The Effect Of Saponin Adjuvant On Expressions Of Several Important Immune Genes And Non-specific Immune Responses In Turbot,Scophthalmus Maximus

Bacteriosis has long-term been a major cause that threatens the health ofaquaculture fishes. Specific to such disease, the means taken for treatment are mainlyby chemical agents and antibiotics, which will not only allow the pathogenic bacteriato enhance the drug resistance, but also lead to indirect damage to human beings dueto the residual drugs in the fish body for a long time. Fish-oriented vaccine isgradually adopted by modern aquaculture industry to replace the drug antibiotics. Inaddition, the immunity way is restricted because of the special living environment forfishes, so neither injection nor oral drug is seen as the optimal method. Under suchcircumstance, soaking becomes the first choice for fish-based vaccine. In order toenhance the immunization efficiency of the soaking, this paper takes the saponinscreened in advance as the adjuvant, the inactivated vaccine of vibrio anguillarumformaldehyde as the vaccine, and turbot as the fish for experiment, to evaluate theadjuvant features of saponin from two aspects including the non-specific immunityand specific immunity of fishes, thereby attempting to explain the immunitymechanism of saponin adjuvant preliminarily from the perspective of molecularbiology.The first chapter is about three important cytokines which about specificimmunity. In order to evaluate the effect of saponin,which combined vaccination withinactivation V.anguillarum on turbot Scophthalmus.Fish was immersed in seawatercontaining saponin at concentration of0，15and35mg/Lrespectively for1h,and thenbathing in seawater containing vaccine for30min.After immunity we detect thedynamic change of three cytokines which contained MHCⅡα, IL-1β,TNF-α in7groups. As is shown by the result, in the turbot gill tissue of the two groups of saponinadjuvant soaked vaccine groups, the MHC II αgene shows significant expression within4to8h of soaking, whose relative expression level is136.3and378.5. After1d, the expression amount was declined quickly. As for the high-concentrationsaponin and sapponin combined vaccine soaking group which are soaked in the turbotskin for8h, the relative expression amount of MHC II αgene shows a dramaticincrease with the relative expression amount being47.1and41.9. Within3to15d, thelow-concentration saponin (15mg/L) combined vaccine soaking group shows thehighest relative expression amount in the skin MHCⅡα gene, whose expressionamount is57.6. After soaking for1d, the injection control group shows the highestrelative expression of MHCⅡα gene in the spleen, and its high relative expressionamount can last for15d in the spleen. In the spleen, the MHCⅡα gene oflow-concentration saponin combined vaccine soaking group for soaking of3d to15dpresents the secondary highest relative expression amount, followed by the injectiongroup, which is significantly higher than other groups (P <0.05). In the head kidney,on the third day of the soaking, the MHCⅡα gene of the two groups of saponincombined vaccine soaking groups both show high relative expression amount withinsignificant difference (P>0.05). In the injection group, the MHCⅡα gene expressionof the head kidney starts from the third day of soaking, which reaches the peek on thefifteenth day. The relative expression amount of that can be up to6.37, whichissignificantly higher than other groups (P<0.05). Through the analysis on thedifferent parts of skin on the back venter of the turbot, the skin of the lower part ofgill shows the highest MHCⅡα gene expression amount, reaching6.56, followed bythe side line of the tail, which reaches6.31. The antigen presentation effect on theventer is unobvious. Based on the analysis on expression of IL-1β gene in the skin,gill, head kidney, spleen and intestine, the result indicates that the IL-1β expression onthe skin it the highest in the adjuvant combined vaccine group, in which, theexpression of low-concentration group (15mg/L) owns higher expression than thehigh-concentration group (35mg/L). The relative expression amount of the two areboth significantly higher than other groups (P<0.05), being14.01and11.02respectively. The expression trend of IL-1β and MHC Ⅱ α on the skin is the same,and the expression of the former in the gill shows the highest value, which also occurs in the adjuvant combined soaking vaccine groups, reaching21.58and32.58respectively. The highest value of IL-1β expression for the head kidney and spleenboth appears in the4h injection group whose expression amounts are169.53and222.1respectively, which is significantly higher than other groups (P <0.05). In thehead kidney and spleen, the expression of adjuvant combined vaccine group issuperior than that of other groups. For the intestine, the highest value of IL-1βexpression appears in the injection groups whose expression amount is24.51, and thesecond highest group is the high-concentration adjuvant combined soaking vaccinegroup whose expression amount reaches20.02.The results have shown thatpresentation of vaccine antigen in tissues such as skin can be improved significantlywith saponin combined anguillarum vaccine soaking for turbot immunization, whichmay lead to the corresponding inflammatory reaction for the tissue at the same time.The second chapter is about the non-specific immunity.In order to evaluate theeffect of saponin, extracted from Quillaja saponaria, on non-specific immunity ofturbot Scophthalmus maximus，fish was immersed in seawater containing saponin atconcentration of0，15and35mg/Lrespectively for1h. No-specific immune parameterincluding leukocytes phagocytic activity, ACH50and total protein of serum, activityof lysozyme, antibacterial andAKP in serum and skin mucus were analyzed at6、12、24、48and72h after the treatment. The results show that the leukocyte phagocyticactivity in the lower concentration group (15mg/L) at12h and in the higherconcentration group (35mg/L) at24h were significantly higher than that in thecontrol group (P<0.05). ACH50in two treatment groups at each testing time weresignificantly higher than that in control group except the ACH50in the lowerconcentration group at12h and24h (P <0.05). The highest lysozyme activities inserum appeared in the lower concentration group at6h，but no ACH50could bedetected in skin mucus. Antibacterial activity in each group changed with time, but itwere improved obviously in the two treatment groups at6h and12h after theimmersion, also no different between the higher and lower saponin groups (P>0.05).Antibacterial activity in serum was too low to be detected. AKP activity of serum intwo treatment groups were significantly higher than that in the control at6h and12h respectively (P <0.05), furthermore, obvious different were found between the twotreatment groups. AKP activity of skin mucus in two treatment groups weresignificantly higher than that in the control at12h (P <0.05).Serum total protein inhigher concentration group reached the highest value at6h, then came to the samelevel with low concentration group and control group at12h. From24h to72h,serum total protein in higher concentration group was remarkably higher than thelower concentration and control group. The results indicate that saponin couldenhance the non-specific immunity of turbot.