Immunogenicity Of Recombinant Adenoviral Vector Expressing STa-K99Fusion Protein Of Enterotoxigenic Escherichia Coli In Mouse

Background:Enterotoxigenic Escherichia coli. is a global major pathogen causing severe watery diarrhea in young animals, causing huge losses to the livestock industry. ETEC is also an serious pathogen in children and travelers’ diarrhea in developing countries, and become an important public health threat to human safety. Adhesion and enterotoxins as the two main types of ETEC virulence factors often as the advantages of enterotoxigenic Escherichia coli vaccine antigens. Adenovirus has many advantages, has become the ideal vehicle for new vaccines. Currently the use of adenoviral vector vaccine immune effects on intestinal infectious disease research has not been reported in detail, especially in the study of the prevention of immunological characteristics caused by the ETEC diarrhea in newborn animals adenovirus vector vaccine has not been reported.Objectives:As for to study its immunological characteristics of recombinant adenoviral vectors of Ad.STa-K99Which were amplified and purified.Methods:The infection adenovirus Ad.STa-K99of293cells for amplification, followed by CsCl density gradient centrifugation and purified, by intramuscular injection, intraperitoneal injection, and intragastrically immunized mice respectively. Meanwhile, cloning ETEC enterotoxin Sta and adhesin K99fusion gene STa-K99, prokaryotic expression vector expressing the antigen fusion protein STa-K99. Then,detected by ELISA in serum specific IgG, intestinal lavage fluid sIgA antibodies, spleen lymphocyte proliferation activity levels and CD4+, CD8+T lymphocyte typing ratio; and evaluated by animal challenge protection test their immunity. Lastly,To observed poisoned mice intestine tissue pathology slice.Results:(1) Successfully amplified, purified recombinant adenovirus Ad.STa-K99, results showed a higher viral titer adenovirus infection efficiency and good biological activity.(2) By prokaryotic expression vector antigen fusion protein was successfully expressed STa-K99. SDS-PAGE, Western Blot and ELISA identification results in line with expectations(3) The Animal immunization experiments results showed that the specific IgG, mucosal sIgA concentration, spleen lymphocyte proliferation activity levels and CD4+/CD8+ratio in immunized mice increased significantly.(4) Animal poison efficacy protective results show that by intraperitoneal injection, intramuscular injection and orally immunized mice,and the animal immune protective efficacy rate reached to67%,70%and80%respectively.(5) Pathological slice observation results show that the Ad.STa-K99immune mice intestine tissue and mucosa were in good condition, microvilli form completely, whereas the control mice intestinal mucosa damage seriously.Conclusion:(1) Ad.STa-K99by intramuscular injection, intraperitoneal injection and orally immunized mice by stimulating the body to produce higher levels of humoral immunity, cellular immunity and intestinal local immune response in mice, effectively protects mice against virulent ETEC strains and its security is good.(2) In three kinds of immune pathways, intragastric immunization is best, intramuscular is better and intraperitoneal injection of the general effect.