Screening Of High Virulent Strains Of Streptococcus Suis Type 2 And Immunizing Protection Of SLY Protein

Swine streptococcal disease is a general term for swine diseases caused by a variety of streptococci.Streptococcus suis（SS）was divided into 35 serotypes（1-34 and 1/2）according to the antigenicity of the surface capsular polysaccharide（CPS）,in which Streptococcus suis serotype 2（SS2）affects the widest and the worst of human and livestock.The serotype of Streptococcus suis、the main virulence factors[CPS、hemolysin（SLY）、muramidase released protein（MRP）、extracellular factor（EF）)]existing regional differences,the cross-immune protection between the strains is poor,and the appropriate vaccine is great significance for control of swine streptococcal disease in a region.There is no standard animal models for the study of SS.In this study,43 strains of SS2 from the clinical cases in Jianghuai area from 2009 to2014 were used as experimental strains,combined with the background of virulence factors（CPS、SLY、MRP、EF）,the pathogenicity of 43 strains of SS2 was evaluated by hemolytic activity（rabbit blood,sheep blood）and two animal models（Kunming,rat,zebrafish）experiments,and the high virulent strains of SS2 were screened out,in which LD₅₀0 were detected and compared with each other.According to the full-length with hemolysin gene（sly）sequence of DQ443533.1 in the GenBank database,a pair of specific primers with BamHI and XhoI cleavage sites were designed and synthesized,construction of pET-30a-SLY Prokaryotic expression recombinant plasmid,induced expression and purification.The activity of SLY protein was detected according to the hemolytic activity of the protein to rabbit blood and the toxicity to Vero cells,then using Western blotting to evaluate its immunogenicity.Two kinds of animal models were used to study the protective effects of SLY protein,the titer of serum antibody in Kunming mice was detected by indirect ELISA method.Through the study of the virulent strains of LD₅₀0 in zebrafish and Kunming mice and the immune protection of SLY protein,evaluating the animal model suitable for SS2.The results:43 strains of SS2 showed that 39 strains ofβhemolysis and 4 strains ofγhemolysis in rabbit blood;41 strains ofβhemolysis and 2 strains ofγhemolysis in sheep blood.The 6 strains of SS2 were screened out as high virulent strains,respectively as HF13-8、BZ13-4、HF10-6、HF09-6、XC09-2、BB14-2,in which the LD₅₀0 of zebrafish were significantly lower than that of Kunming mice.Enzyme digestion and sequencing verified recombinant plasmid of pET-30a-SLY was successfully constructed,induced and expression by IPTG,SDS-PAGE showed that the target protein was about 60 kDa and appeared in inclusion body;The SLY protein was denatured and renatured,was purified by nickel column（NI-NTA）,which showed hemolytic activity and cytotoxicity;Western blotting showed that there was specific target band at 60 kDa,and SLY protein could react with SS2 positive serum.The relative protection rates of SLY protein were 40%in Kunming mice and 84%in zebrafish;The relative protection rates of SS2 inactivated whole cells were 80%in Kunming mice,92%in zebrafish.The ultrasonic SS2 virulent strain of whole cell lysate as antigen to detect SS2 virulent strain of inactivated whole cell immune serum antibody titers to IgG after three times was 1:819200,and to detect the SLY protein immune serum antibody titers to IgG after three times was 1:6400;The SLY recombinant protein as antigen to detect SS2 virulent strain of inactivated whole cell immune serum antibody titers to IgG after three times was 1:51200,and to detect the SLY protein immune serum antibody titers to IgG after three times was 1:102400.Conclusion:The 6 strains of SS2 were screened out as high virulent strains（HF13-8,BZ13-4,HF10-6,HF09-6,XC09-2,BB14-2）could be used as the reserve strain for the preparation of SS2 inactivated vaccine.Successful expression of SLY protein has good reactivity and immunogenicity,which can lay the foundation for further development of SS2 gene engineering subunit vaccine.Through experimental animals to study the pathogenicity for SS2,immunoprotection and other aspects of research,zebrafish is suitable as animal model for Kunming mice.