| The purpose of this study is to develop a universal and effective recombinant subunit vaccine against highly pathogenic serotypes of Streptococcus suis（SS）,such as SS2,SS3 and SS9.Firstly,this study used a variety of bioinformatics software to comprehensively analyze three virulence factors of Highly conserved secretory DNA nuclease A（Ssn A）,Dihydrolipoamide dehydrogenase（DLDH）and Zinc transporter（Znu A）of highly pathogenic SS,predicted and obtained three B/Th cell epitopes with hydrophilic and good antigenicity.Then the amino acid sequence of multi epitope recombinant protein SDZ was designed by connecting the dominant epitopes in series;The amino acid sequence of multi epitope recombinant protein DC3pep-SDZ with targeted binding potential to dendritic cells（DCS）was designed by connecting dodecamer dendritic cell targeting peptide（DC3pep）with dominant epitope in series.Protein physicochemical analysis of two amino acid sequences,the results showed that the molecular weights of SDZ and DC3pep-SDZ proteins were about 42 ku and 44 ku,the theoretical isoelectric point（PI）was 4.5 and 4.65,and the average hydrophilicity（gravy）was-0.4436 and-0.462,respectively.The Recombinant Prokaryotic expression vectors p ET28a-SDZ and p ET28a-DC3pep-SDZ were constructed and transformed into competent cells.After IPTG induction,a large number of the two recombinant proteins were expressed in the supernatant and purified by 200 mmol/L imidazole flow through affinity chromatography nickel column.SDS-PAGE results showed that 42 ku and 44 ku of the target proteins were obtained,The concentration of recombinant protein after ultrafiltration concentration reached4.90 mg/ml and 5.60 mg/ml respectively.The hemolysis test of 2% porcine erythrocyte suspension was carried out in vitro with different concentrations of recombinant protein,and the detection results showed that within5～500 μg/m L protein concentration,the hemolysis rate of recombinant protein SDZ and DC3pep-SDZ to porcine erythrocyte suspension was less than 5%;PK15 and IPEC-J2 cells were incubated with different concentrations of recombinant protein,and the results were detected by CCK-8 reagent that the relative increment rates of PK15 and IPEC-J2 cells incubated with 500 μg/m L recombinant protein SDZ and DC3pep-SDZ were 97.3% and 99.8%,98.5% and 99.6% respectively,indicating that the two recombinant proteins had no obvious toxic effect;The recombinant proteins SDZ and DC3pep-SDZ were immunized to healthy zebrafishs and mice for safety evaluation.After 7 days,zebrafishs and mice immunized with the two recombinant proteins had no adverse reactions and were in good health.The recombinant protein SDZ and DC3pep-SDZ are safe.After the two recombinant proteins were immunized to adult healthy zebrafishs for 3 times,they attacked highly pathogenic types 2,3 and 9 SS（SS2,SS3 and SS9）,calculated the immune protection rate and observed the pathological sections of fish brain tissue.Finally,q PCR was used to detect the expression difference of inflammatory cytokines in equine brain tissue after SS3 attack.The results showed that the protective rates of recombinant protein DC3pep-SDZ against zebrafishs infection with SS2,SS3 and SS9 were 50%,65% and 60% higher than that of SDZ.The recombinant protein DC3pep-SDZ was better than SDZ and significantly reduced the pro-inflammatory cytokines IL-6 and TNF-α gene compared with the control group,up regulation of anti-inflammatory factors IL-10 and TGF-1β gene expression level.Finally,the recombinant protein SDZ was mixed with DC3pep-SDZ and Freund’s adjuvant to prepare subunit vaccine.After 3 immunization,the antibody titer and cytokine concentration of mice were detected by ELISA,and the mice were attacked with SS3 to detect the bacterial load of organs and blood and the survival of mice.The results showed that both vaccines produced immune protection in mice.The serum antibody titer of mice immunized with sub DC3pep-SDZ+CFA/IFA was higher than that of mice immunized with SDZ+CFA/IFA,reaching 1:102400.Compared with the control group,immunization with DC3pepSDZ+CFA/IFA significantly increased the levels of cytokines IL-6 and IFN-γ,the concentrations reached 34.7 pg/m L and 228.6 pg/m L,and the protection rate of SS3 challenged mice was higher than that of DC3pep-SDZ+CFA/IFA,reaching 75%.In conclusion,the results showed that the recombinant protein DC3pep-SDZ targeting DCs had better immunogenicity than the epitope recombinant protein SDZ,and DC3pepSDZ+CFA/IFA vaccine could better stimulate humoral and cellular immunity than SDZ+CFA/IFA vaccine,laying the foundation for becoming an effective universal subunit vaccine of highly pathogenic SS. |
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