Association Of Chitosan Oligosaccharide And Oil Improves Immune Response Of Vibrio Harveyi FlgE Subunit Vaccine And DNA Vaccine In ♀Epinephelus Fuscoguttatus×♂ Epinephelus Lanceolatus

Vibrio harveyi is the main pathogen of vibriosis in grouper,which often causes deep skin ulcers,gastrointestinal diseases and ocular lesions,leading to high morbidity and mortality of grouper and great economic losses.At present,antibiotics are mainly used to treat vibriosis in grouper culture,but the abuse of antibiotics leads to the emergence of drug-resistant bacteria and drug residues and other ecological problems,affecting the food safety of grouper.Vaccination is an alternative choice to control vibriosis in grouper and reduce the use of antibiotics.Therefore,the development of safe and effective V.harveyi vaccine will have a good market prospect.In this study,the immunogenicity of flagellum matrix protein FlgE of V.harveyi was studied,and the efficacy of the vaccine was further evaluated by adding chitooligosaccharides and oil adjuvants.The research results will lay a foundation for the development of commercial anti-Vibrio vaccine in the future.The results of this study are as following:(1)The flgE gene was cloned from the whole genome of V.harvey with designed primers.Bioinformatics analysis showed that the total length of flgE gene was 1200bp,encoding 399 amino acids,with a relative molecular weight of 41.44ku,theoretical isoelectric point p I of 4.58,and no signal peptide.Subcellular localization predicted that FlgE is an extracellular protein,and the N-C terminal is a FlgE superfamily domain.The amino acid sequence of FlgE was conserved by BLAST analysis,and the homology with other Vibrio species reached more than 85%.The prokaryotic recombinant expression vector of flgE gene was constructed using pET28a vector,namely pET28a-flgE,and transferred into Escherichia coli BL21(DE3)for prokaryotic expression.The optimal time,temperature and Isopropyl-beta-D-thiogalactopyranoside(IPTG)concentration condition of expression was screened.Finally,The recombinant FlgE was overexpressed and purified by using nickel column affinity chromatography,and evaluated by SDS-PAGE and Western blotting analysis.(2)The recombinant protein FlgE diluted with PBS and combined with COS and OIL adjuvants and subunit vaccines were prepared.Five groups of pearl gentian grouper(♀Epinephelus fuscoguttatus×♂Epinephelus lanceolatus)were divided:PBS group(control),FlgE group,FlgE+COS group,FlgE+OIL group and FlgE+COS+OIL.All experimental fishes were intraperitoneal injected with the final protein concentration of25μg/fish,COS of 10μg/fish.The serum of grouper was extracted at 7,14,21,28,35and 42 days after immunization,and the activities of Catalase(CAT),Superoxide dismutase(SOD)and lysozyme(LZM)in the serum were measured.The antibody titer was detected by ELISA.Total m RNA was extracted from liver,head kidney,spleen and thymus tissues,and the transcription levels of CD8-α,TNF-αand My D88 were determined by qRT-PCR.Finally,100μL 5.7076×10~8cfu/m L)of V.harveyi was used to challenge the experimental groupers on the 6 week after immunization and the death was observed within 14 days.The results showed that in the experimental group immuned by vaccine the activity of CAT,SOD,LZM and the titre of Ig M antibody were significantly up-regulated,and there was significant difference between FlgE+COS+OIL and other groups.The qRT-PCR results showed the expression levels of the related immune genes in the immune group were significantly up-regulated compared with the control group,and there was significant difference between FlgE+COS+OIL and other groups.The relative percent survival(RPS)of FlgE group,FlgE+COS group,FlgE+OIL group and FlgE+COS+OIL were 50%,66.6%,66.7%and 77.7%,respectively,which indicated that the recombinant subunit vaccine FlgE could enhance the immune protection of grouper against V.harveyi and the addition of COS and OIL adjuvant could further improve the immune protection of the recombinant subunit vaccine.(3)The eukaryotic expression vector pcDNA3.1(+)-flgE was constructed.Five groups of pearl gentian grouperwere divided:pcDNA3.1 group(control),p FlgE,p FlgE+COS,p FlgE+OIL and p FlgE+COS+OIL.All experimental fishes were intramuscularly injected with the final plasmid concentration of 25μg/fish,COS of 10μg/fish.After 6 w of immunization,100μL 5.7076×10~8cfu/m L)of V.harveyi was used to challenge the experimental groupers and the death was observed within 14 days.The results showed the RPS of p FlgE,p FlgE+COS,p FlgE+OIL and p FlgE+COS+OIL was56%,68%,76%and 88%,respectively.The results showed that the vaccine could effectively improve the activities of CAT,SOD and LZM and Ig M antibody titers of serum in vaccinated groups,and there is significant difference between p FlgE+COS+OIL group and other groups.The qRT-PCR results showed that the expression level of the related immune genes was significantly up-regulated in the vaccinated groups compared with that of the control.Among them,the expression levels of immune genes in plg GE+COS+OIL reached the highest level during the whole experiment.These results indicate that COS and oil adjuvants can enhance the specific and non-specific immunity of nucleic acid vaccine.