| Objective:Infection ratio of Helicobacter pylori which severly endanger human being is very high. Around the world reports about gene vaccine against H.pylori are seldom reported and most resoults are not satisfied. In this study, mucosal H.pylori gene vaccines enclosed with chitosan were constructed while using cytokine co-expression with antigen to enhance and regulate immune response. Relationship between immune response and anti-H.pylori reaction caused by vaccine was observed for concealling probable protection mechanism.Methods:1. hpaA and ureB gene were amplifed from genome of H.pylori standard stain NCTC11637 by PCR, then were subcloned into vector pMD18-T. After identified by digestion with restriction enzyme and sequencing, both antigen genes were separately inserted into eucaryotic expresstion vector pTCAE and named pT-H and pT-U. Plasmids were tranfected into CHO cells, and expressing protein was detected by indirect immunoinfluroscense and Western blot. Mice were intramusclly vaccined with pT-H or pT-U at quadriceps femoris. Specific IgG level in serum were assayed.2. Gene GM-CSF and IL4 were amplifed by RT-PCR from spleen cells,then subcloned into vector pMD18-T. After identified by digestion with restriction enzyme and sequencing, both were separately cloned into expression vector pT-U and named pT-GU and pT-IU. Plasmids were tranfected into CHO cells, and expressing protein was detected by Western blot and cytokine relied cells.3. DH5α(pT-U) were fermented.Plasmid pT-U was purifed with the progress: bacteria lysis—capatured on Q Sepharose XL—polished on Source 30 Q.Concentration for gene vaccine nanoparticles was determined according to shape,size and stability.4. Nanoparticles were made with chitosan and plasid pT,pT-GU,pT-IU which were purified from fermented bacteria. BALB/c mice were intranasally or orally vaccined with naked plasmid pT-U, chitosan enclosing pT,pT-U,pT-GU, pT-IU. IgG,IgG1,IgG2a,IgA in...
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