Expression And Immunogenicity Research Of Five Antigens Of Streptococcus Suis Type 2 Combine With LTB And FLIC

In the present study,the prevalence of SAO,SLY,RFEA,MAC and Pullulanase genes in clinical Streptococcus suis serotype 2（SS2）isolates were investigated.The results showed that the positive rates of these genes in 110 SS2 isolates were 70.91%,83.64%,74.55%,100%and 100%comparing with the positive rates among SS2isolates whose complete genomes were included in GenBank being 88.89%,59.26%,51.85%,100%and 100%,respectively.The amino acids homology of SLY,RFEA,MAC and Pullulanase from Genbank were 99.0-100%,97.0-100%,82.0-100%and98.0-100%,respectively（SAO has three variants and therefor didn’t analysed）.SLY,RFEA,partial SAO（SAO-N）,partial MAC（MAC-N）and Pullulanase（Pullulanase-N）were amplified and cloned into pET-28a（+）,and these genes were further fused with LTB and FLIC,respectively.Fifteen recombinant plasmids were constructed and 13 recombinant proteins were successfully expressed and purified.Mice were injected with recombinant proteins adjuvanted with Alum three times and the serums of each mouse were collected at 2 weeks after last vaccination.The mice were challenged with 2LD₅₀0 of strain CZ-XNN228.These 5 recombinant proteins were reacted with sampled sera by Western blot and antibodies titers of the sera were measured.The results were listed as follows:（1）All of mice in negative group dead within3 days and 83.33%of mice vaccinated with both of inactivated and commercially available vaccines survived;protection rates conferred by recombinant SAO-N,SLY without LTB or FLIC were 66.67%and 100%,respectively,and the remaining 3recombinant proteins protected 83.33%mice.（2）Recombinant proteins fused with LTB or FLIC further decreased the mortalities（14.82%）and morbidities（33.33%）of challenged mice compared with non-fused proteins.Nevertheless,the antibodies titers between fused and non-fused recombinant proteins have no obvious correlation.（3）MAC-N and Pullulanase-N can induce protective immunity in mice but these 2proteins didn’t reacted with sera raised by immunizing whole cells.Taken together,fusion LTB and FLIC with SAO,SLY,RFEA,MAC,and Pullulanase can induce enhanced protective immunity for mice.Especially MAC and Pullulanase are widely existed and conseverd and the truncated proteins induced complete protection in mice in current study,suggesting that they are potential candidates of subunit vaccine development.