Proteomics Analysis The Differential Expression Of Outer Membrane Proteins In Aeromonas Hydrophila Under Iron Starvation And Related Immunoprotectivity Evaluation

Aeromonas hydrophila is a typical human-fish-beast disease conditional pathogen that can cause fish to suffer from hemorrhagic sepsis.Each year,due to the bacteria caused by epidemic diseases to the aquaculture industry caused huge economic losses,a serious impediment to the development of aquaculture.In the aquaculture industry,for the disease caused by the disease,the common prevention and treatment measures are antibiotic control,resulting in the growing antibiotics resistant bacteria strains.Due to the rapid development of vaccine-related technology,and its efficient antibacterial or bactericidal effect,it is becoming one of the preventive measures to replace antibiotics.However,there are only a few types of vaccine,especially for the bacteria vaccine,restricting its widespread use.Therefore,it is of great practical significance to find the protective protein with immunogenicity and develop new vaccine,which is of great practical significance to prevent and cure the disease caused by the bacteria and its drug resistance.It has been reported that the outer membrane protein component of bacteria can be used as a candidate protein for fish vaccine in the literature,but it is concentrated on the study of a few outer membrane proteins with high expression in vitro nutrient enrichment medium.The immunogenicity of the outer membrane protein is less studied.The bacteria are often in a local iron deficiency environment when they enter the host,and express a specific outer membrane protein and play an important biological role in competing with the host for iron ions or in the intracellular iron balance.The study of the expression and immunoprotective properties of these related outer membrane proteins in iron deficiency environment is expected to be a candidate protein and provide effective candidate antigens for the development of new vaccines.In this study,we used quantitative proteomics and molecular biology methods to simulate the iron deficiency environment of natural conditions,and analyzed the differential expression of the outer membrane proteins in Aeromonas hydrophilia between normal and iron deficiency medium.A total of 683 proteins were identified by the method of TMT labeling based mass spectrometry.91 of them were up-regulated and 83 were down-regulated including 21 and 10 outer membrane proteins,respectively.In combination with bioinformatics analysis,it was found that most of the down-regulated proteins were ribosomal proteins,and their main function was involved in RNA polymerization.Most of up-regulated proteins were outer membrane proteins such as iron receptor protein,iron pigment carrier receptor and heme receptor.Additionaly,some proteins involved in the synthesis of antibiotics and secondary metabolites.In this thesis,five outer membrane proteins of AHA₀972?A0KGW8?,AHA₄275?A0KQZ1?,AHA₃963?AOKQ46?,AHA₀461?A0KFG8?and AHA₁663?A0KIU8?were selected to verify proteomics results.The corresponding recombinant plasmids were constructed by pET-32a prokaryotic expression vector.The above proteins were successfully over-expressed and purified,and the mice were immunized to obtain the corresponding antibodies,and the titer was above 1:8000.Western blotting analysis showed that these OMPs were low-abundance proteins under normal medium whereas significantly increased under iron stravation in A.hydrophilia ATCC 7966 and virulent strain LP-1.That is consistent with the results of proteomics,indicating that they may play an important role in the iron homeostasis.Finally,in order to test whether the iron-related outer membrane proteins have immunoprotective characters to fish,we immunized zebrafish from the outer membrane proteins of A.hydrophilia ATCC 7966 and LP-1 in normal and iron-deficient conditions,and challenged the attenuated virulent strain LP-1.The results showed that the survival rates of zebrafish immunized with normal and DIP treated OMPs and challenging with A.hydrophilia ATCC 7966 were 60%and 63.3%,respectively,and the relative immunoprotective rates?RPSs?were 53.8%and 57.6%,respectively.The survival rates of zebrafish immunized with normal and DIP treated OMPs and challenging with A.hydrophilia LP-1 were 63.3%and 66.6%,respectively,and the RPSs were 57.6%and 61.5%,respectively.The control group?BSA + adjuvant?,the final survival rate was 13.33%,suggesting that the OMP components in the iron deficiency state were higher than those in the normal state.In addition,the protective effects of five iron related OMPs?A0KGW8,A0KQ46,A0KQZ1,A0KIU8 and A0KFG8?were further evaluated.The survival rate of these proteins was 46.7%,63.3%,63.3%,73.3%and 73.3%,respectively,and the survival rates were 46.7%,63.3%,63.3%,73.3%and 66.7%,respectively while the control group?BSA + adjuvant?was 16.7%;the RPSs were 36%,56%,56%,68%and 60%,respectively.The results indicated that these OMPs have a powerful immunoprotective effect on zebrafish except for A0KGW8 and could be used as candidate proteins for the Aeromonas species vaccine.It is also suggested an effective strategy to use quantitative proteomics technologies for candidate vaccines screening.