| Salmonella is a common food-borne pathogen with a wide range of hosts,which has posed a serious threat to the health of people and animals and caused great economic losses around the world.The diseases caused by Salmonella infection are mainly divided into two categories:invasive typhoid Salmonella and non-typhoid Salmonella.Invasive Salmonella typhi was mainly caused by Salmonella Typhi（S.Typhi,Vi capsular polysaccharide）and Salmonella paratyphi（S.Paratyphi A,SA,O antigen main serotype O2）,presenting with intestinal fever,bacteremia and gastroenteritis;the main pathogen of non-typhoid Salmonellosis are Salmonella typhimurium（S.Typhimurium,ST,O antigen main serotype O4）and Salmonella enteritidis（S.Enteritidis,SE,O antigen main serotype O9 serotype）,which usually cause localized enterocolitis.In recent years,the drug resistance of Salmonella has become more and more serious,and multi-drug resistant strains have emerged,making vaccination the first choice for the prevention of Salmonella infection.However,there is currently no licensed vaccine for the prevention of Salmonella paratyphoid A infection.In addition,the effect of the non-typhoid Salmonella vaccine is limited to one group or one serotype,and it cannot or can only provide partial cross-protection against other types of non-typhoid Salmonella infection.Therefore,this study intends to construct new recombinant strains that can co-express the antigens of Salmonella paratyphi A O2,Salmonella typhimurium O4 and Salmonella enteritidis O9 O,and evaluate the immunogenicity of the attenuated vaccine strains through animal infection models and cross-immune protection effects,providing a theoretical and experimental basis for the development and application of vaccines.The research content of this paper is mainly divided into two parts.The research methods and results are as follows:1.Construction of novel recombinant attenuated strains co-expressing serotypes o4&O9O and O4&O9 O of Salmonella at genome levelIn this section,two recombinant strains ST-ddhc::prt＿A1（O4&O2）and SE-ddhc::abe＿B1（O9&O4）were successfully screened by gene recombination technique.The two recombinant strains can co-express the O antigen main serotypes O4 and O2 of ST and SA,and the O antigen main serotypes O9 and O4 of SE and ST respectively.The biological characteristics of the two recombinant strains showed that the co-expression of O antigen had no significant effect on the strain,then the new vaccine strains ST-ddhc::prt＿A1（O4&O2）-?cya-?crp and SE-ddhc::abe＿B1（O9&O4）-?cya-?crp were constructed by deletion of virulence genes cya and crp of the two recombinant strains respectively.In animal infection models:Both novel attenuated vaccine strains can induce specific Ig G antibodies against the corresponding homologous and heterologous O antigen structures in mice.ST-ddhc::prt＿A1（O4&O2）-?cya-?crp can effectively protect mice against wild virulent ST.Mice immunized with SE-ddhc::abe＿B1（O4&O2）-?cya-?crp could resist the invasion of homologous wild virulent strain SE and heterologous wild virulent strain ST.2.A novel recombinant attenuated strain co-expressing serotypes of Salmonella O2,O4 and O9 O antigens was constructed by using the double equilibrium lethal system and O antigen polysaccharide expression plasmidthe suicide plasmid homologous recombination and gene traceless deletion technology were firstly used to construct the double equilibrium lethal system of ST,namely BG mutant strain（S.Typhimurium?asd?dad B?alr?rec F?ca）,and then the overlapping PCR was used to construct respectively The polysaccharide plasmid p SC101-asd-O2 expressing the tyv-prt＿A1-wzx＿A1-wbav＿A1 gene in the SA O antigen gene cluster and the polysaccharide plasmid p15a-dad B-O9 expressing the tyv-prt＿D1-wzx＿D1-wbav＿D1 gene in the SE O antigen gene cluster.Then the plasmids p SC101-asd and p15a-dad B were electroporated into BG competent cells to obtain a recombinant strain BG-01（O4）.The plasmids p SC101-asd-O2 and p15a-dad B-O9 were electroporated into BG competent cells to obtain the recombinant strain BG-01（O2&O4&O9）,and also the p SC101-asd-O2,p15a-dad B plasmids and p SC101-asd,p15a-dad B-O9 plasmids were electroporated into BG competent cells to obtain recombinant strain BG-01（O2&O4）and BG-01（O4&O9）,in which BG-01（O4）,BG-01（O2&O4）and BG-01（O4&O9）were all used as the control group for BG-01（O2&O4&O9）.The results of silver staining and serum agglutination experiments both proved that BG-01（O2&O4&O9）strains can successfully co-express SA main serotype O2,ST main serotype O4 and SE main serotype O9 O antigen.Subsequently,the biological characteristics of the recombinant strain were evaluated and it was proved that the transfer of p SC101-asd and P15A-dad B plasmids would not affect the strain.In order to further explore the immune effect of attenuated recombinant strain BG-01（O2&O4&O9）,New attenuated vaccine strains S.T-AS-01（O2&O4）,S.T-AS-01（O2&O4）,S.T-AS-01（O4&O9）and S.T-AS-01（O2&O4&O9）were constructed by deletion of virulence genes cya and crp of BG-01（O2&O4&O9）,then the colonization ability of each attenuated strain in mice was detected by in vivo experiment,and also verified the safety of each attenuated strain.Finally,the immunogenicity and cross-immunoprotective effect of S.T-AS-01（O2&O4&O9）attenuated strain were evaluated in combination with animal infection model.The results of ELISA showed that S.T-AS-01（O2&O4&O9）attenuated strains could stimulate the production of anti-S.Paratyphi A LPS,anti-S.Typhimurium LPS,anti-S.Enteritidis LPS and anti-S.Typhimurium OMP in mice Ig G-specific antibodies also produced specific Ig G2a and Ig G1 antibodies against the corresponding O antigen structure,and the level of Ig G2a antibodies was generally higher than that of Ig G1antibodies.The results of immune protection experiment showed that the mice immunized with S.T-AS-01（O2&O4&O9）could resist the challenge of 100 times LD50 ST and SE virulent wild strains.In addition,mice immunized with S.T-AS-01（O2&O4&O9）survived the longest when subjected to abdominal attack by a virulent wild SA strain compared to all other groups.The results showed that the modified ST attenuated strain S.T-AS-01（O2&O4&O9）could well establish specific immune responses to heterologous and homologous O antigens.This study explored the feasibility of co-expression of O2,O4 and O9 O antigens and their potential impact on the bacteria itself,and proved that the new recombinant attenuated strains co-expressing O2,O4 and O9 O antigens can produce cross-protective reactions to prevent The mixed infection of ST and SE double Salmonella provides experimental and theoretical basis for the development of Salmonella multivalent vaccine. |
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