Construction Of Orally Administered Attenuated Recombinant Salmonella Gallinarum Vaccine Expressing Type Ⅰ Fimbriae Of Avian Pathogenic E.Coli And Related Immunology Study

Chicken colibacillosis is caused by a group of pathogens designated avian pathogenic Escherichia coli (APEC), and is one of the major endemic disease afflicting the poultry industry worldwide. Much more attentions have been attached to the biosafety problems of the avian colibacillosis due to the abuse of antibacterial drugs recently. Combination of application of antibiotics legitimately and preventive vaccine were recognized as the most useful measure in preventing avian colibacillosis. However, there is still no genetic engineering vaccine which has high immunity specificity and wild spectrum. In this study, the chromosome-plasmid lethal system was established which expresses antigenic type Ⅰ fimbriae gene of APEC in attenuated S. gallinarum SG9R Δasd strain, which lost the nutritious asd gene to investigate the protection opotential of this orally administered recombinant vaccine in preventing avian colibacillosis.1. Cloning, expression and bioactivity of type Ⅰ fimbrial operon gene from avian pathogenic Escherichia coliThe fim operon gene cluster encoding the type Ⅰ fimbriae was amplified by PCR using the genomic DNA template from APEC. The PCR products were cloned into the expression vector pBR322. The recombinant plasimd pBR322-fim containing whole fim gene clusters was confirmed by PCR and sequencing.The positive recombinant plasmid was electroporated into non-fimbriae E.coli SE5000 strains. Expression of type Ⅰ fimbriae was confirmed by mannose-sensitive agglutination and electromicroscope.6-week-old ICR mice was inolculated with the recombinant strain, subsequently, to get specific serum. The type Ⅰ pili single factor serum was obtained using cross absorption test. The recombinant E.coli expressing type Ⅰ fimbriae possesses better adherence lung adenocarcinoma cells in vitro than wild type E.coli CE2. The D-mannose can efficiently inhibit type Ⅰ fimbriae-mediated adherence upon lung adenocarcinoma cells in E.coli SE5000(pBR322-fim) and wild type. These results provided important experimental data for pilus vaccine development.2. Construction of chromosome-plasmid balance lethal system based on Salmonella Gallinarum 9R asd deletion mutantSalmonella Gallinarum 9R strain as an attenuated vaccine strain has been reported to successfully prevent fowl typhoid in poultry industry. In order to develop SG9R as an potential oral administrated vaccine vector carrying heterologous antigen gene with its original immunogenicity based on SG9R, in-frame gene asd deletion mutant SG9RΔasd had been constructed through Red system, which lost the capability of growth without DAP. The deletion mutant could recovered the capability of growth on LB plate when DAP component was complemented or plasmid expressing asd gene was transformed into the auxotrophic strain. Therefore a complemented plasmid pYA3342 expressing β-aspartic semialdehyde dehydrogenase was transformed into the auxotrophic strain to set up a chromosome-plasmid balanced lethal system. The GFP gene was cloned into prokaryotic expression plasmid pYA3342 and electroporated into asd SG9R. The plasmid stability of pYA3342-GFP was assessed in vitro by passage on LB medium under fluorescence microscopy observation and flow cytometry analysis. The growth stability and capability of the SG9R(pYA3342-GFP) to colonize the organs of chicken after oral administration were analyzed. The SG9R(pYA3342-GFP) could keep on expressing GFP in vitro for 40 generations, and could be detected in spleen of chicken 2 to 3 weeks post oral administration, which demonstrated it can persist in vivo for a long time and keep stimulating the host immune response. The recombinant attenuated S.Gallinarum expressing GFP is a good platform for further development of attenuated recombinant avirulent vaccines as well as for other potential applications.3. Construction of the attenuated Salmonella Gullinarum 9R strain expressing type Ⅰ fimbriae from APEC and related immunology studyThe fim operon gene was amplified by PCR, and then inserted into expressing vector pYA3342 which is containing asd gene. The recombinant plasmid was introduce into SG9RΔasd mutant and obtained recombinant attenuated S.gallinarum vector vaccine SG9R (pYA3342-fim). The expression of type I fimbriae was confirmed by mannose-sensitive agglutination.All chickens could survive for 3 weeks after orally inoculated with 1.75× 10 cfu of SG9R (pYA3342-fim) which confirmed the safety of the recombinant vaccine. Immune group were orally inoculated with recombinant vaccine strain SG9R (pYA3342-fim), empty vector control group were inoculated with recombinant strain SG9R (pYA3342) and control group were fed with PBS buffer. In contrast to control groups, the SG9R (pYA3342-fim) could induce specific antibody responses and the level of antibody was significantly increased(P<0.05) at the end of the two weeks after primary immune. All chickens were challenged with 8×107cfu of APEC challenge strain QD2 by the airsac routeon day 29 after primary immune. The mortality rate of chickens vaccinated with SG9R (pYA3342-fim), SG9R (pYA3342) and PBS were 40%,73.3% and 80%,respectively. To sum up, the recombinant vaccine strain can express type I fimbriae of APEC stably and is a promising candidate vaccine.