Construction And Activities Research Of ScFv-PE40 Recombinant Immunotoxin Against Eimeria Acervulina

Coccidiosis is a kind of worldwide protoziasis.It is one of the most serious diseases in the intensive hennery.Medicine is used to control coccidiosis for many years.For the drug-fast coccidians appeared constantly and the anti-coccidia medicine is remnant in the body of cattle and fowl, lmmunotoxin is used to control coccidiosis.To Recombinant plasmid pET22-ScFv-PE40 was constructed and specific McAb-PE40 immunotoxin was obtained to study immunotoxin's kill action to Eimeria acervulinaHealthy male chicken was inoculated with the supernatant of the ATCC27853 culture which included the exotoxin A, the chicken was dissected after death . The pathologic changes in heaet ,liver, spleen,lung,kidney,stomach show the exotoxin A produced in ATCC27853 is very toxic.Based on the published nucleotide sequence of Pseudomonas aeruginosa exotoxin A structural gene,a pair of primer was designed and synthesized to amplify the PE40 gene by PCR.The purified PCR product was cloned into pMD18-T simple vector to construct a cloned plasmid pT-pE40.The plasmids were transformed into competent E.coli JM109 cells.Colonies were selected on ampicillin-containing agar plates and plasmids were isolated from several independent clones.The recombinant plasmid was identified with HindIII and. an objective gene fragment with the length 1101bp was obtained.After pT-PE40 and expression vector pET22(b)+ were digested with Hindlll and purified on an agarose gel,the PE40 gene was ligated into expression vector pET22-ScFv to construct a recombinant plasmid pET22-ScFv-PE40,and then transformed into competent E.coli JM109 cells.The recombinants grown on the amp plate. The aimed clone was identified with SalI and NotI. The objective gene fragments with the length 1101bp and 6194bp were obtained.The plasmid pET22-ScFv-PE40 was sepuenced to select the plasmid in which 5' terminus of the PE40gene follows 3' terminus of the ScFv gene.Recombinant gene expression was induced by the addition of IPTG for five hours. SDS-PAGE and western blot showed that a new protein of 68KD was expressed in E. coli which was in agreement with the expected molecular mass of the fusion protein of ScFvand PE40. SDS-PAGE showed that the expression products existed in form of soluble fraction.The expression ptoducts were evaluated the cytotoxicity to E.acervulina sporozoites. The results showed that the immunotoxin has specific cytotoxicity to E.acervulina sporozoites.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 E.acervulina sporozoites...