| The research of safe and effective protein carrier is very important.First,it can work out the problem of carrier diversification;Second,TT as the protein carrier has so many abuse,in need of exploiting new-style protein carrier;Mucosal delivery vaccine is the new trend and direction.Considering these,this paper analyse the feasibility of rCTB as the protein carrier candidate.Moreover,we primarily discussed the immune effects of TT as the protein carrier in mucosal delivery vaccine.Based on the gene sequence of CTB in genebank,we cutoff the signal peptide of CTB,which is consists of 21AA.We optimize the CTB gene based on the E.coli prefer code,and constructed the cloning vector and expressing vector.At the same time we optimized the condition of expressing protein.Latter we confirmed the form of expressing protein is inclusion body.We purified the inclusion body by Ion exchange chromatography and gel filtration chromatography.By dialyzing and renaturing of the protein,we get the renatured pentramer protein.We wipe out the monomer from the pentramer.Then we examine the character of renatured protein by GM1-ELISA.With the chemicial methods(ADH),we coupled the renatured protein to GASP.Via the route of intranasal and subcutaneous,we immunized the Balb/c.Moreover,we intranased the mice with GAMP-TT vaccine that TT as protein carrier,then examined the level of IgG in serum and IgA in intestine mucous membrane.The results of the experiment indicats the conjugates of polysaccride -potein with the rCTB protein carrier can induce high level of GAMP specific IgG antibody in serum and specific IgA antibody in intestine mucous membrane.This results indicate that rCTB and TT both are protein carrier for polysaccride - potein vaccine in mucosal delivery systerm.If we optimized the coupling efficiency of polysaccride and protein,rCTB maybe superior to TT as a protein carrier in mucosal deliverying.We also primarily researched the mucosal delivery of the chitosan.The results of the experiment is the chitosan boost up the immunity of polysaccride -potein conjugate in the mucosal delivery systerm.It stimulates the body to produce the higher level of polysaccride specific IgG antibody in serum and stronger mucosal immunity response.It primarily shows that chitosan can effectively deliver the antigen to the mucosal systerm.It as a novel mucosal carrier can also promote the local mucosal immune response.Mucosal immunity of the polysaccride - potein conjugate vaccine with rCTB as a potein carrier indicats the carrier protein can induce mucos- al immune response via intranasal route.The conclusion is rCTB as a protein carrier can stimulate anti-CTB antibody in serum and intestine mucous membrane via mucosal immune.This results provide thought use for reference in the exploiting multi-vaccine. |
