Functional Studies Of Streptococcus Suis Serotype 2 05ZYH33 Surface Protein SSU05₀630

Streptococcus suis?S.suis?is an important zoonotic pathgoen.Currently,the S.suis has 29 serotypes,in which the S.suis serotype 2?SS2?were reported as the mainly serotype associated with diseases in pigs and humans worldwide.SS2 can cause a variety of diseases and deaths in pigs and humans,posing a serious threat to the pig industry and human life,and has attracted attention from researchers worldwide.In this study,the function of the surface protein glycoside hydrolase SSU05₀630 of S.suis type 05ZYH33 was studied.First of all,domain analysis of SSU05₀630,gene cloning,protein expression and purification were performed to obtain the purified proteins of domains Ss CBM,Ss GH16,Ss GH20 and Ss CBM+GH16.Then,polyclonal antibodies against Ss CBM,Ss GH16 and Ss GH20 domain proteins were prepared by immunizing rabbits,and the antibody titers were 1:204800.And the ssu05₀630 gene deletion strain was constructed by pheromone GE9-induced DNA fragment transformation and homologous recombination,the growth characteristics and virulence of 05ZYH33 wild type?WT?and ssu05₀630 gene deletion strain were determined by measuring growth curve and mouse infection experiment.The results showed that there was no significant difference in the morphology and growth curve of the two strains.The mouse infection experiment showed that the WT group are the same as su05₀630?group of the survival rate.The activity of the purified Ss GH20 domain protein was further determined.It was confirmed that the Ss GH20 domain protein has NAG activity,with NP-Glc NAc as substrate.The activity of Ss GH20 was 371 U/mg protein.The optimum temperature for Ss GH20 activity was 50?,and the optimum p H was 5.5.The Km for Ss GH20 toward NP-Glc NAc was 0.69.By measuring the NAG activity of whole cells of ssu05₀630?strain and the WT strain,it was found that the NAG activity was only shown on the cell surface of the WT strain,but not on the cell surface of ssu05₀630?strain,indicating that SSU05₀630is the only protein having the NAG enzyme activity on the cell surface of the SS2 05ZYH33.Using transferrin as substrate,it was found that the Ss GH20 was able to cleave the?-1-2-N-acetylglucosamine residue in the complex N-glycan.The enzyme activity analysis of Ss CBM+GH16 and Ss GH16 domain proteins indicated that Ss GH16 could not hydrolyze the laminarin substrate-Laminarin,?-D-Glucan from barley,and ?-1,3-Glucan from Euglena gracilis.However,the Ss CBM+GH16 domain proteins were able to remove the heterologous antigenic determinant ?Gal of erythrocytes on rabbits,bovine and porcine with clearance rates of 99.9%?pigs?and 98.7%?cattles?.Therefore,the Ss CBM+GH16 and domain proteins are the same as Gal C and are endo-?-galactosidase.In this study,we preliminary explored the protein crystallization of Ss GH16 domain protein by gas diffusion diffusion method.A diamond-shaped crystal of 0.21×0.17×0.33 mm was obtained,the surface of the crystal was flat,three-dimensional structure was obvious,and was reproducible.In summary,the study lays the foundation for the role of SSU05₀630 in SS2 pathogenesis and the mechanism by SS2 evading host immune response,and provides a new means to reduce allogeneic transplant rejection.