| Streptococcus suis(S.suis)and porcine circovirus disease spread widly and has caused huge economic loss to swine industry.The vaccine research of these two dieases is of great significance.Salmonella,a kind of intracellular bacteria,has great power of invasion and can survive in host cell,which means it could be an excellent bacterin to stimulate cellular and humoral immunity effectively once it was attenuated.On the basis ofpreviously construction ofthe live attenuated Salmonella enterica serovar Choleraesuis(S.Choleraesuis)with regulated delayed attenuated and regulated delayed heterologous antigen synthesis,which named rSC0016,the immunogenicity of S.Choleraesuis rSC0016 expressing the three heterologous antigens of Streptococcus suis serotype 2(SS2),and two Cap proteins of porcine circovirus type 2(PCV2)were evaluated in BALB/c mice in this study.The results would provide theoretical basis for the construction of bivalent vaccines of S.Choleraesuis and SS2,or S.Choleraesuis and PCV2.1.Selection of antigens from S.suisEno is enolaseof SS,and a kind of virulent factor of Streptococcus suis and involved in the invasion and transmission of S.suis to host cells;6-phosphogluconate-dehydrogenase is higly conserved and can provide certain protection to Streptococcus suis;SaoA is a newly discovered surface protein of S.suis and participate in the coordination of various virulent factors.Our team has construced a live attenuated Salmonella enterica serovar Choleraesuis with regulated delayed attenuated and regulated delayed heterologous antigen synthesis,which named rSC0016,two prokaryotic expression vector pYA3493 carrying heterologous antigens named pS-6PGD and pS-SaoA.In this study,the prokaryotic expression vector pS-Eno need to be constructed pS-Eno,and the immunogenicities of rSC0016 delivering Eno,SaoA and 6PGD dereived from S.suis were evalutated in mice,respectively.1.1 Clone and expression of SS2 enolaseThe primers of amplifying eno gene of SS2(GT:8151617)were designed and the 1305bp of eno gene were amplified and cloned into the prokaryotic expression vector pET28a.After sequenced,the pET28a with eno gene was transformed into E.coli BL21,and eno protein was purified by western blotting.The New Zealand white rabbits were immunized with eno protein,then euthanized to make serum against eno protein.The eno gene was also inserted into prokaryotic expression vector pYA3493 with asd+,named pS-Eno.Meanwhile,plasmids pS-Eno,pS-6PGD and pS-SaoA were transformed into rSC0016,called rSC0016(pS-6PGD)?rSC0016(pS-SaoA)and rSC0016(pS-Eno).The proteins of 6PGD,SaoA and Eno could be expressed in rSC0016 strain detected by Western-blotting,respectively.1.2Evalution of protective efficacy of rSC0016(pS-6PGD)?rSC0016(pS-SaoA)and rSC0016(pS-Eno)First the growth curve of rSC0016(pS-Eno)in LB media was detected;Then the colonization ability of rSC0016(pS-Eno)was displored in liver,spleen and Payer's patches;Finally,the immunogenicities of rSC0016 delivering Eno,SaoA and 6PGD dereived from S.suis were evalutated and compared in Balb/C mice,respectively.The results showed that rSC0016(pS-Eno)growed slightly slower than that of rSC0016(pYA3493)and rSC0018(pYA3493)in LB media.As the time passed,the colonies of rSC00 16(pS-Eno)in liver or spleen decreased gradually and turned to 0 after 2 weeks,while rSC0018(pYA3493)in liver and spleen could still be isolated rSC00 18(pYA3493),suggesting the virulence of rSC00 16(pS-Eno)was lower than that of n rSC00 18(pYA3493).At 3 weeks after inoculation,the number of rSC00 16(pS-Eno)was 104CFU,in contrast,rSC0018(pYA3493)could not be isolated any more in Payer's patches of mice.The IgG and IgA titer inmice immunized with rSC00 16(pS-Eno)?rSC00 16(pS-SaoA)and rSC00 16(pS-6PGD),respectively,were higher than those of rSC0018(pYA3493)mearsured by ELISA.(P<0.01)At 3 weeks and 5 weeks after first vaccination,the IgG titer in mice immunized with rSC00 16(pS-Eno)and rSC00 16(pS-SaoA)were strongly higher than that of rSC0016(pS-6PGD)(P<0.01).The IgA titer in mice immunized with the three strains at 5 weeks post inoculation were higher than those at 3 weeks after prime vaccination,however,the IgA respose in mice immunized with the three strains presented similar level.On day 7 after boost,the IL-4 and IFN-y level in lung and spleen of mice inoculated with rSC00 16(pS-Eno)?rSC00 16(pS-SaoA)and rSC00 16(pS-6PGD)were significantly higher than that of empty vector rSC00 16(pYA3493)(P<0.01).The IL-4 and IFN-y titer in mice immunizaed with rSC00 16(pS-SaoA)were significantly higher than that of rSC00 16(pS-Eno)and rSC00 16(pS-6PGD),and the IL-4 and IFN-y titer in mice immunizaed with rSC00 16(pS-Eno)were significantly stronger than that of rSC00 16(pS-6PGD)(P<0.05).The protective efficiency provided by rSC00 16(pS-Eno)and rSC00 16(pS-SaoA)against challenge of 24×LDso SS2 were 100%,whereas all mice immunized with rSC00 16(pYA3493)or PBS were dead in 7 days after challenge.2.Construction and evaluation of recombinant attenuated Salmonella vaccine carring Cap and dCap protein of porcine circovirus type 2Cap,a kind of protective protein encoded by ORF2 of porcine circovirus type 2(PCV2),can induce antibody against virus.It means that cap gene is the major candidate of vaccine and diagnostic reagent of PCV2.2.1Clone and expression of cap and dcap PCV2The primers of amplifying cap gene(702bp)and the deleted nuclear localization signal cap(dcap,579bp)gene of porcine circovirus type 2 were designed.The cap and dcap were amplified and cloned into the prokaryotic expression vector pET28a.After sequenced,the pET28a with cap or dcap gene was transformed into E.coli BL21,and Cap or dCap protein was purified by western blotting.The New Zealand white rabbits were immunized with Cap or dCap protein,then euthanized to make serum against eno protein.The cap or dcap gene were also inserted into prokaryotic expression vector pYA3493 with asd+,named pS-Cap or pS-dCap.Meanwhile,plasmids pS-Cap or pS-dCap were transformed into rSC0016,called rSC0016(pS-Cap)or rSC0016(pS-dCap).The proteins of Cap or dCap could be expressed in rSC0016 strain detected by Western-blotting,respectively.2.2 The evalution of protective efficacy and biology of rSC0016(pS-Cap)and rSC0016(pS-dCap)First the growth curve of rSC0016(pS-Cap)and rSC0016(pS-dCap)in LB media were detected;Then the colonization ability of rSC0016(pS-Cap)and rSC0016(pS-dCap)were detected in liver,spleen and Payer's patches;Finally,the immunogenicities of rSC0016 delivering cap ans dcap gene dereived from PCV2 were evalutated and compared in Balb/C mice,respectively.The results showed that the growth speed of rSC0016(pS-dCap)was faster than that of rSC0016(p pS-Cap)in LB media.The distributed ability of rSC0016(pS-Cap)and rSC0016(pS-dCap)in mice were similar.As the time passed,the colonies of rSC0016(pS-Cap)and rSC0016(pS-dCap)in liver or spleen decreased gradually and turned to 0 after 2 weeks.At 3 weeks after inoculation,the number of rSC0016(pS-Cap)and rSC0016(pS-dCap)was 104CFU in Payer's patches of mice.The IgG and IgA titer in mice immunized with rSC0016(pS-Cap)and rSC0016(pS-dCap)achieved similar level,and were higher than those of rSC0018(pYA3493)and PBS tested by ELISA(P<0.01).At 14 day after challenge by intraperitoneal(i.p.)injection with 106TCID50 PCV2,the PCV2 virus loads in sera of mice immunized with rSC0016(pS-Cap)and rSC0016(pS-dCap)were detected by Real-time PCR.The results showed that the virus loads in sera of mice immunized with rSC0016(pS-Cap)and rSC0016(pS-dCap)were reduced 67%and 83%compared to that of control group or rSC0016(pYA3493)(P<0.05).Specifically,the virus loads in sera of mice immunized with rSC0016(pS-dCap)were lower than that of rSC0016(pS-Cap),suggesting that strains rSC0016(pS-Cap)and rSC0016(pS-dCap)could provide high protection against PCV2,and rSC0016(pS-dCap)might be better than that of rSC0016(pS-Cap)in reducing viral load.In summary,Recombinant attenuated Salmonella vaccine candidate rSC0016 expressing Eno protein of SS2,Cap and dCap protein of PCV2 respectively were constructed by molecular cloning.The evaluation of protective effiecy of rSC0016 expressing 6PGD,SaoA,Eno protein of SS2 and Cap,dCap of PCV2 was done in Balb/C mice.The results showed the effiency of SaoA and Eno was higer than 6PGD and both of Cap and dCap could induced considerable immunological response. |
PDF Full Text Request