Biological Characteristics And Immunogenicity Of Recombinant Clostridium Septicum Alpha Toxin Mutants

As the major pathogen that causes human gas gangrene and animal malignant edema,Clostridium septicum is also the pathogen of sheep braxy.Clostridium septicum alpha toxin,CSA,is the main pathogenic factor and immunogen.Sheep braxy caused by Clostridium septicum often leads to death since the acute onset makes it too late to be treated.As a result,vaccination is the only effective way to prevent this disease.Conventional vaccines against this disease are bacteria-toxoid vaccines made from Clostridium septicum culture inactivated and detoxicated by formaldehyde.Biosafety hazard comes out since the vaccine’s producing process needs to cultivate a mass of virulent culture,which makes the non-toxic recombination CSA vaccine a new research focus.This research optimized and synthesized CSA whole gene containing signal peptide and combined it with pEZ-clone vector according to Escherichia coli preferred codon.Using the vector as the template,we adopted OEPR（Overlap Extension PCR and Recombination）technique to construct a recombination vector pET28a-csa expressing CSA.By point mutation with pET28a-csa,we constructed 7 different CSA mutant expression vectors successively,including pET28a-csa_C86L,pET28a-csa_Y118T,pET28a-csa_TMD△_212-222,pET28a-csa_C86L/TMD△_212-222,pET28a-csa_Y118T/TMD△_212-222,pET28a-csa_C86L/Y118T86L/Y118T and pET28a-csa_{C86L/Y118T/TMD}△_212-222.The pET28a-csa and 7 different CSA mutant expression vectors were transformed into BL21 and then 8 recombinant bacteria were obtained.Results of inducing expression and SDS-PAGE revealed that 8 recombinant proteins molecular weight were accordance with anticipation.The protein was expressed in both inclusion body and soluble ways.The relative expression quantities amounts of 8 recombinant proteins rCSA,rCSA_C86L,rCSA_Y118T,rCSA_TMD△_212-222,rCSAC86L/TMD△212-222,rCSAY118T/TMD△212-222,rCSAC86L/Y118T and rCSAC86L/Y118T/TMD△212-222 were 7.11%,5.86%,6.56%,15.4%,12.7%,12.9%,4.26%and 11.9%respectively,which showed that partial deletion of transmembrane domain would increase the protein expression.The western-blotting results suggested that all the recombinant proteins could be recognized by CSA hyper-immune serum.Toxicity test in mice showed that lethal dosage of rCSA was 0.1μg per mouse while that of rCSA_Y118T was 1μg.The lethality decreased by 10 times.This result suggested that although rCSA_Y118T118T could decrease the lethality compared with CSA,its toxicity still existed and was not eliminated.In the meantime,18μg rCSA_C86L,18μg rCSA_C86L/Y118T,80μg rCSA_Y118T/TMD△_212-222,120μg rCSA_TMD△_212-222,120μg rCSA_C86L/TMD△_212-222,and 120μg rCSA_{C86L/Y118T/TMD}△_212-222 were injected into mice respectively and they all did not cause mice death.The data showed that compared to rCSA,lethality of rCSA_C86L86L and rCSA_C86L/Y118T decreased at least 180 times,lethality of rCSA_Y118T/TMD△_212-222 decreased at least 800times and lethality of rCSA_TMD△_212-222,rCSA_C86L/TMD△_212-222,and rCSA_{C86L/Y118T/TMD}△_212-222 decreased at least 1200 times.These 6 CSA mutants lost their lethality to mice.Cytotoxicity experiments showed that cytopathy occurred when Vero cells were inoculated with 0.001μg/mL of rCSA and 0.5μg/mL of rCSA_Y118T,while 1μg/mL of rCSA_C86L/Y118T,10μg/mL of rCSA_C86L,10μg/mL of rCSA_Y118T/TMD△_212-222,30μg/mLofrCSA_C86L/TMD△_212-222,50μg/mLofrCSA_TMD△_212-222and50μg/mLof rCSA_{C86L/Y118T/TMD}△_212-222 did not cause cytopathy.Comparing with rCSA,cytotoxicity of rCSA mutants decreased at least 500 times for rCSA_Y118T,1000 times for rCSA_C86L/Y118T,10000 times for rCSA_C86L86L and rCSA_Y118T/TMD△_212-222,30000 times for rCSA_C86L/TMD△_212-22212-222 and 50000 times for rCSA_TMD△_212-222and rCSA_{C86L/Y118T/TMD}△_212-222.These data suggested that among the 7 rCSA mutants,the other 6 rCSA mutants all lost cytotoxicity except rCSA_Y118T,which accorded with the toxicity test in mice.Hemolysis test revealed that rCSA had hemolytic activity while 7 rCSA mutants lost this activity.Soluble expression products of rCSA and 7 rCSA mutants were prepared as aluminum hydroxide vaccines respectively.The rabbits were divided into 8 groups and then 8 vaccines were injected in rabbits with a dosage of 200μg per rabbit on day0 and day21 respectively.Blood samples were collected on vaccination day0,day21 and day35 and then these rabbits were challenged with 1MLD Clostridium septicum toxin on day35.The other 6 immunized-challenged groups were all 5/5 protected except the CSA_C86L/TMD△_212-222 group（4/5）and rCSA_Y118T/TMD△_212-222 group（2/4）.Results of neutralizing titer assay showed that after the first immunization,neutralizing titers（quantity of Clostridium septicum toxin mice MLD neutralized by 0.1mL mixture immune serum）of rCSA_C86L,rCSA_Y118T,rCSA_TMD△_212-222,rCSAC86L/TMD△212-222,rCSAY118T/TMD△212-222,rCSAC86L/Y118T,rCSAC86L/Y118T/TMD△212-222 and rCSA group were 3,7,2,2,1,2,4 and 10 respectively.After the second immunization,neutralizing titers of rCSAC86L,rCSAY118T,rCSATMD△212-222,rCSAC86L/TMD△212-222,rCSAY118T/TMD△212-222,rCSAC86L/Y118T,rCSA_{C86L/Y118T/TMD}△_212-222 and rCSA group were 20,36,34,20,2,60,24 and 60 respectively.This result also indicates that the 118th Y and transmembrane region of CSA is not only related to the virulence of the toxin,but also to the immunogenicity of the toxin.Based on the above results,we chose E.coli BL21(pET28a-csa_TMD△_212-222)to conduct the expression conditions optimization of rCSA_TMD△_212-22212-222 to improve the expression quantity of soluble protein.We also tested the immunogenicity of purified rCSA_TMD△_212-222 further.The optimum expression condition we found was that add 0.5mM IPTG into the culture when OD₆₀₀ was 0.8 and then induce culture at 15℃for 16h.The soluble protein expression could reach to 24.7%at this condition and purity could reach to 85%after purification.Mice stayed alive after injected intravenously with purified 140μg of rCSA_TMD△_212-22212-222 while cytopathic did not occur after the Vero cells were inoculated with 80μg/mL of rCSA_TMD△_212-222.This indicated that rCSA_TMD△_212-222 lost both its lethality against mice and cytotoxicity.We prepared the purified expression products into aluminum hydroxide vaccines.Then rabbits were divided into 3 groups and then each group was immunized with a dosage of 25μg per rabbit,50μg per rabbit and 100μg per rabbit subcutaneously respectively on day0 and day21.Blood samples were collected on day0,day21 and day35 and then these rabbits were challenged with 1MLD Clostridium septicum toxin on day35.Results showed that after the first immunization,neutralizing titers of the three groups were 1,4 and 6 respectively while after the second immunization,those of the three groups were 68,72 and 58 respectively.Besides that,the three immunized-challenged groups were all 5/5 protected.In conclusion,E.coli BL21(pET28a-csa_TMD△_212-222)constructed in this research has the advantages of high expression of target protein,nontoxicity of expression products and good immunogenicity.Thus this strain could be a candidate strain of recombination Clostridium septicum vaccine’s research and development.