Preparation And Immunogenicity Of Subunit Vaccine And C3d Molecular Adjuvant DNA Vaccine Of Salmonella

The Salmonellae comprise over 2300 serovars, many of which are known as the intracellular pathogens of mammals, birds, and reptiles. Salmonella continues to be a major cause of food poisoning throughout the world. The oral toute through the consumption of contaminated food or drink is the usual cause of Salmonella infection. Thus, through the rearch the immunology function of invH of salmonella, it is important that development new vaccian against Salmonellae. This study clone the invasion H of type III secretion system of Salmonella by molecular biology techniques, then express the protein of invH by the prokaryotic expression, and construction a DNA vaccine encoding fusion protein of murine complement C3d and invH gene of salmonella. This study explored the immune function of invH by cell transfection and animal experiments, and achieved the desired results. This study was divided into three parts:Part one: Sequence analysis and Detection of invH gene from separate sources of Salmonella spp.The method of DNA sequence analysis was used to find the difference in DNA sequence of invH of separate sources of salmonella spp., and to develop a molecular method for the detection of salmonella spp., and to analysis sequence and antigenicity of autoantigen invH. A pair of oligonucleotide primer was synthesized according to the invH gene nucleotide sequence of salmonella spp.. And the invH gene was amplified from 9 strains of salmonella and other 4 strains of bacterium by PCR. And then the amplified invH gene was also cloned and sequence analyzed. Based on bioinformatic techniuque, the antigenicity of T lymphcytes in invH protein was screened. 519 bp specific bands was amplified from 9 strains of salmonella bacaterium by PCR, and no specific product was amplified from other 4 strains of bacterium. The homology among three strains of S.Typhinurium was 99.8% verified by nucleotide sequence analysis. And homology among the three strains of S.Typhinurium and Salmonella pollorum and Salmonella Enteritidis was 98.6%. And the homology of the three S.Typhisuis and three chicken-derived Salmonella was low, and the two derivations of Salmonella were not in the same phylogenetic tree. There were five higest repeated T cell epitope on invH protein. The method based on PCR for detecting salmonella spp. shows high specificity and sensitivity. There are genus-species differences on invH between separate sources of salmonella. The amino acids sequence of invH protein determines its may have immunogenicity and it have the molecular basis that stimulate the immune responses which is mediated by T lymphocytes.Part two: Expression and Immunogenicity Study of invH Gene of S.TyphinuriumThe objective of the present study is to obtain subunit vaccine of invH gene of S.Typhinurium to further reveal its immunogenicity function. Gene of invasionH (invH) from S.Typhinurium DT104 amplified by polymerase chain reaction (PCR) was cloned into PGEX-4T-3 getting the recombinant PGEX/invH, and the invH was coexpressed with GST. The bioactivity of antisera was detected by adherence inhibition assay. Western-blotting analysis indicated that the fusion protein was 44KD in molecular weight and had immunological activity. We immunized mice with there different doses of fusion protein invH, all groups of mice were challenged with 100 times LD50 amount of S.Typhinurium DT104 (4.2×107cfu),the survive rate of subunit vaccine groups more than 60%(p<0.01),and raised levels of mice serum IL-4 and IFN-γ(p<0.05). The antiserum of expressed protein can inhibit adherence activity of S.Typhinurium DT104 to cell surface by adherence inhibition assay. These results proved that invH has certain immunogenicity and can offer partial protection against high dose challenge. The study provides a theoretical base for the prevention of S.Typhinurium.Part three: Construction and immunogenicity of a DNA vaccine encoding fusion protein of murine complement C3d and invH gene of salmonellaObtain a safe and effective DNA vaccine against salmonella and the enhancing effect of molecular adjuvant C3d-p28 was proven, in order to explore a new way to enhance the immune effect of DNA vaccine. The gene fragment (p28) coding for the molecular adjuvants complement protein, C3d (mC3d) from BALB/c mouse was cloned and the gene of invasionH (invH) from S.Typhinurium DT104 amplified by polymerase chain reaction (PCR). One vaccine construct units was engineered to contain six copies of mC3d-p28 coding gene linked to the invH gene (pcDNA3.1-invH-P28.6) and another vaccine expressing invH alone (pcDNA3.1-invH) was constructed. In additions, indirect immuno-fluorescence proof could be found within the cells after Mark145 cells tranfected by recombinant plasmids. Furthermore, vaccine trials and challenge protection experiments were performed on BALB/c mouse.50 mice were randomly divided into five groups: control group (I), inactivated vaccine group (II), pcDNA3.1 group (III), pcDNA3.1-invH group (IV), pcDNA3.1-invH-P28.6 group (V). All mice were challenged with 100LD50 of S.Typhinurium DT104 in 7th week and invH-specific ELISA antibody, IFN-γlevel and IL-4 level in the sera were detected. The bioactivity of antisera was detected by adherence inhibition assay. It was showed that pcDNA3.1-invH and pcDNA3.1-invH-P28.6 were successfully constructed and express in Mark-145 cell. Serological analysis showed that the result of Group (V) was significantly higher than Group (IV) (P<0.05).The results of Group (V) was higher than Group (I) and Group (III). The survive rate of Group (V) is 60%. The antiserum of expressed protein can inhibit adherence activity of S.Typhinurium DT104 to cell surface by adherence inhibition assay. These results proved that invH has certain immunogenicity and can offer partial protection against high dose challenge. The complement C3d–p28 can significantly enhance the nucleic acid vaccine immune effect as molecular adjuvant.