The Study On Virulence Factors Of Streptococcus Suis Type 2 Using The Method Of Insertional Inactivation

Streptococcus suis is a gram-positive,facultatively anaerobic cocci that have beenimplicated as the cause of a wide range of clinical disease syndromes in swine andother domestic animals,and also can infect human beings.In 1968,there is the firstreport about human being infecting SS in Denmark.So far,there are a lot of countriesreporting this desease,such as China,American and Canada.SS has been paid closeattention to because it harms to human body’s health and has resulted in economicloss.Among the 35 known serotypes,SS2 is the most prevalent serotype,and itspathogenicity is the most doughtiest with the hallmarks of meningitis,endocarditis,arthritis and septicaemia,etc.Moreover,SS2 has been recognized as an etiologicalagent for toxic shock syndrome(TSS)in the desease exploded in China.The pathogenicity of SS2 is concerned with its virulence factors,and somevirulence factors has been known,such as capsular polysaccharide,muramidase-released protein,extracellular protein factor,suilysin,adhesion,etc.However,the pathogenic mechanism has not been known,and the virulence factorsare very complex.After SS2 was broken out in our country in 2005,the SS genomes(98HAH12,05ZYH33)were sequenced by the researchers of Beijing Genomics Institute.And apathogenicity island(PAIs),89kb,was detected when the genomes of 98HAH12,05ZYH33,05HAS68 and P1/7 were compared,and it has been named 89K.This PAIonly exists in the Chinese popular strains,and maybe is the reason that the SS2 causesTSS and has powerful virulence.Therefore,we chose 0910,0913,0936,0962 and0975 in the 89K which comes from the genome of 05ZYH33 as the objects,constructed the mutant stains 98HAH 12△0910,05ZYH33△0910,05ZYH33△0913,05ZYH33△0936,05ZYH33△0962 and 05ZYH33△0975 which deactivated thepurpose genes using the method of insertional inactivation,and then those mutantstrains’ virulence were checked through animal experiments.Through comparingwith the wild strains,the virulence of the mutant strains 98HAH12△0910 and 05ZYH33△0910 is lower than other strains.The gene fragment 0910 codes an ABC transporter.In order to further understandthe pathogenic mechanism of the 0910,we constructed the 0910-pET-32a(+)which isa expression vector of fusion gene through the method of molecular biology.Then the0910-pET-32a(+)was been translated into the BL21 competent cells,expressed andpurified to prepare the fusion protein of 0910.And after this fusion protein was usedto immune rats three times,the serum was taken and the antibody titer was measured.And we used the method of whole-cell ELISA and identified the 0910 coding cellsurface protein.The results established the fundament for further researching thefunction and effectiveness of 0910 in the SS2.