Identification And Functional Studies On The HtrA Gene From Streptococcus Suis Type 2

Streptococcus Suis is caused by a variety of pathogenic Streptococcus, capable of transmission to humans from pigs. Streptococcus suis type 2 is the most important of all, showing a global distribution, Not only cause a huge damage on the pig, but also It can lead people get meningitis, arthritis, sepsis and endoearditi, even acute death of human death. An outbreak of Streptococcus suis emerged in 2005 in Sichuan Province and in 1998 in jiangsu Province, arossed a mixed infection of pigs and humans, causing a hign degree of concern about the country.Therefore, from the pig industry and human health, the control of the disease have a major public health significance. To Streptococcus Suis type 2, at present it focused on the study of virulence factors, Pathogenicioulty Island, the study of two-component system, but its pathogenesis has not yet accurately stated.High temperature requirement A (HtrA) protein family as a heat shock-induced serine proteases both have serine protease and the dual function of molecular chaperone. HtrA genes found in animals, plants, bacteria, fungi, yeast, mycoplasma, and human.In these organism, can help these species to avoid changes of the environment when supressde the oxidative stress and high temperature. It showed the impact of bacterial virulence and colonization in cells; in human, the homologous gene HtrA2 showed affect to apoptosis and cell cycle. However, in Streptococcus Suis type 2 HtrA has no been reported.This study was designed to research on the HtrA gen identification of Streptococcus suis type 2 gene and its function, cloning thre HtrA gene of SC-19, recombinant protein and gene deletion mutants, research links with the pig bacteria in the pathogenicity of type 2 on the relevance of the study, including:1. Identification, protein expression and purification and immunological studies of HtrA gene on in Streptococcus suis type 2.Throμgh the comparece of the publicaed sepuence of P1/7 strain HtrA gene and protein sequence, we identified the HtrA gene of Streptococcus suis type 2 virulent strain 05ZYH33 from people(CP000407), use Streptococcus suis type 2 SC-19 as a template And we construct pET-32a(+)-HtrA plasmid with prokaryotic expression plasmid,then transform it into Rosetta (DE3),37℃shaking to OD600 reached 0.6,0.2mM/mL IPTG induction at 16℃with 15 hours, and then use Ni-NTA Agarose to obtian purified protein. Finally we use SDS-PAGE and Western Blotting to verify the target protein.Through Western Blotting, immunologic tests, immune with different adjuvant we detected serum antibody and IgG1 and IgG2a.we do protection test with three imMunity,then use 2.5LD50,1.8×109CFU/mL SC-19 strain infect mice to identify that the recombinant protein has a protective force.In order to know the serotype distribution of HtrA gene in Streptococcus Suis, and study the conservative of sequence and correlation of virulence, we carried out PCR with 32 serotypes, from the results we can see that most of the 32 serotypes can get the HtrA gene amplification. In order to study the functions of HtrA gene, we have cell experiments in vitro. Though the indirect immunofluorescence experiments(Hep2 cell), opsonophagocytosis experiments (RAW264.7)and cell adhesion experiments, we have confirmed that the HtrA protien can adhesion in the cell surface; the recombinant protein antibody has a good conditioning effect in the agocytosis. Meantime we constructed eukaryotic expression vector to study the cell cycle and apoptosis, the experimental results showed that HtrA plasmid in eukaryotic cells can be well expressed and impact the cell cycle and apoptosis, and promote cell apoptosis.2. Construction of HtrA gene mntant strain of Streptococcus suis type 2 of and study on the characteristics of biologicalTo further clarify the correlation of HtrA gene and the pathogenicity of Streptococcus Suis type 2.We construct its gene deletion strain and complementary strain. We use temperature-sensitive suicide vector pSET4s and pSET2s to construct homologous recombinant plasmid and the complementary arm plasmid, then electric it into Streptococcus suis type 2 strains SC-19 orΔHtrA, and using homologous recombination technology to get the deletion strainsΔHtrA and complementary gene deletion strain cΔHtrA.Though Biological experiment of hemolytic activity tests, stress tests we confirmed that the gene ofΔHtrA in growth characteristics, hemolytic activity, stress reactions hasve changed compared with wild type; but the presence of deletion strainsΔHtrA on bacterial morpHology, virulence did not change significantly compared with wild type. Therefore the effect of HtrA gene to the virulence of Streptococcus Suis type 2 to be further researched.