Construction Of Eukaryotic Expression Plasmids Of Clostridium Perfringens EF-Tu And PFO Gene And Their Immune Evaluation

Avian necrotic enteritis is an enteric disease that is caused predominantly by Clostridiumperfringens type A and to a lesser extent by type C strains, and has significant economic impact onthe global poultry production. This disease is usually controlled by antimicrobial drugsadministered at prophylactic doses either in water or in feed. Since the European Union has bannedthe use of antimicrobial growth promoters, the disease was reported much frequently, vaccinationoffers an alternative approach to antimicrobial drugs in control of the disease. In this study, weexpressed the immunogenic protein elongation factor Tu (EF-Tu) and pyruvate ferredoxinoxidoreductase (PFO), prepared and characterized of their polyclonal antibodies. Furthermore, theeukaryotic expression plasmids were constructed with pcDNA3.1(+) vector, and evaluated theimmunogenicity for experimental necrotic enteritis.Based on the gene sequence of Clostridium perfringens ATCC13124strain, EF-Tu and PFOgene were amplified by PCR with specific designed primers, which were1194bp and3516bp inlength, respectively. Then two recombinant expression plasmids pGEX-EF and pGEX-PFO wereconstructed and expressed, with MW of69kDa and154kDa of each recombinant protein. Thepurified expressed protein was injected into rabbit and polyclonal antibody was prepared, TheELISA titer of polyclonal antibody was approximately218and221. Western blot and indirectimmunofluorescence showed that the antiserum had specific affinity for the recombinant expressedprotein, bacterium lysate of Clostridium perfringens and Clostridium perfringens in the bacteriumsmear and in intestine of bacterium infected mice.Then EF-Tu and PFO gene were cloned into pcDNA3.1(+) to construct two eukaryoticexpression plasmids of pcDNA-EF and pcDNA-PFO. Transient transfection of these two plasmidsin BHK21cells were demonstrated successfully by their antibodies, respectively. The chicken inimmunoprotection experiment were divided into4groups, EF group with pcDNA-EFimmunization, PFO group with pcDNA-PFO, pcDNA group with empty pcDNA3.1(+) vector, thegroup of non-vaccinated and non-challenged as control. The chicken except the group ofnon-vaccinated and non-challenged were immunized on14and24days of age, and7days later, allchicken were challenged with Clostridium perfringens ATCC13124(10~8cfu) for5days consecutively. Body weight, the change of antibody titers, intestinal lesions, bacterial colonizationin intestine, the immune protection efficacy of pcDNA-EF and pcDNA-PFO was assessed. Resultsshowed that before the challenge, there was not significantly difference of their body weight in allgroups. However, after challenge, the average body weight gain of chickens in group EF and PFOwas significantly higher than the group of pcDNA. The levels of special antibody in blood werehigher than those of control group. The intestinal lesions score of EF and PFO was significantlylower than the group of pcDNA. Colonization of Clostridium perfringens in the intestine alsosignificantly lower than in the pcDNA group. This demonstrated the certain efficacy of eukaryoticexpression plasmid in immunity of chickens to NE. The studay provides theoretical and practicalbasis for development of new vaccine for NE.