Construction Of Streptococcus Suis Serotype2Fbps Gene Deletion Mutant And The Study On Its Biological Traits

Streptococcus suis is an important pathogen of swine, and33serotypes have been identified based on their capsular antigen, of which the Streptococcus suis serotype2is the most important and frequently isolated from the clinical samples. SS2can cause a lot of serious diseases, such as meningitis, septicemia, arthritis, endocarditis, pnuemonia and even acute death in swine. SS2can also infect humans who were associated with pigs or swine industry directly; the serious cases would have STSS which leads to multiple organ failure or even death. It is an important zoonosis pathogen and is paid worldwide attention. Recent years several outbreaks of SS2have seriously harmed the swine industry and human health, then it was paid great attention by researchers of China or other countries.The adhesion of SS2to the host maybe a key step in the pathopoiesis processes, so many works about adhesins have been carried out. But the adhesion of SS2to the host cells is not a simple way, many different components of SS2or the host cells were involved in it, so to find out which one play the major role in this process is usefully and necessarily to furter explain how SS2do harm to the hosts.In the SS2genome there is a gene fbps encoding a surface membrane protein FBPS, which has the Fn binding sites, those who were homologus with it in other streptococcus played a major role in the process of infection so FBPS was assumed to be an important involving protein in the way of the adhesion of SS2to host.Under this background, we used SS2high virulent strain SC19as the parental strain, take fbps as the study object, construct a fbps deletion mutant, and study its biological traits and virulence changes compare to the wild type strain. More detailed imformation about our research are listed below:1. construction of temperature-sensitive recombination suicide plasmidUse SS2high virulent strain SC19isolated from Sichuan province as the parental strain, PCR amplifies the780bp upstream sequnces and810bp downstream sequences. Then use fusion PCR to amplify the whole upstream and downstream fragment of fbps and link to the pMDx-18T vector to construct clone vector pMDx-18T-f. Use restriction enzeymes EcoRI and BamHI to digest both pMDx-18T-f and pSET4s then link them to construct the suicide plasmid pSET4s-fbps.2. construction of the SS2fbps gene deletion mutant The suicide recombinat plasmid pSET4s-fbps was transformed by electroporation into the SS2wildtype strain SC19. Through a double seletion of temperature and antibiotics, select the fbps gene deletion mutant. Then use PCR to futher verify.3. research of the SS2fbps gene deletion mutant’s biological traits Study the genetic stability, in vitro growth and proliferation situation of the SS2fbps deletion mutant. The results showed that afer cultured more10generation the mutant was still stable. The growth curves found that the fbps deletion don’t effect the growth of SS2in vivo.4. the study of the effects of how fbps works when SS2adhesion, invation and do cytotoxicity to host cellsUse Hep-2cells to be the cell model, to compare the ability of adhesion and invasion of SC19and SS2fbps deletion mutant, evaluate the LDH concentration to compare the two strains’ability of cytotoxicity. The results showed tha the ability of SS2fbps deletion mutant adhesion to the Hep-2cells is apparently descent about78%, and the ability of invasion also decreased obviously about23%, test the cytotoxicity of mutant to cells compared with the wildtype SC19strain showed it decreased a lot about32%, too.5. study the impact of deletion of fbps on the pathopoiesisUse wildtype SC19and fbps deletion mutant intraperitoneal inject KM mice as animal models with the dosage of10cfu/ml,108cfu/ml and109cfu/ml, after injection observe the clinical symptoms each12h. The results showed that the death rate was much lower in the mutant injection group to the wildtype injection group. Dissect mice at different to screen the bacteria showed deletion of fbps leads to the decline of field planting. Histopathology observation showed that SS2fbps deletion mutant and wildtype SC19have the similar pathopoiesis areas, but the former has much lighter pathological change levels. The virulence of mutant is decreased.