| 1. Background Helicobacter pylori (H. pylori) is a major cause of chronic superficial gastritis, chronic active gastritis and peptic ulcer disease, and has a close relation with gastric mucosa associated lymphoid tissue lymphoma and gastric cancer. Although significant progresses have been made in treating H. pylori infection with current triple or quadruple therapy based on antibiotics and proton pump inhibitor, the limitations of pharmacological therapy such as side effects, poor compliance, high cost, and most importantly, rapid emergence of antibiotic resistance have set the stage for the development of less costly and more efficient means to prevent and control H. pylori infection. Ample precedence from previous experiences suggests that vaccination may be an alternative. DNA vaccine has shown large potential in protecting and treating many diseases since it was born. It can induce complete immune responses, provide heterologous cross protection, and can be easily prepared as multivalency vaccine. On the other hand, compared with traditional vaccines, live attenuated Salmonella typhimurium (S. typhimurium) used as neotype of vector releasing system for heterologous antigens does not require antigen purification, and it can not only protect antigen from degradation and denaturation in stomach but also express adjuvant activity preventing induction of oral tolerance. Massive trials have proven that attenuated S. typhimurium is safe to immunize human as vaccine bearer. More recently, a new major virulence factor of H. pylori was identified and termed neutrophil activating protein (HP-NAP) for its ability of inducing adhesion of neutrophils to gastric endothelial cells and production of reactive oxygen radicals. Little is known about its immunogenic and immunoprotection, and no study on HP-NAP was reported domestically. The purpose of the present study was to establish a recombinant live attenuated S. typhimurium strain harbouring HP-NAP encoding gene, napA, as an oral DNA vaccine. 2. Objectives To construct a live attenuated S. typhimurium strain carrying HP-NAP gene as an oral recombinant DNA vaccine. And research its immunotherapy against H. pylori infection. 3. Materials and methods Part I The genomic DNA of H. pylori was extracted as template by standard procedures. The complete napA gene of H. pylori was obtained by PCR amplification using the primers NPF (5?-GTC CTC GAG ATG AAA ACA TTT GAA ATT TTA AAA CAT TTG CAA GCG-3?, containing Xho I site) as upstream primer, NPR (5?-GTC ACG CGT TTA AGC CAA ATG GGC TTG CAA CAT CC-3?, containing Mlu I site) as downstream primer. PCR product was T-A cloned into pBT vector giving rise to pBT-napA. The recombinant plasmid pBT-napA was digested by Xho I and Mlu I and inserted in the corresponding sites of eukaryotic expression vector pIRES giving birth to pIRES-napA. The bionomics of cloned gene napA translated protein was predicted by software ANTHEPROT Ver. 5.0 and DNAStar-Protean? Ver. 5.0. The recombinant plasmid pIRES-napA was transformed into live attenuated S. typhimurium strain LB5000 for methylation modification. Then extracted and electroporated into SL7207. Identification by PCR and endonuclease digestion followed. Part II Long-term infection BALB/c mice model of H. pylori strain SS1 was built as reference described. Fifteen of them were randomly divided equally into 3 groups 30 weeks later. The treatment group was lavaged with 109 cfu/0.4 ml of oral DNA vaccine, once a week for 3 times. While the two control groups were lavaged with identical volume of normal saline or vacant pIRES, respectively. Mice were sacrificed 4 weeks after last immunization, rapid urease test of gastric antrum mucosa followed. Serum antibody titer was measured by ELISA immunoassay. 4. Results Part I A 435 bp product was amplified by PCR, which was consistent with the complete sequence of H. pylori-napA gene confirmed by sequencing analysis. BLAST analysis showed that the nucleotide homology between the cloned napA gene and that of SS1 strain in Genban... |
PDF Full Text Request