特异性单抗—PE40重组毒素对隐孢子虫杀灭作用研究

Cryptosporidiosis is a worldwide zoonstic protozoiasis.So far there are no good methods to treat the disease althrogh more than 200 drugs had been tested.To observe the immunotoxin action to Cryptosporidium parvum,the study was conducted. A single chain antibody ScFv against Cryptosporidium parvum sprozoite envelope glycoprotein was constructed by using the antibody variable region (V) genes of murine McAb against Cryptosporidium parvum, in which the VH and VL gene were amplified by RT-PCR from hybridomas 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. To express ScFv containing chimeric immunotoxins , we constructed a immunotoxins expressing plasmids by replacing the receptor binding domain of PE genes with ScFv gene.The plasmid pET28-ScFv-PE40 encodes for a hybrid protein consisting N terminus of ScFv and C terminus of PE. After transformed these plasmids into E.coli strain BL21(DE3) and induced by IPTG ,. immunotoxins (ScFv-PE40) were expressed successfully . The expression ptoducts were renatured and preliminary purified to investigated the cytotoxicity to Cryptosporidium parvum sprozoites, The results show that the expression immunotoxins have specific cytotoxicity to Cryptosporidium parvum sprozoite, it would provide a benefit way for the clinical treatment of cryptosporidiosis.

首先制备隐孢子虫特异性单克隆抗体单链抗体基因，然后与绿脓杆菌外毒素A第II、III功能段的完整基因PE40连接并装入表达载体PET-28a中，获得重组毒素后，观察其对隐孢子虫的杀灭作用。进而为隐孢子虫病治疗研究奠定基础，同时亦为其它寄生虫病的生物治疗开辟新的途径。