Immune Protection Research Of Muramidase-released Protein, Extracellular Factor Protein And GroEL From Streptococcus Suis Serotype 2

Streptococcus suis serotype 2 is an important cause of meningitis, arthritis,Septicemia,hydrymenitis,endocarditis and polyarticular,it severely threatens the practitioners working in the field of animal husbandry,and also causes serious diseases like meningitis and Streptococcal Toxic shock syndrome,STSS).(1) Purification of Muramidase-released protein,Extracellular factor protein and hemolysin from Streptococcus suis serotype 2Recently the research on Streptococcus suis serotype 2 is mainly concentrating on virulence-related factors,Discovery and Identification of novel virulence-related factors are still The focal point of research at home and abroad.The pathogenesis of Streptococcus suis serotype2 is unknown,The generally received virulence-related factors include Capsular-bolysaccharides,Muramidase-released protein,Extracellular-factor protein,hemolysin and adhesion,Nevertheless the research on Muramidase-released protein and Extracellular factor protein is mainly focus on epidemiology and are seldomly involved in other aspects.One reason is that there are certain difficulties in obtaining the whole fragments of these two proteins,which Restricted the Functional and pathogenesis research of these two virulence-related factors.In this experiment,we divided the whole open reading frame of Muramidase-released protein gene into three parts,and cloned each part to pET-28a and pGEX-KG expression vector Accordingly,then we did the gene expression and protein purification.We also purified Extracellular factor protein using ammonium sulfate precipitation method.Then the Balb/c mice were immunized with purified MRP,EF and SLY proteins,and challenged with mice 5LD₅₀ of Streptococcus suis sero-type 2 high-virulent strain,we estimated the relative percent survival rate(RPS) of MRP,EF and SLY fragments in the immune protection experiment and also compared and analyzed the effectual protective Ingredients of MRP.This experiment optimized the skill of Protein affinity chromatography with(HIS and GST tag)And modified ammonium sulfate precipitation method for protein purification.(2) Heat shock proteins from Streptococcus suis serotype 2Heat Shock Proteins are a group of stress proteins which extensively existed in bacterium and mammalian,and are highly conservatived in amino acid sequence.They are present in cells under normal conditions to maintain the basic forms and functions of cells,but are expressed at high levels when a cell undergoes various types of environmental stresses to protect cells by repairing injured ones.Heat Shock Proteins can be classified into five categories according to their molecular weight,they are HSP100, HSP90,HSP70,HSP60 and mall HSPs.HSPs in eukaryotic had been commonly studied and understood,however there are few researches on procaryotic HSPs.In this experiment we have cloned and expressed five HSP genes from SC-19 including GroEL,Dnak,GrpE,Hsp33 and GroEs.Choosing GroEL and Dnak as our subjects,we did immunoprotection experiment on mice,monitored the antibody levels of mice after immunization for 7 weeks using the ELISA assay,and also identified the subclass antibodies of the mice.At last we detected the changes on the proinflammatory factors(IL-10,IL-4,IF-Nå’ŒIL-6,) of mice induced by GroEL in mRNA level.The experimental result shows that:Dnak and GroEL can induce relative high titer antibody within two weeks after primary immunization,GroEL can cause IL-10 up-regulated in mRNA level in spleen cell.