Study On Preparation And Immune Efficacy Of A Alginate-Chitosan Oral Vaccine Of Srr-Protein From Streptococcus Iniae

Channel catfish is one of the main aquaculture species in China.The disease occured frequently with large-scale farming and Streptococcus iniae become a main pathogenic bacteria in recent years.In order to control the disease caused by S.iniaeon channel catfish,vaccine was prepared to prevent and control the bacterium in this study.TheSrr-protein of S.iniae was inner core to make microparticles with alginate-chitosan by emulsion-based methodology.Based on the Design-Expert 8.0.6 of response surface methodology to obtain the optimal fabrication conditions,we analyzed the swelling ratio characteristics of microparticles,acid-baseproperty,immunogenicity and security of microparticles.The safety,immunological protection and valence of this vaccine were studied,and an oral vaccinne was carried out in healthy channel catfishes.During the preliminary study,it lays the foundation for the study of the oral vaccine of S.iniae and the development of new vaccines.This experiment covers three main chapters listed below:1.Experimental design and optimization of alginate-chitosan Srr-protein microspheresThe Srr-protein of S.iniae was used as antigen model,and the natural macromolecule polymer sodium alginate and chitosan were used as protein carrier,and the response surface methodology was the way to optimize the preparation conditions.Firstly,the effects of sodium alginate concentration,the water/oil ratio,the concentration of calcium chloride and the concentration of chitosan were investigated by single-factor experiments through the encapsulation efficiencies.Then,on the basis of single-factor experiments,the relationships among main preparation conditions were modeled using a 4-factor,3-level Box-Behnken experimental design.The established model was analyzed by response surface methodology to obtain the optimal preparation conditions.The results showed that the optimal preparation conditions were as follows:sodium alginate concentration was 1.68%,water-oil ratio was 3.6:6.4,the concentration of calcium chloride was 9.64%,and the concentration of Chitosan was 0.95%.The encapsulation efficiency of microspheres were 92.38%,the drug-loaded rate were 19.41%,and its diameters were(4.26±1.13)?m,the oral vaccine be appropriate for fish.2.Swelling,safety and Immunogenicity of of alginate-chitosan Srr-protein microspheresIn order to explorethe swelling properties of microspheres,pH,temperature and ionic strength of the swelling medium had been changed in 3 hours.At the same time,the experiment was carried out to investigate the Srr-protein in vitro release withsimulated gastrointestinal tract.Then we tested its'immunogenicity and safety.All the results showed that when pH was 1.2 and 7.4,there was no significant difference between the swelling property and the stability of the microspheres and the highest swelling rate were 1.15,1.24,respectively.However,the swelling property of the microspheres was larger in the alkaline environment,swelling rate was 3.74 and the stability of the microspheres was poor.With the increase of temperature and ionic strength,the swelling rate of microspheres increased,swelling rate was 0.41,0.65 and 0.96 when temperature at 16?,28?,37? respectively.And swelling rate was 9.53 and 13.74 whenionic concentration was 0.3 mol/L,0.6 mol/L respectively.These suggested that the microspheres were more sensitive to pH,temperature and ionic strength of the swelling medium.The microspheres were relatively more stable under alkaline conditions than under acidic conditions,and the same statuation was found in simulated gastrointestinal conditions.It demonstrated that the microspheres to drug targeting transport with a wide range of potential applications.The immunogenicity integrity was confirmed byWestern-blot test and the safety of microspheres were confirmed by safety test.The theoretical basis for drug delivery and safety had be provided in the next animal experiment.3.The immune efficacy of alginate-chitosan Srr-protein microspherevaccineHealthy fish were divided into four groups and immunized Srr,Srr-microcapsules,empty-microcapsules and PBS as control.1-8 weeks after immunization,the serum antibody level was detected by the indirect ELISA method,and serum non-specific index was detected by reagent kit.The healthy channel catfish were challenged with 50×LD5Os.iniae at 4 weeks after immunization,and the relative percent survivals(RPSs)of various groups were calculated.The influence on the immune relativity genes transcription after oral immunizing withs.iniaewere detected by real-time PCR.The results showed thatSrr-microcapsules groupdetectedserum antibody titer from 2 weeks to 8 weeks after immunization and it had a peak value(0.99)at 4 weeks.The Srr-protein microsphere vaccine could produce antibody for a long time.Nonspecific immune index in experimental group were increased,and theSrr-microcapsules groupextremely significant(P<0.01)higher than the control group in a number of indicators.After challenging by S.iniae,the relative percent survivals of the Srr group and Srr-microcapsules group were 35.0%and 60.0%respectively.Real-time quantitative PCR analysis showed that IFN-??TNF-??MHC-???CD4L-2 of Srr-microcapsules group extremely significant(P<0.01)higher than the control group and significantly higher than other groups.Thus the alginate-chitosan microparticles vaccine can effectively enhance the channel catfish immune function,which is a good vaccine to prevent S.iniae disease of channel catfish.