Construction And Characterization Of Dbp Gene Knock-Out Mutant In Streptococcus Suis 2

Streptococcus suis (SS) is an worldwide and major zoonotic pathogen that causes a wide range of disease of human or swine. Among 35 known serotypes, Streptococcus suis type 2(SS2) is the most virulent and the most frequently isolated from diseased pigs. Two outbreaks of human SS2 infections in China in 1998 and 2005 were reported. The prevention and control of the SS2 infection has turned into an imperative task. Though the discovered factors has contributed to the understanding of SS2 pathogenesis, the pathogenesis of SS2 infection is still limited, and knowledge concerning virulence factors of S. suis remain obscure. Our studied showed that some genes only profiled in virulent strain after comparative genomics assay between virulent strain ZY05719 and the avirulent strain T15 by marve software. Dbp is one of these genes that identified using this approach. To investigate the contribution of Dbp to the virulence of S. suis 2, An isogenic SS2 mutant of Dbp, ADbp, was constructed and its biological characteristics were studied. All these help us to understand its pathogenic mechanism.1 Comparative genomics assay between virulent strain ZY05719 and the avirulent strain T15, and Distribution of the dbp in SSObjective to identify virulence factors, some differential genes that only profiled in virulence were disclosed between the virulent strain ZY05719 and avirulent strain T15 of S.suis 2 using comparative genomics assay by marve software.92 genes were identified and a novel gene, Dbp, was revealed by this method above. PCR detection for evaluating the distribution of the Dbp gene in 95 different SS displayed that the Dbp gene was present in most of the SS2(88%), but it were seldom detected in other serotypes of SS(17.8%), indicating it may be a relatively conserved gene of SS2. Primary sequence analyses of Dbp unusual modular organization with a region homologous to members of the HTH-XRE family of transcriptional regulators.2 Construction of Dbp gene mutation of SS2To research the function of Dbp gene, the isogenic Dbp mutant were constructed, using the genomic DNA of ZY05719 as the template, upstream Dbp arm and downstream Dbp arms were amplified,667bp and 668bp, respectively. The ad gene (1335bp) was amplified according to the template above. The ad gene was linked to shuttle vector, pSET4S. Then the products were transferred into DH5a competent cells and the vectors ad-pSET4S were constructed. Subsequently, the acquired knockout plasmid ad-pSET4S was electroporated into the ZY05719 competent cells so as to obtain the isogenic mutant Adbp. Spc resistance colonies were selected on THB plates containing Spc(50μg/ml).The resultant mutants were verified by PCR and further confirmed by DNA sequencing of the PCR product. The results showed that ADbp was successfully constructed.3 Analysis of biological characteristics and pathogenicity of ADbp of SS2To investigate the function of Dbp gene, the biological characteristics and pathogenicity of △Dbp and wild type ZY05719 was compared. The results showed, compared with wild type, growth and biochemical characteristics had changed. Zebrafish pathogenicity test showed that the LD50 of ADbp (3.25×10O6) had a significant decrease as compared with wild type ZY05719 (LD50=5.97×105). In conclusion, Dbp gene was found to play an important role in pathogenicity of SS2.4 Cloning, expression and identification of the Dbp gene of SS2A novel gene, Dbp, was revealed by comparative genomics assay, which may be the virulence factor of SS2. According to the sequence of Dbp, Dbp genes were optimized and total synthesized. Then gene of Dbp was inserted into expression vector PET32a(+), and the recombinant plasmid PET32a-Dbp was transformed into E.coli BL21, and induced by IPTG to express recombinant Dbp. The Dbp protein was analyzed with SDS-PAGE and Western blotting and the recombinant protein was probed with rabbit immune sera. The experimental results showed that the the Dbp gene can be highly expressed in prokaryotic system, and the recombinant protein showed specific antigencity.