Construction Of A Bivalent Candidate Vaccine Strain Expressing Virulence Factor Genes Ef And Sly From Streptococcus Suis Type2

Streptococcus suis serotype2(Streptocuccus suis2, SS2) distributes widely in nature, it belongs to Streptococcus which was reported that could cause a extensive range of diseases during pigs, and those holding high infectious could even infect humans. According to recently studies, the virulence factors of Streptococcus suis includ hemolysin (Streptolysin, SLY), extracellular protein factor (extracellular proteins factor, EF), muramidase released protein (Muramidase released protein, MRP), capsular polysaccharide (Capsular polysaccharide, CPS) and so on. The synthesis of these types of virulence factors were controlled by sly, ef, mrp gene, in order to study the relationship between these types of virulence factors and pathogenicity of Streptococcus suis, and to prepare recombinant bacterias which could synthesize these types of virulence factors, and further to compose a vaccine against Streptococcus suis strains, we conducted this experiment.By means of molecular cloning, one of SS2's virulence factor ef gene was ligased into project plasmid pGEX-6P-1to construct recombinant plasmid pZH01containing the ef gene fragment, then pZH01was transferred into E. coli BL21(DE3). After that asd gene was ligased into pZH01to form recombinant plasmid pZH02, and pZH02was transferred into E. coli X6097. Asd gene controls the synthesis of DAP. DAP is an important component of cell wall, and the X6097is an auxotrophic bacteria which could not synthesize DAP, recombinant plasmid pZH02containing asd gene and the host strain X6097constitute the host balanced lethal system, which can effectively carry out the reorganization of colony screening. Sly gene further was connected to pZH01to construct recombinant plasmid pZH03, and pZH03was following transferred into E. coli DH5a. In addition, sly gene was individual connected to the project plasmid pGEX-6P-1transformated from plasmid pGEX-6P-lA to construct recombinant plasmids pGEXSLY containing only sly gene, pGEXSLY was transferred to E. coli JM109. Apply the recombinant bacteria in blood agar hemolysis test to the determine its hemolytic activity. Through those steps, respectively, the recombinant plasmids containing ef, asd or ef, sly, or separately sly gene were constructed. By Amp antibiotics and host balanced lethal systems approach, the recombinant colonies were screened, further identify of the recombinant plasmid was amplified. Finally, the rabbit immune tests were pocessed, accomponied with determination of inactivated Streptococcus suis antisera and titer of the antiserum.Trials ultimately construct the recombinant plasmid containing the virulence factors of both ef and sly gene, as well as to provide effective technical support for following ef, sly protein expression, identification and preparation of the vaccine strains of Streptococcus suis virulence factors.