Cloning, Expression, And Purification Of A Nontoxic Mutant Of Diphtheria Toxin-CRM₁₉₇ And Preliminary Application In The Glycoprotein Conjugate Vaccines As A Carrier Protein

Neisseria meningitis is one of the leading causes of bacterial meningitis andsepticemia. It is an effective measure to prevent the infection by immunization.Vaccines containing the purified polysaccharide capsule from the organism, a T cellindependent antigen, can provide short-lived and moderate protection in adults, butusually are poorly immunogenic in infants under two years of age. The enhancement ofthe immunogenicity of polysaccharides can be achieved by coupling polysaccharide tocarrier proteins, a process which converts the polysaccharide from a T cell independentantigen to a T cell dependent antigen. In china, the majority of carrier used in conjugatesis tetanus toxoid, derived from tetanus toxin with formaldehyde treatment. It isconceivable, however, that the tendency for certain preparations of formalized toxoid tospontaneously regain toxicity, might be increased by sacchride substitution. A nontoxiccarrier is necessary, and can avoid the problem of handling large volumes of toxicsupernatants. CRM₁₉₇, a nontoxic mutant of diphtheria toxin, has been widely used as acarrier in the glycoconjugates abroad, which is safe and immunogenitic in humans. Bynow, no glycoconjugates using CRM₁₉₇ as carrier protein were developed in ourcountry.The recombinant CRM₁₉₇ was expressed in the prokaryotic expression system by themethod of gene engineering. A glycoconjugate of Neisseria meningitis serogroup A wasprepared, using rCRM₁₉₇ as the carrier. The immunogenicity of the conjugate wasevaluated and analyzed in mice.The genomic DNA of the C7/β₁₉₇ strain as the template, oligonucletides designedaccording to the Diphtheria toxin gene sequence (accession no. K01722) as primers, the CRM₁₉₇ gene (crm197) was amplified by PCR. The amplified fragment was cloned intothe PCR2.1-TOPO vector. After identified by the restriction endoneuclease digestion,the positive clonings were sequenced and analyzed. The crm197 was then cloned intoexpression vector-pET28a. After induced and expressed in E.coli BL21 (DE3), therecombinant CRM₁₉₇ was analyzed by SDS-PAGE. The results showed the proteinexpressed as insoluble inclusion body in cytoplasm, and accounted for about 25ï¼…of thetotal proteins in the E.coli. After purified by Nickel chelate affinity chromatography, thepurity of the recombinant protein was above 95ï¼…. The results of western blottingshowed excellent antigenic property of the protein.The rCRM₁₉₇ was then applied for covalent binding with Neisseria meningitidesgroup A capsular polysaccharide (GAMP) to prepare conjugates of GAMP andrCRM₁₉₇. The conjugate was synthesized by carbodiime-mediated synthesis with adipicacid dihydrazide (ADH) as the linker. Hydrazide groups were introduced into GAMPby treatment with cyanogen bromide (CNBr) and ADH. The resultant adipic acidhydrazide derivative (GAMP-AH) was bound to rCRM₁₉₇ by treatment with EDAC.The conjugate was purified by Sepharose 4 Fast flow gel column and tested. The resultsshowed that the yield rate of the conjugate was 26.9ï¼…, and the ratio of thepolysaccharide/protein was 0.31. Double immunodiffusion demonstrated that thebinding was successful. Solutions of 2.5μg polysaccharide, alone or mixed withrCRM₁₉₇ or as a conjugate, were injected subcutaneous into Balb/c mice (18-20g), withthree doses, to evaluate immunogenicity of the conjugate. The immunoglobulin G (IgG)anti-GAMP was tested by the enzyme-linked immunosorbent assay (ELISA). IgGinduced by the conjugate (GAMP-rCRM₁₉₇) were significantly higher than the GAMPand GAMP+rCRM₁₉₇ after each dose. In the conjugate group, it demonstrated theconjugate primed immunologic memory and led to good response to booster dosessince the second dose is significantly higher than the first dose and the thirdsignificantly higher than the second. The rCRM₁₉₇ can be used as a carrier ptotein sinceit makes GAMP convert to a T cell dependent antigen.In summary, we obtained a recombinant E. coli strain that can yield recombinantCRM₁₉₇ by gene cloning and expression, successfully prepared a conjugate of GAMP and rCRM197, and evaluated the immunogenicity of the conjugate in mice. The resultsindicated that the conjugate had higher immunogenicity and behaved as a T celldependent antigen. Our study contributes to the preparation of other conjugates usingrCRM₁₉₇ as a carrier protein in the future.