Exploration Of Streptococcus Suis Surface Display Systems For Multivalent-Vaccine Development

Gram-positive Streptococcus suis is an important zoonotic pathogen that seriously endangers the development of the pig industry and human health.It often appears clinically with multiple serotypes,multiple pathogens mixed or secondary infections.Therefore,the development of swine streptococcus multivalent vaccine is of great significance for preventing mixed infections and reducing the immune burden on pig farms.The display of heterologous antigens on the surface of bacteria through surface presentation is a promising method for the preparation of multivalent vaccines.However,there are no reports of exogenous proteins present on the surface of S.suis.According to the principle of surface anchoring of LPxTG protein of Gram-positive bacteria,the fusion of LPxTG protein transport and anchoring signals with exogenous proteins can achieve the display of exogenous proteins on the surface of gram-positive bacteria and provides a new idea for the development of S.suis multivalent vaccine.In order to construct the protein surface display system of S.suis,the LPx TG protein and signal peptide?SP?and cell wall motif?CWA?of S.suis type 2 05ZYH33 strain were identified through sequence analysis.By amplifying and fusing the DNA fragments of P_eno-SP,GFP,and CWA,the recombinant DNA fragments encoding the SP-GFP-CWA fusion protein controlled by strong promoter P_enono was constructed.The recombinant DNA fragments were ligated into the vector pSET2,resulting the protein surface display plasmids.The plasmids were transformed into S.suis to obtain the protein surface display systems,with GFP as reporter protein.The presenting protein were preliminarily observed by Western Blot.The results showed that 10 LPxTG proteins and their SP and CWA were identified from S.suis,and 10 pSET2 surface display plasmids containing P_eno-SP-GFP-CWA fusion fragments were respectively constructed,namely p SsPSD1 to pSsPSD10.Seven of them were successfully transformed into S.suis.Western Blot and preliminary quantitative analysis showed that all seven transformed positive strains effectively express GFP protein.Taking mature GFP band as indicators,all of them showed a certain level of exogenous GFP surface display.This indicated that the protein surface display system of S.suis was successfully constructed.They were named as SsPSD1,SsPSD2,SsPSD4,SsPSD7-SsPSD10.The surface display levels of SsPSD1,Ss PSD2,SsPSD8 and SsPSD9 showed better surface display potential.In order to further confirm the role of sortase A?Srt A?in the processing of LPxTG protein of S.suis,this study established srtA gene deletion strain?srtA^??,and the pSsPSD4 plasmid was transformed into 05ZYH33 wild type?WT?strain and 05ZYH33 srtA^?strain to construct SsPSD4-WT and SsPSD4-srtA^?strains.The total bacterial protein of SsPSD4-WT and SsPSD4-srtA^?strains were extracted to compare the state of GFP protein produced by the two strains using Western Blot.The results show that there are three kinds of GFP states in SsPSD4-WT,namely GFP precursor protein,GFP intermediate protein and GFP mature protein.The SsPSD4-srtA^?strain has no expression of SrtA and only produces GFP precursor protein and GFP intermediate protein,indicating that the GFP mature protein does indeed come from the processing of SrtA.This study is the first attempt to use the anchoring principle of LPxTG protein to establish protein surface display systems for S.suis,providing a new strategy for the display of exogenous proteins or heterologous antigens on the surface of S.suis.