Study On Triple Genetic Engineering Vaccine Of Salmonella Enterica Serovar Choleraesuis And Swine Streptococcus

Streptococcus suis type2 and Streptococcus equi subsp,zooepidemicus are important pathogens of swine,causing meningitis,septicemia,arthritis,endocarditis,pneumonia, and substantial economical losses in the swine industry worldwide.They are also zoonotic agents for humans in contact with diseased pigs or their products,causing life threatening diseases.Salmonella as the vector of vaccine has received wide attention in medical and veterinary medicine.The major advantage of Salmonella is that it can be administered by mucosal route(oral or intranasal),easily genetically manipulated and cause little stimuli to animals.Recent research shows that the Sao protein,0197 protein of streptococcus suis type2 and M like protein SzP of streptococcus equi subsp. zooepidemicus are vital important virulence factors and protective antigens,respectively. In this work,based on molecular cloning methods,we cloned and integrated the genes into non-resistance expressing plasmid,successfully constructed non-resistance recombinant Salmonella strains efficiently expressing the fragments integrated in.Further more,we evaluated the immuno-genicity and protective capacity of the recombinant Salmonella strains using a vaccination and challenge trial in mice.1.Construction and bio-characterics of the recombinant Salmonella enterica Serovar Choleresuis strains.Refer to the reported sao gene,0197 gene sequences of streptococcus suis type2 strain 05ZYH33 and szp gene sequence of streptococcus equi subsp.Zooepidemicus ATCC35246,we designed primers to amplify the fragment A of sao gene(saoA, 121bp-897 bp),0197 gene(121bp-1593bp) and the fragment A of szp gene(szpA, 238bp-789bp),then constructed non-resistance expressing plasmids pYA-saoA, pYA-0197and pYA-szpAsaoA.The plasmids were electrotransformated into C501 competent cells to construct the recombinant Salmonella strains C501(pYA-saoA),C501 (pYA-0197) and C501(pYA-szpAsaoA) respectively.At last,we idenficated bio-characterics of the strains.The results showed that we successfully constucted the recombinant Salmonella strains C501(pYA-saoA),C501(pYA-0197) and C501 (pYA-szpAsaoA) which could highly expressed the gene integrated in and got back the ability to grow in LB culture.Although highly expressing heterologous fragments,the recombinant Salmonella strains did not change their bio-characterics.The recombinant Salmonella strains have lower virulence than the parent strain C500. 2.The immunogenicity and protective capacity of the recombinant Salmonella strains.We prepared the recombinat stain C501(pYA-saoA) expressing rSao and the recombinat stain C501(pYA-szpAsaoA) expressing rSao and rSzP as new recombinat vaccines to evaluate the immunogenicity and protective capacity of the recombinant Salmonella strains using a vaccination and challenge trial in mice.The results showed us that the recombinant Salmonella strain C501(pYA-saoA) induced the imuunized mice a high titer of serum IgG and gut mucus secreted IgA against the antigens of Salmonella and rSao,confering protection against Streptococcus suis SC19 Infection;The recombinant Salmonella strain C501(pYA-szpAsaoA) induced the imuunized mice a high titer of serum IgG and gut mucus secreted IgA against the antigens of Salmonella, rSao and rSzP,confering protection against Streptococcus suis SC19 and Streptococcus equi subsp,zooepidemicus C55138 Infection.Furthermore,IgG subclasses were induced in mice immunized by C501(pYA-saoA) and C501(pYA-szpAsaoA) with the IgG2a titer being higher than IgG1,indicating that the recombinant Salmonella strains C501 (pYA-saoA) and C501(pYA-szpAsaoA) could induce cell mediated immunity response except for humoral immunity and mucosal immunity responses.