Alginate-Chitosan Microcapsules Delivery Of Oral Lactobacillus Casei Expressing VP56_310-500 Vaccine To Prevent Grass Carp Haemorrhagic Disease

The development of grass carp cultivation industry is hindered seriously by hemorrhagic disease caused by grass carp reovirus（GCRV-II）.To prevent the outbreak of grass carp haemorrhagic disease and reduce economic loss,oral vaccines are often used as an effective means of control.Therefore,it is imperative to develop a new GCRV-II vaccine that is effective,safe,and stress-free.Lactobacillus casei is generally recognized as safe（GRAS）food-grade microorganisms,which can maintain microecological balance in gastrointestinal tract and regulate mucosal immune system function.For the development of gene expression system of L.casei,it is regarded as living carriers that present antigens to mucosal sites of host body in the study.And,SA/CS encapsulation is used to improve tolerance.In addition,we also explored the ability of microencapsulated probiotic vaccines to improve the prevention of GCRV-II infection in grass carp.The main findings are as follows:1.VP56_310-500was the optimal fragment against GCRV-IIVP56 is a key outer capsid protein of GCRV-II.In this study,DNA star software was used to predict the antigenicity and hydrophobicity of VP56 fragments,and the gene sequence of the full-length fragment and four truncated fragments(VP56,VP56_1-210,VP56_210-310,VP563_10-500,and VP56_100-209)were amplified and inserted into p PG612.1,the recombinant plasmid was constructed and introduced into L.casei for expression to form r Lcs.Then,by feeding r Lcs mixed with fodder to immunized grass carp,corresponding anti-VP56 fragments of sera was produced,and VP56_310-500was screened as optimal fragment of GCRV-II by adopting enzyme linked immunosorbent assay（ELISA）.2.SA/CS microcapsule can significantly improve the tolerance and colonization ability of probiotic vaccines in the digestive environmentIn this study,SA/CS was used as the encapsulating material of L.casei live vaccine expressing VP56_310-500,thereby preparing oral microcapsule live vaccine SCL-56_310-500.With good resistance to bile（0-0.4%）,Na Cl（0-8%）,simulated intestinal fluid,and simulated gastric fluid（p H=1.5-4.5）,as well as colonization in the intestinal tract.The results showed that SCL-56_310-500had good resistance and colonization ability.Also,it had the ability to maintain high viability after colonizing intestine of grass carp in 7 days.3.Oral SA/CS microcapsule vaccine can effectively improve the immune protection of grass carp against GCRV-II infectionIn addition,Flagellin C（Fla C）was adopted as an immune adjuvant,and r Lc was constructed to express VP56_310-500-Fla C(56_310-500C),thereby enhancing immune effect of oral live vaccine.After oral administration of encapsulated L.casei vaccine,its protective effect on GCRV-II infection was evaluated.As indicated by the results,SCL-56_310-500C effectively up-regulated the activity of serum biochemical enzymes（including TSOD,lysozyme,and complement 3）,and induced m RNA expression of important immune genes（including IL-1β,IFN1,MHC-IIα,Ig M,and Ig T in immune tissues）.Simultaneously,SCL-56_310-500C greatly suppressed GCRV-II replication in head kidney,spleen,and intestine.Correspondingly,grass carp vaccinated orally with SCL-56_310-500C have distinctly higher survival rate（58%versus 24%in control）against GCRV-II infection.In conclusion,SA/CS microcapsules preparation method was developed,and immune effect of vaccine was explored.Subsequently,test was conducted regarding tolerance of encapsulated r Lc to simulative digestive environments and ability of probiotic strains to colonize intestine of grass carp.Afterwards,protective effect of encapsulated L.casei vaccine(SCL-56_310-500C)was evaluated by oral administration.This study provided a feasible strategy for preventing fish viral diseases and promoted application of encapsulated r Lc vaccine in GCRV-II prevention and control in aquaculture.