| Vaccine can be classified as traditional vaccine and new vaccine due to itsfunctional characteristics. Among these, polysaccharide conjugate vaccinebelongs to the new vaccines. Polysaccharide conjugate vaccine is made througha chemical process by bounding polysaccharide to the protein carrier, and isused to enhance the immunogenicity of the polysaccharide antigen of bacterialvaccine. The domestic production of conjugate vaccine is mainly using thetoxoid. Unfortunately, the use of toxoid can result in a systematic or localimmune response of the human body, like the mucosal adjuvant effect, and islimited to certain extent. Toxoid with Formalin or glutaraldehyde detoxificationwill change its protein structure, which may cause damages to the importantT-cell antigenic sites, and thus affect its immunogenicity. Moreover, thepresence of toxoid in the human body may affect the immune response toconjugate vaccine. Therefore, most of the present studies are focusing on theprotein carrier CRM197.CRM197(cross reacting material197) is a mutant of diphtheria toxin，whose52nd amino acids Glu mutates to Gly. This mutation leads to a change inthe activity of the diphtheria toxin’s enzyme, and thus can not produce toxiceffects on cells. But the antigenicity and immunogenicity are still the same withthe natural diphtheria toxin. Therefore, the present experiment is going to usethe genetic engineering techniques to get the induced expression of CRM197byusing the E. coli. Then followed by a study about the feasibility of therecombinant protein CRM197with Streptococcus pneumoniae capsularpolysaccharide after its purification. Lastly, a preliminary study is given to itsimmunogenicity.Firstly, codon optimization, secretion signal peptide enhancement, and genesynthetion are performed according to the information of gene sequences ofdiphtheria toxin given by GenBank (accession numbers: CQ967258). Then， primers will be designed based on CRM197gene without signal peptide whichwas amplified using PCR technology. After that, two sets of plasmidpBAD-DEST49/CRM197and*pBAD-DEST49/CRM197will be constructed. Then theyare transformed into E. coli to obtain induced expression after doubledigestion. The results show that secretion of type strains of recombinant proteinCRM197are found in the cell gap, some are in the form of inclusion bodies inthe cytoplasm.Secondly, to separate the expressed protein for purification. The proteinSecreted into the intercellular space, which has enhanced the permeability of thecell wall, has caused a release of the protein in the cell gap. For inclusion bodyform protein, the bacteria are been crushed and undertaken centrifugalprecipitation. After several steps of protein denaturation and refolding, thepassing of DEAE anion column is able to achieve recombinant protein CRM197purity at above95%. By immunoblotting to verify the activity of recombinantCRM197, it shows that the recombinant protein CRM197has good antigenicity.Finally, to use cyanogen brominated to activate Streptococcus pneumoniaecapsular polysaccharide, by using ADH as a coupling agent and recombinantCRM197as protein carrier, and prepare Streptococcus pneumoniae capsularpolysaccharide-CRM197conjugate under the action of the EDAC. Byconducting immune double-diffusion test to prove that the conjugates cangenerate precipitation line with both Streptococcus pneumoniae and diphtheriaantitoxin. This indicates the successful coupling and good antigenicity of theconjugates,In this study, genetic engineering techniques have been used to obtain theexpression of recombinant protein CRM917. The purified recombinant proteinCRM197is further utilized to couple with Streptococcus pneumoniae capsularpolysaccharide.And finally Streptococcus pneumoniae capsular polysaccharide-CRM197conjugate is constructed. It has provided a foundation for a further industrial production of combined vaccines which is based on the use recombinant proteinCRM197. |
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