| Disease is one of the important factors restricting the green,sustainable and healthy development of aquaculture.The subunit aquatic vaccines not only have no pollution to the water environment and no residues in aquatic organisms,but also can effectively reduce the pathogenicity risk of pathogenic bacteria.It is the best effective disease prevention and control measure.As high-quality aquatic food,microalgae with high nutritional value can enhance the immune protection of aquatic organisms.The development of recombinant microalgae subunit vaccines has the dual effect of "medicine and food homology",and therefore has broad application prospects in the aquaculture industry.First of all,this article mainly explores the antioxidant and innate immune protection effects of the zebrafish in response to diets including three marine microalgae.The nutrient compositions of three marine microalgae were analyzed.The results showed that Dunaliella salina contained 36.72%carbohydrates,0.51%total chlorophyll and 1.20%total carotenoids of dry weight.Isochrysis sp.contained 1.33%total chlorophyll and 0.91%total carotenoids of dry weight.The total fatty acid content of Phaeodactylum tricornutum and Isochrysis sp.were 60.77 μg/mg DCW and 76.85 μg/mg DCW,respectively.The DHA content of Isochrysis sp.was 8.57 μg/mg DCW.The zebrafish were fed with different diets for 30 days.Compared with control group,the relative percent survival of the D.salina and Isochrysis sp.group challenged with Edwardsiella tarda EIB202 were 17.39%and 26.09%,respectively.The relative expression levels of the antioxidant factors Cat and GSH-PX in the Isochrysis sp.fed group increased by 1.5-2.5 times.The Isochrysis sp.group activated the TLR2 signaling pathway,which relied on the MyD88 pathway to activate the signal transcription factor NF-κB and promoted the expression of cytokines.The relative expression of defensins in Isochrysis sp.group had also increased by 1.26 times,which helped to strengthen the innate immune defense system.The above results showed that the Isochrysis sp.was promising as an excellent objective for the development of recombinant microalgae subunit vaccines.Secondly,based on the key epitopes GAP345 and FBA4 of glycolytic housekeeping enzymes GAPDH and FBA in Edwardsiella tarda,recombinant plasmids were constructed in different combinations such as single,multiple repeats,and combinations to induce protein expression,identification and purification.As a result,2GAP345,3FBA4,GAP345-FBA4 recombinant proteins were successfuly expressed.3FBA4 was soluble protein,and 2GAP345,GAP345-FBA4 were inclusion bodies.These proteins could lay the foundation for the next step of protein immunization.Finally,in order to better simulate the infection mode of pathogens in natural water,Vibrio anguillarum was used to soaking challenge.The relative percent survival of recombinant epitope peptides 2GAP345,3FBA4,and GAP345-FBA4 were 85.00%,50.00%,and 35.00%,respectively.It could be seen that epitope peptides had immune protection.At the same time,the relative expression of MHC I in zebrafish in the 2GAP345 immunization group was significantly increased,which promoted the expression of IL-1β by two times.The 3FBA4 protein immunization group simultaneously activated the TLR2 and TLR4 signaling pathways,relying on MyD88 to increase the expression of NF-κB by 1.8 times,as well as relying on MHC II and CD4+for antigen presentation.Also,TNF expression increased 5.51 times,and defensin expression increased 14.35 times.They significantly promoted innate immunity and acquired immunity,and enhanced its protective ability.The research in this article has successfully screened the key epitope recombinant proteins(2GAP345,3FBA4)based on housekeeping enzymes in the glycolytic pathway,which can be used as candidate proteins for the development of aquatic vaccines.In the future,these are used for heterologous expression in Isochrysis sp.and other marine microalgae hosts,in order to,prevent and control bacterial aquatic diseases. |
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