| Streptococcus agalactiae was previously considered as the pathogen of bovine mastitis and neonates.However,recent evidences showed that S.agalactiae has caused overwhelming infections and deaths in fish.S.agalactiae has the capable of interspecies transmission and risk of crossover contagion a mong human beings,animals and fishes.Furthermore,consumptions of fish,for human beings,ha ve been proven to increase the risk of exposure to Ia and Ib serotypes of S.agalactiae significantly.Since 2004,China has been the largest tilapia farming country in the world,with an output accounted for 1/3 of the world total production.However,high incidences of S.agalactiae diseases have made serious impacts on tilapia industry of C hina.Since 2009,S.agalactiae,replaced S.iniae as the representative of streptococcal disease in tilapia,has becoming the primary pathogen in China with a miniaturized trend and increasing areas of pathogenetic tilapia.In recent years,as the new infected fishes including coldwater fishes and seawater fishes have been reported,most of the fish production enterprises in our country will be affected by S.agalactiae without proper preventive measures.Scophthalmus maximus whose?trade name turbot?is an important commercial mariculture fishin China with the main producing a rea as Shandong peninsula.There have been some reports about exophthalmos disease caused by S.agalactiae with the serious harm to torbut aquaculture in recent years.But so far,no research on vaccines of turbot against S.agalactiae has been reported.Vaccine is an effective way to prevent streptococcal disease.However,there are 10 serotypes in S.agalactiae.Inactivated vaccines made by a certain serotype strain of S.agalactia with no cross protection to other serotypes are greatly limited on the application.There are also some problems of attenuated vaccines,such as safety and immune efficacy.So,looking for a safe,stable and efficient vaccine to S.agalactiae has becoming the common concern of human medicine,animal husbandry industry and aquaculture industry.Furthermore,there is of great significance to human health,economic development and public safety.Genomes of S.agalactiae isolated from human,cattle and fishes had been analysed by bioinformatic methods,and some conserved antigen prote ins had been predicted as vaccine targets.Functional analyses of most of these proteins were not reported.Compared with other types of proteins,the surface proteins of bacteria are candidates for developing outstanding vaccines to prevent the disease with characteristics of sufficient exposure,large quantity,easy to be approached with antibodies,and higher level of immune response.This study selected 3 cell surface proteins predicted as immune function proteins of S.agalactiae,analysed their gene sequences by bioinformatics software,and cloned their epitopes isolated from tilapia.Proteins were expressed and purified by gene engineering technologies,and indirect ELISA detection methods were established as well.What's more,immunoprotection effects of subunit vaccines made by these 3 recombinant proteins to tilapia?Oreochromis aureus?and turbot?Scophthalmus maximus?were studied.The main research contents and results were as follows:1.Regions of Bepipred linear epitope,antigenicitiy,beta-turn,surface accessibility,flexibility and hydrophilicity in two cell wall surface anchored protein gene sequences?GenBank ID: CP000114.1,SAK1503,SAK0896?and one lipoprotein gene sequence?GenBank ID: CP000114.1 SAK0321?of S.agalactiae isolated from human were analyzed and predicted by bioinformatics software,respectively.B cell epitopes of three cell surface proteins were calculated based on the above parameters,and specific primers were designed based on the sequences from 29 to 183 amino acid s,113 to 457 amino acids and 31 to 144 amino acids of these genes,respectively.Objective fragments of 3 cell surface proteins of S.agalactiae BX2012 isolated from tilapia were cloned.First,p ET-32a-CWSAP465,pET-32a-CWSAP1035 and pET-32a-LIP342 prokaryotic expression plasmids were constructed successfully,and their sequences were uploaded to GenBank with ID number KU759322,K U759323 and KU759324,respectively.Soluble recombinant proteins with molecular mass of 36,54 and 30 k Da were expressed efficiently in BL21?DE3?cells,respectively.Then,recombinant proteins were purified by His-Trap FF affinity chromatography,the purity of CWSAP465,CWSAP1035 and LIP342 proteins were increased from 52.89%,36.40% and 41.75% to 94.25%,58.61% and 87.23%,respectively.The results of Western blotting showed that three proteins were distinguished specifically by high immune serum of rabbit anti-S.agalactiae with good immunogenicities.2.Compared with the similar sequences in GenBank for homology analysis,the results showed that sequences in CWSAP465,CWSAP1035 and LIP342 protein of S.agalactiae from all known species?including human,cattle,camel and tilapia?or different serotypes isolates?Ia,II,III,IV and V?were highly conserved,with homology of 96.13%-100%,99.13%-100% and 90.35%-100%,respectively.While their homologies were 98.06%-100%,99.71%-100% and 100% among tilapia isolates,respectively.It is inferred that three proteins may have the similar or same immune protective characteristics to other species of fish and animals who susceptible to S.agalactiae,even to the human being.3.The detection methods of indirect ELISA with CWSAP465,CWSAP1035 and LIP342 were established.The optimal package concentrations of CWSAP465 and LIP342 were confirmed as 10 ng/?L,while CWSAP1035 as 15 ng/?L.The optimal serum dilution ratios to all of them were 1:40.The optimal sealing fluids to CWSAP465 and CWSAP1035 were 10% bovine serum,and 5% BSA to LIP342.The optimal sealing times and reaction times to all three proteins were 0.5 h.While CUT-OFF values of CWSAP465-ELISA,CWSAP1035-ELISA and LIP342-ELISA were 0.243,0.309 and 0.288,respectively.Negative reactions were tested by 3 ELISA methods applicated in detecting O.spp.anti-Edwardsiella tarda serum,anti-Aeromonas hydrophila serum,anti-A.sobria serum and swine anti-S.suis type 2 serum.The results showed that coefficient of variations of intra-plate repetitive tests was from 0.29% to 5.33%,inter-plates was from 0.96% to 7.13%,and inter-batches was from 0.35% to 8.91% of 3 ELISA methods.So,CWSAP465-ELISA,CWSAP1035-ELISA and LIP342-ELISA were specific and repeatable and can be applied to the vaccine potency tests and epidemiological investigations of S.agalactiae serotype Ia.4.In order to test the immune protective effects to tilapia and turbot,subunit vaccines of CWSAP465,CWSAP1035 and LIP342 recombinant proteins were made with Freund's incomplete adjuvant.5 experimental groups were set as CWSAP465 immune group,CWSAP1035 immune group,LIP342 immune group,CWSAP465 + CWSAP1035 immune group?mixed by two CWSAP recombinant proteins with equal doses?,and CWSAP465 + CWSAP1035 + LIP342 immune group?mixed by three recombinant proteins geometric with equal doses?.Three control groups were set as inactivated vaccine control group,PBS control group and negative control group,respectively.Injection dose was 4 ?L/g fish weight?recombinant protein immune dose of 2 ?g/g fish weight?with once intrapleural injection.Serums antibody levels of tilapia and turbot were detected by ELISA on 0,7,14,21,28,35,and 42 d right after immunizing,respectively.The results showed that CWSAP465,CWSAP1035 and LIP342 recombinant proteins could induce tilapia and turbot to raise antibody level significantly?P<0.05?,and higher than inactivated vaccine control group significantly?P<0.05?.Antibody levels of 5 immune groups reached the peak on the fourth week and fell with the challenge of S.agalactiae5.Challenge tests at 28 d after immunization showed that tilapia and turbot of groups immunized with CWSAP465,CWSAP1035 and LIP342 recombinant proteins and also inactivated vaccine were protected from a challenge by a virulent S.agalactiae at a dose of 1×109 colony forming units/mL?P<0.05?.Results of Kaplan-Meier tests,for tilapia,showed that cumulative survival rates of CWSAP465 + CWSAP1035 + LIP342 immune group and CWSAP465 + CWSAP1035 immune group were higher significantly than that of inactivated vaccine control group?P<0.05?.For turbot,CWSAP465 + CWSAP1035 + LIP342 immune group was higher significantly than inactivated vaccine control group?P<0.05?.Relative percentage survivals of CWSAP465 + CWSAP1035 + LIP342 immune groups of tilapia and turbot were higher significantly than that of inactivated vaccine control groups?P<0.05?,respectively.There were no significant differences among groups immunized by CWSAP465,CWSAP1035,LIP342 and CWSAP465+CWSAP1035?P>0.05?,but RPS vaules of these groups were also higher than that of inactivated vaccine control groups.In a conclusion,immunoprotective effects of vaccine mixed by three recombinant proteins geometric with equal doses were most outstanding.In summary,three truncated cell surface proteins of S.agalactiae,CWSAP465 and CWSAP1035 and LIP342,were expressed in prokaryotic systems,three indirect ELISA detection methods were established,and they were evaluated as potential vaccine candidates against the S.agalactiae.Both tilapia and turbot immunized with recombinant proteins produced higher levels of antibody than those immunized with inactivated vaccines,and displayed resistance capacity when challenged by a virulent S.agalactiae strain.It is proved that 3 cell surface proteins can be used as substitutes for inactivated vaccine against S.agalactiae for tilapia and turbot.This study filled the blank of turbot on vaccine research of S.agalactiae,and proved that subunit vaccines and inactivated vaccine were immuneprotective to turbot.It even filled the blank of the immuneprotective research of these cell surface proteins,layed the foundation for structure and function analyses in the future.It also proved that CWSAP465,CWSAP1035 and LIP342 could be potential candidates in the pre vention of S.agalactiae disease,and maybe used as vaccines with widely utilization in fishes. |
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