 3D6, 4G4 were of the IgG1 subclass,while the 2E9were of the IgM class.All McAbs were reacted with Cryptosporidium sporozoites by indirect immunofluorescent assay, two of which were reacted with surface of sporozoites.The number of chromosome of the hybirdoma cell line was 90 to 98. The ELISA liters of acites were 1:409, 600, 1:819, 200, 1:819, 200 and 1:1, 638, 400, and the ELISA liters of cell supernatant were 1:160, 1:320, 1:320 and 1:640 respectively.Two single chain antibody ScFv directed against Cryptosporidium parvum sporozoite envelope glycoprotein was constructed by using the antibody variable region (V) genes of murine McAb against Cryptosporidium parvum, the VH and VL gene were amplified by RT-PCR from a hybridoma cell line 3D6 and 4G4, producing mouse McAb against Cryptosporidium parvum sprozoite envelope glycoprotein protein. The heavy and light chain variable regions were connected by a flexible Linker(Gly4Ser)3. The fusion gene (ScFv) were cloned into pMD-18T vector and sequenced. The nucleotide sequence results showed that the VH and VL genes were homologous with the published mouse antibody variable region gene sequences and were confirmed as functionally rearranged mouse immunoglobulin variable region genes. The 3D6 ScFv gene were 720bp, consisting of VH, Linker and VL genes , VH gene was 351bp,encoding 117 amino acids ,The VL gene was 324bp,encoding 108 amino acids .The 4G4 ScFv gene were 717bp, consisting of VH, Linker and VL genes , VH gene was 348bp,encoding 116 amino acids ,The VL gene was 324bp,encoding 108 amino acids.To express ScFv containing chimeric immunotoxins , we conslructed a immunotoxins expressing plasmids by replacing the receptor binding domain of PE genes with ScFv gene, plasmid pET28-ScFv-PE40 encodes for a hybrid protein consisting Nterminus of ScFv and C terminus of PE, After transformed these plasmids into E.coli strain BL21(DE3) and induced by IPTG, both immunotoxins (3D6 ScFv-PE,4G4 ScFv-PE) were expressed successfully.Expressed recombinant immunotoxins 3D6 ScFv-PE take 14% of the total cell proteins , while the expression level of recombinant immunotoxins 4G4 ScFv-PE were 12%.. After cell lysis and SDS-PAGE analysis , the expression ptoducts exist in forms of inclusion body and soluble fraction.The expression ptoducts were evaluated the cytotoxicity to Cryptosporidium parvum sprozoite, The results showed that the expression immunotoxins have specific cytotoxicity to cryptosporidium parvum sporozoite.The number of wandering sporozoites in treatment group is much less than control group, while the mobility of the sporozoite in treatment group is much less than control group.These results showed the chemeric toxin has good killing effect on C.p sporozoite.