The Study On The Prophylactic And Therapeutic Preparations For The Clostridium Perfringens Type A Alpha-toxin

In this study,the fusion protein of rCPA and HSP65 were expressed in order to boost the immunizing effect of Clostridium perfringens Alpha—toxin vaccine.In addition,a human scFv against the alpha-toxin was obtained by screening human phage antibody library(Tomlinson I+J)in the previous experimental work of our laboratory.On a basis of it,we further explored the the biological activity of the scFv and constructed the specific bivalent scFv against CPA to improve immune binding activity.The above measures conducted could lay the foundation to control various diseases caused by CPA.In this study,the recombinant rCPA gene with its encoding gene of phospholipase C(PLC)and lethal activities knocked out was amplified by PCR using whole CPA gene as a template and inserted into expression vector pET-28a(+)-HSP65 with by gene-recombination technology.The recombinant plasmid pET-28a(+)-rCPA-HSP65,which was confirmed by enzyme digestion and sequence determination and analysis,was transformed to E.coli BL21(DE3)for expression with5 hours of induction by IPTG at 34?.The expressed product identified by SDS-PAGE was soluble form of proteins,which showed an apparent molecular mass corresponding to the predicted size(90 kDa)and the objective protein accounted for about 30.17% of total bacterial proteins.The conditions of inducement including medium type,induction temperature,and induction time were optimized,then the fermentation with high cell density and high expression culture of engineering bacteria was conducted.After one-step on column purification and refolding in DEAE ion exchange chromatography,then the preliminary obtained fusion protein was further purified with metal chelating chromatography.The purity of the recombinant protein was about 95%.The purified fusion protein rCPA-HSP65 was used to immunize mice by routine immunization schedule with sole rCPA or HSP65 as negative control and the antibody titers in peripheral blood of mice were determined and compared by indirect ELISA method.The results showed that the level of serum neutralizing antibody in mice immunized with rCPA-HSP65 vaccine continued to increase and after the third immunization,the average neutralizing antibody titerreached about 212.The serum antibody titer of rCPA-HSP65 group was significantly higher than that of rCPA group.The challenge test results showed that immunization with rCPA-HSP65 yielded a significant protection rate(above 80%)when mice were challenged intravenously with 2×LD50.In addition,the hyper-immune sera can obviously neutralize the lethal effect of a toxin in mice.In the study,high cell-density fermentation of engineering bacteria was conducted to obtain large quantity of scFv against CPA after induction with IPTG for6 hours at 28?.After centrifugation,the supernatant of fermentation products was treated by 60% saturated ammonium sulfateprecipitation method.Precipitation dissolved by PB was dialyzed overnight in PBS.The sample was preliminarily purified with metal chelate affinity chromatography and further purified by rProtein A affinity chromatography.The final scFv reached a purity of more than 97% after purification with a relative molecular weight of approximately 31 kDa.Western blot analysis showed that there was a specific protein band at the site of 43 kDa,Which confirmed that scFv could specifically associate with CPA.The precise binding affinity of scFv to CPA was measured by surface plasmon resonance.Kinetic analysis suggested that the equilibrium association constant(KA)was 2.02×1010(1/M).The equilibrium dissociation constant(KD)was 4.94×10-11(M).Based on this property that the phospholipase C(PLC)and hemolytic activities of CPA could be neutralized by scFv in vitro,CPA were preincubated(37°C,2 h)with different amounts of scFv.The mixtures were added to egg yolk emulsion,mRBC suspension or blood agar plates.Following incubation for 2 h at 37°C,PLC activity was indicated by an increase in absorbance at 620 nm with the development of turbidity.Hemolytic activity was determined by measuring the absorbance of the centrifuged mRBC suspension at 540 nm or by the haemolysis result on blood agar plates.The final test results showed that the scFv could inhibit PLC activity and hemolytic activity,and both kinds of inhibition were dose-dependent.In vivo neutralization test,mice were intravenously injected with preincubated mixtures of 2×LD50 of CPA and various doses of scFv.Compared with negative control,no deaths occurred at a CPA to scFv mass ratio of 1:10 or 1:15.However,the protective effect was decreased at a mass ratio at or below 1:8.The results initially showed that the dosage required for neutralizing 2 × LD50 of CPA was about 18.69mg/kg,denoted as m(scFv)for convenience.In passive protection assays,mice were intravenously injected with 2× m(scFv)at different time prior to intravenous challenge with 2 × LD50 of CPA.The result showed that the passive protection rate reached 80% when mice were challenged 1-h post scFv injection.In the emergency rescue assays,the treatment effects of scFv was detected when using the dose of 2 × m(scFv)injected intravenously at different time intervals after each group of mice was challenged with2×LD50 of CPA.The survival rate reached 80% for mice treated with scFv within 30 min of being challenged with a 2×LD50 dose of CPA.None of the mice intravenously injected with this dose of CPA survived if the interval between challenge and scFv treatment was more than 1 hour.The result showed that accurate diagnosis and timely treatment were very important for treatment of CPA poisoning with this single chain antibody.Pathological observation showed that lesions of major organs in survival mice treated by scFv were slighter than that of dead mice untreated after challenged.However,in comparison with the IgG forms of monoclonal antibodies(MAb),the binding force of this scFv with single binding site was relatively low.In addition,another characteristic of the scFv was its instability in vivo because of its small molecular weights that would be subject to a fast renal clearance.So,the specific bivalent scFv against CPA was constructed and expressed using molecular cloning technology,together with preliminary study on its biological activity.Methods:with scFv gene as a template,to introduce connecting peptide G4 S or(G4S)3 by primer design and amplify two single chain antibody fragment by PCR,then clone into the prokaryotic expression vector pET-28a(+).The recombinant plasmids sc(Fv)-G4S-sc(Fv)and sc(Fv)-(G4S)3-sc(Fv),denoted as sc(Fv)2-5 and sc(Fv)2-15 for convenience,were confirmed by enzyme digestion and sequence determination and analysis,then transformed and expressed in Ecoli BL21(DE3)by IPTG induction for 5 hours at 30?.The analysis conducted by 12 % SDS-PAGE showed that proteins was expressed in a high level as inclusion bodies and the molecular weight consistent with theoretical value.Inclusion bodies was preliminary purified by chelate affinity chromatography after washing and denaturation.The target protein eluent was subjected to renaturation by step-wise dialysis,then purified by rProtein-A and by molecular screen chromatography(S-75).The target proteins with high purity were obtained in final.The binding ability of bivalent single chain antibody to CPA was detected by indirect ELISA.The result showed that the binding ability of sc(Fv)2-15 to CPA was higher than that of scFv and sc(Fv)2-5.To prove the neutralizing potential of the bivalent single chain antibody in vitro,equimolar amounts of scFv,sc(Fv)2-5 or sc(Fv)2-15 was preincubated with alpha-toxin and subsequently tested for theirlecithinase activity in an egg yolk diffusion turbidity(EYDT)assay,their hemolytic activity in a hemolysis test.The result showed that three specific antibodies could neutralize the phospholipase C(PLC)and hemolytic activities of CPA,and the neutralizing ability of sc(Fv)2-15 was higher than that of sc(Fv)2-5 and scFv.Passive protection assays compared the protective rate of mice intravenously injected with sc(Fv)2-15 or scFv at different time prior to intravenous challenge with 2×LD50of CPA.The result showed that the passive protection rate of sc(Fv)2-15 was higher than that of scFv.In this study,Clostridium perfringens alpha—toxin recombinant vaccine protein rCPA-HSP65 was successfully obtained,simultaneously,its immunogenicity was evaluated.In addition,the biological activity of a human scFv against the Clostridium perfringens type A alpha-toxin was thoroughly studied and human bivalent single chain antibody against this toxin with much higher binding capacity than scFv was successfully constructed.All these lay a good foundation of further study on control measures of the alpha-toxin of Clostridium perfringens type A.