Construction And Evaluation Of Attenuated Salmonella Choleraesuis Presenting Streptococcus Suis Protein Antigen

Salmonella enterica serovar Choleraesuis(S.Choleraesuis)is a non-typhoid Salmonella,a major pathogen that induces paratyphoid in piglets,and has brought huge economic losses to the Chinese pig industry.The most effective vaccine against S.Choleraesuis in China is C500.C500 is a strain with good immunity and genetic stability obtained by chemical mutagenesis.Although the piglet paratyphoid fever in China has been effectively controlled since the use of the live C500 attenuated vaccine,the attenuated strain still has high residual virulence and its genetic background is unclear.Therefore,C500 needs to be optimized to obtain more safe live attenuated vaccine.By sequencing the C500 strain,it was found that the multiple genes were deleted or inactivated in C500,including asr,ydgF,ydgD,ydgE,rpoS,and ptsG;analysis of these genes and animal experiments revealed that the inactivation of the rpoS gene was the major reason to reduce its virulence in C500.This method provides a research basis for the development of a safer S.Choleraesuis vaccine.In this study,the C500 vaccine strain was used as the background strain,and its virulent rpoS gene function was restored to make it capable of invading cells.Then,attenuated strains that had restored rpoS gene activity were used as background strains,which lacked crp,fur,phop and aroA genes,then from these four recombinant attenuated strains to screen an optimal vaccine strain with good immunogenicity and small side effects.The optimal vaccine strain was used as a carrier for presenting the protein antigen of Streptococcus suis(SS)to achieve the purpose of preventing both S.Choleraesuis and Streptococcus suis.1.Construction of recombinant attenuated strains of Salmonella Choleraesuis Objective: To construct a recombinant Salmonella Choleraesuis vaccine strain with good immune effect and small side effects.Methods: Firstly,the Salmonella choleraesuis strain C500 was used as the parent strain,and the rpoS gene was restored by using the suicide plasmid pRE112-mediated homologous recombination technology to make it capable of invading the body and colonizing the body.By analyzing the whole genome sequence of C500,we found that the rpoS gene was not completely mutated,but retained the original 103 bp of the gene.Therefore,when constructing a suicide plasmid that restores the rpoS gene,we first amplified the complete 993 bp rpoS gene using wild-type Salmonella Choleraesuis C3545 as a template;then we used the C500 gene as a template to amplify the 293 bp homologous arm gene upstream of the gene.The two fragments were fused as upstream homology arms to restore the gene.For the downstream homology arm,a 350 bp sequence downstream of the rpoS gene was selected,and finally the upstream and downstream homology arms were fused to construct a suicide plasmid.On the basis of reverting the rpoS gene mutant strain,the suicide plasmid-mediated homologous recombination technology was also used to introduce crp,fur,phop,and aroA mutations.Conclusion: The suicide plasmid-mediated homologous recombination method was used to successfully restore the rpoS gene,and four recombinant attenuated strains C5001,C5002,C5003,and C5004 were constructed.2.Study on the biological characteristics of mutants Objective: To compare the recombinant attenuated Salmonella choleraesuis strains C5001,C5002,C5003,and C5004 constructed in this study with vaccine strain C500 in order to screen out a strain that can colonize host lymphoid cells like wild-type strains when invading the organism The ability of tissues to maintain a high degree of immunogenicity while at the same time having fewer side effects of vaccine strains.Methods: First,the growth curve of each attenuated strain in LB liquid medium was detected,then the mobility of each attenuated strain in semi-solid medium was detected,and the colonization of the attenuated strain in mice and the immunity to mice were detected.Protection effect.Conclusion: By comparing the attenuated strains,a better vaccine strain C500 was finally selected.3.Construction of the attenuated Salmonella choleraesuis presenting Streptococcus suis STK,GDH,ccpA protein and its immunogenic Objective: To construct a C5001 vector,and then present STK,GDH,ccpA protein antigens of Streptococcus suis,and evaluate the immunogenicity of vaccines.Methods: Using suicide plasmid-mediated homologous recombination technology,the asd gene of C5001 was deleted to construct a balanced lethal system for presenting protein antigens such as STK,GDH,ccpA.The stk,gdh,and ccpA genes were ligated to the expression plasmid pYA3493 by enzymatic digestion,respectively,to construct pYA3493-stk,pYA3493-gdh,and pYA3493-ccpA expression plasmids.Each expression plasmid was transferred into vector C5001,and then the protection rate of the recombinant strain on mice was examined.Conclusion: A recombinant vaccine strain was successfully constructed.The mice were immunized with the vaccine strain and then challenged with the Streptococcus suis strain SC19.All the mice died within 24 hours.