Single-walled Carbon Nanotubes As Delivery Vehicles To Evaluate The Outer Membrane Protein Subunit Vaccine Of Aeromonas Hydrophila

At present,the scale of China's aquaculture industry has reached 70 % of aquaculture industries in the world,and its proportion has increased year by year.It can be seen that the aquaculture industry has a significant impact on China.While the aquaculture industry is developing at a rapid pace,the diseases?the spread and infection of pathogenic bacteria?have caused more and more serious losses to it.Aeromonas hydrophilia is a common opportunistic pathogen that can cause sepsis in fish.The main symptoms are hemorrhagic sepsis,eyelid cellulitis and eyeball rupture,which eventually lead to the death of fish.In addition,it is a human-animal-fish commensal pathogen,and it not only infects aquatic organisms,but even endangers human life and health.Vaccine immunization is an effective way to inhibit the harm of A.hydrophila.At present,the vaccine immunization has problems such as adjuvant,transportation,and immunization methods,which limits the scale of production and use of the vaccine.Therefore,there is urgent need to find effective immunoprotective candidate vaccines and a vaccine delivery adjuvant to control the spread of the disease.Our previously researches have shown that several outer membrane proteins of A.hydrophila with high expression in iron deficiency can significantly enhance the immunity of zebrafish,suggesting that the iron-related outer membrane proteins may be used as a candidate vaccine for bacteria.However,the immune effects of these candidate vaccines may be limited by problems such as adjuvants and immune methods.Therefore,it is a hotspot in the current research to find a more sustainable,stable and efficient adjuvant of fish vaccines.In recent years,single-walled carbon nanotubes?SWCNTs?in nanomaterials can be covalently bound to proteins by functional modification,and the SWCNTs have the advantages of cell permeability,low toxicity or non-toxicity,having a better application in biomedical fields,and having a preliminary application in aquaculture vaccines.It can enhance the efficacy and duration of action of the drug in the recipient by the addition of an adjuvant.At the same time,an efficient and simple vaccine for the preparation of aquatic organism products by using nanomaterials to penetrate the cell membrane effect is a novel strategy for current vaccine development.In order to screen highly efficient vaccines under the bath immune mode for fish of commercial vaccine,we seven proteins localized on the outer membrane of A.hydrophila?six iron-related differential proteins P55870?A0KGK5?A0KIY3?A0KPP0?A0KLQ6?A0KH89 and one outer membrane protein OmpAII as control?were selected from our previous research.High expression and purification of proteins were obtained by prokaryotic recombination,and a chemical modification method was utilized to link proteins and functionalized SWCNTs to prepare a novel SWCNTs-protein vaccine.Combined with bacterial challenge and host immune factor test,we compared the immunoprotective effects of outer membrane proteins and SWCNTs-proteins on zebrafish by intraperitoneal injection and bath immunization,respectively.The research results are as follows:Firstly,The PCR production of AHA₁279?protein name A0KHR9??AHA₁512?P55870??AHA₀854?A0KGK5??AHA₁698?A0KIY3??AHA₃793?A0KPP0??AHA₂699?A0KLQ6??AHA₁098?A0KH89?were obtained from the A.hydrophila ATCC 7966 genome and then combined with prokaryotic expression vector to obtain high expression recombinant bacteria.The obtained high expression protein is collected by ultrasonication,and the proteins were purified by Ni-NTA resin column,then were immunized to rabbits to obtain immune serum.At the same time,we used western blotting to confirm the differential expression of target proteins of A.hydrophila ATCC 7966 and virulent strain LP-2 with or without DIP treatment,the results were consistent with the mass spectrometry.The immunogenicity of the target proteins was further verified using Western blotting with primary mice antibodies against live and inactivated A.hydrophila,respectively.Our results suggest that these outer membrane proteins play an important role in the iron homeostasis and may be promising vaccine candidates against pathogenic A.hydrophila infection.Secondly,SWCNTs-proteins vaccines were prepared by condensing an acylated reaction between the purified protein and the functionalized SWCNTs by chemical synthesis.We select four SWCNT-encapsulated proteins to detect the connection by the Flourier transform infrared spectrometer and the scanning electron microscope.The results show that the purified proteins are tightly and uniformly attached to the surface of the SWCNTs.That indicates the SWCNTs-encapsulate protein samples are successful and can be used in the next experiment.Finally,the iron-related protein vaccines and the SWCNTS-protein vaccines were evaluated via both intraperitoneal injection and bath immunization,BSA+adjuvant and SWCNTs were used as controls.The expression of seven immune-related gene?Lyz,MHCI,MHCII,IL-1?,IL-8,IL-10 and IL-15?was detected by qPCR.For intraperitoneal injection,the protein and SWCNTs-protein vaccine were immunized to zebrafish at four doses?1,2,3,5 ?g?,respectively.It was found that compared with the control group,the SWCNTs-protein can significantly increase the expression of immune-related genes.Further challenge experiments with A.hydrophila homologous strain LP-2 showed that the relative immune protection rates of A0KHR9,P55870,A0KGK5,A0KIY3,A0KPP0,A0KLQ6 and A0KH89 proteins were 61.6 %,42.86 %,47.37 %,57.67 %,94.15 %,65.39 % and 84.96 % at the highest dose of 5 ?g,respectively.The relative immunoprotective rates?RPSs?of immunized SWCNTs-protein vaccines of SWCNTs-A0KHR9,SWCNTs-P55870,SWCNTs-A0KGK5,SWCNTs-A0KIY3,SWCNTs-A0KPP0,SWCNTs-A0KLQ6,and SWCNTs-A0KH89 were 68.10 %?50.00 %?55.56 %?66.67 %?94.44 %?77.50 % and 90.43 % of the relative immune protection rates,respectively.For bath immunization,the protein and SWCNTs-protein vaccine were immunized to zebrafish at four doses?10,20,30,40 mg/L?,respectively.When compared with the control group,the SWCNTs-protein significantly increased the expression of immune-related genes.The further challenge experiments with A.hydrophila LP-2 strain showed that the RPSs of A0KHR9,P55870,A0KGK5,A0KIY3,A0KPP0,A0KLQ6 and A0KH89 proteins were 30.80 %?20.00 %?22.22 %?22.22 %?29.29 %?25.85 % and 37.80 % at the highest dose of 40mg/L,respectively.The RPSs of the immunized SWCNTs-protein vaccines of SWCNTs-A0KHR9,SWCNTs-P55870,SWCNTs-A0KGK5,SWCNTs-A0KIY3,SWCNTs-A0KPP0,SWCNTs-A0KLQ6,and SWCNTs-A0KH89 were 63.60 %?52.94 %?55.56 %?61.11 %?86.11 %?73.74 % and 68.49 %,respectively.These results indicate that the RPSs of A0KPP0,A0KLQ6 and A0KH89 proteins and SWCNTs-encapsulations of these proteins were more than 60 % in the injection immunization,which is higher than the outer membrane protein OmpA control,indicate that they are potential candidate subunit vaccine.In bath immunization,the immune protection of protein is not obvious,and the highest RPS is 37.80 %.After SWCNTs-encapsulated with protein,all the experimental groups showed enhanced RPS when compared with the control group.The effects of SWCNTs-A0KPP0,SWCNTs-A0KLQ6 and SWCNTs-A0KH89 were better,reaching over 68 %.The results showed that the differential outer membrane protein was prepared as a subunit vaccine under the restriction of iron limitation of A.hydrophila,which could have better immune response and RPS in zebrafish.Intraperitoneal injection of zebrafish with SWCNT-encapsulated vaccine can increase the immune response and RPS.The SWCNTs can significantly enhance the efficacy of the subunit vaccine in the bath immunization route,and can effectively alleviate the problem that the subunit vaccine has a lower titer in the bath immunity due to degradation.In summary,the outer membrane proteins A0KPP0,A0KLQ6 and A0KH89 can be used as potential vaccines against A.s hydrophila,and SWCNTs can significantly enhance host immunity by both inject immunization and bath immunization.It is a suitable immersion nanocarrier for vaccine.The above studies provide a theoretical basis for the search for efficient candidate strains of A.hydrophila subunit vaccines and adjuvants.