Cloning, Prokaryotic Expression And Purification Of Haemophlilus Influenza Protein D Gene And Preliminary Application In The Glycoprotein Conjugate Vaccines As A Carrier Protein

Neisseria meningitis is one of the most common bacterial that can cause bacteria meningitis in the world. And it is the only one that can induce large-scale epidemic of meningitis. Vaccination is the effective measure to prevent the infection, but polysaccharide vaccine are not able to produce a protective effect under two years of age.Experiments show that conjugate vaccine which combines with bacterial polysaccharide and protein by chemical ways can overcome the shortcomings by polysaccharide immunization, and it can induce cellular immunity and strengthen the immune response, it is also protect people from all age groups. In china, the conjugated vaccines usually use tetanus toxoid as a carrier, which is derived from tetanus toxin with formal dehyde treatment. But it has some limitations, such as recovery, or produce toxic effects like carrier suppression problems. So it is one of the topics areas to look for the safe and effective carrier protein to conjugate vaccine. Haemophilus influenzae protein D has good immunogenicity and safety, and as a carrier has been applied to pneumococcal conjugate vaccines in the foreign market. Currently, there is no glycoconjugates using protein D as carrier protein in our country.Recombinant plasmids pET30a/hpd were constructed via amplifying the gens of protein D from Haemophilus influenzae. Then screning and save clones bacteria after the plasmid was being transformed into E.coli DH5a. The plasmid pET30a/hpd was transformed into E.coli BL21 (DE3), and the protein D was induced expressing by isopropyl-β-D-thiogalactoside (IPTG) as insoluble inclusion body in cytoplasm and counted for about 40%,. Purify the interesting protein and obtain the recombinant protein which's purity was about 95%, and identify it.The protein D was then applied for covalent binding with Neisseria meningitides group A and group C capsular polysaecharide (GAMP and GCMP) to prepare conjugates of GAMP-D and GCMP-D. The groups were introduced into GAMP and GCMP by treatment with CDAP. The derivative was bound to protein D by treatment with TEA. The conjugate was purified by Sepharose 4 fast flow gel column and tested.Components of polysaccharide alone or mixed with protein D or as a conjugate, were injected Balb/c mice subcutaneously with three doses, which is objective to evaluate the immunogenicity of the conjugate preliminaryly. The IgG, IgG1 and IgG2a in the mice was tested by the enayme-linked immunosorbent assay (ELISA). IgG, IgG1 and IgG2a induced by the conjugate were significantly higher than the GAMP, GCMP, GAMP+D and GCMP +D after three doses. It is an initial proof that the protein D can be used as a carrier protein in the conjugated vaccines.In summary, this study obtained a recombinant strain that can yield recombinant protein D by molecular cloning techniques, prepared a conjugate of GAMP and GCMP with protein D successfully, and evaluated the immunogenicity of the conjugate in mice. The results indicated that the conjugate have good immunogenicity and protein D can be used as a carrier protein. The research contributes to the preparations of other conjugated vaccines using protein D as a carrier protein in the future.