Haemophilus Influenzae Type B Polysaccharide Preparation Process Optimization And Combined With D Protein Research

Haemophilus influenzae type b (Hib) conjugate vaccines can protect children from Hib, which is the leading cause of serious invasive infections in children younger than5years old. Today, tetanus toxoid is used as the carrier of the conjugate vaccine in domestic market. However it has limitations such as the phenomenon of immunosuppression and toxicity response. Therefore, finding safe and effective new carrier becames the new orientation of vaccine research. H.influenzae D protein as vaccine carrier has been applied in10valence of conjugate pneumonia vaccine in abroad and has safety and immunity. At present, there is no D protein as the carrier of Hib conjugate vaccine in the domestic.The first part:technology research about the polysaccharide of haemophilus influenzae type b and D protein carrier preparation.The pH is very important to the yield of capsular polysaccharide (CP) obtained by precipitation with the cationic surfactant cetyltrimethylammonium bromide (CTAB).Twelve samples obtained from three batches of culture supernatant were adjusted to different pH values (5.25-8.0). The purification of CP was performed following the Gotschlich procedure. The1H NMR analyses of CP under pH values5.25,7.0and8.0were carried out. The P-values of all threeparallel experiments were<0.01(for crude:0.001,0.001and0.001, respectively; for refined:0.002,0.01and0.001, respectively), indicating that pH values had significant influence onthe precipitation harvests. Polysaccharide recovery was highest and its structure was constant when the pH of the supernatant was6.5. The1H NMR spectra showed that the structure of CP changed at pH8.0. The yield and quality of CP were affected by the pH of the supernatant.This article amplify Hib D protein gene by PCR, remove the signal peptide HPD gene and insert pET43.1a into expression plasmid.Transformed E. coli BL21(DE3) can express D protein by IPTG. The protein is precipitated by ammonium sulfate and purificated by DEAE sepharose Fast Flow anion exchange chromatography. The purified protein has good immunogenicity, which has been proved by Western blot method.The second part:Hib polysaccharide and protein conjugate vaccine preparationIn this paper, CDAP was used to activate the purified Hib capsular polysaccharide antigens under different level of pH. Though Oxalyl hydrazine (ADH), Hib capsular polysaccharide binds covalently to D protein. Hib-D conjugation mix solution is purified by Sepharose4FF gel chromatography column. And then glycoprotein conjugation is harvested. The BALB/c mice were immunized with these conjugations. the conjugation of three runs are tested successively on different indexes (polysaccharide, protein, and polymer binders, molecular weight, EDAC residual content, immunogenicity, asepsis check and endotoxin, etc.The level of IgG in serum was analyzed by ELISA. Results show that the above indexes of three glycoprotein conjugations conform to the requirements of the pharmacopoeia. ADH content in activated polysaccharide has important influence on immunogenicity of glycoprotein conjugations. Hib capsular polysaccharide was activated, and was bonded with carrier D protein. The polysaccharide derivatives and combination concentrate are measured and their quality according with the requirement of Chinese Pharmacopoeia2010edition. D protein works as the carrier protein of the conjugate vaccine preparation,and make a good basis for five league vaccine in the future.