Design,Expression And Immunoprotection Effect Of The Recombinant Tandem Epitope Antigen Of Actinobacillus Pleuropneumoniae

Porcine contagious pleuropneumonia(PCP)caused by Actinobacillus pleuropneumoniae(APP)is a contagious respiratory disease,which brings great economic loss for the pig industry in the worldwide.PCP is often mixed with other diseases or secondary infection,making the disease more complex,and immune prevention becomes an important way to control PCP.However,the APP is divided into 16 serotypes,and the lack of cross-immune protection between major serotypes leads to slow progress in vaccine research.APP vaccine research mainly focus on the subunit structure,which has complex structure,large molecule,few effective antigen epitopes on the molecular surface.Few studies have been reported on the APP epitope-based vaccine,and the studies are rare about the changes of the cytokines after immunization of the epitopes.Therefore,it is of great significance for prevention PCP to design an APP epitope vaccine and study the changes of cytokines after immunization and challenge.Previous study of our group found that the head of trimeric atuotransporter adhesin(TAA)of APP,named ADH,was the key functional area and played an important role in the bacterial adhesion,invasion,the formation of biofilm and evasion of the host immune responses,and also,ADH had immunological protection to the infection of APP.In addition,our group found that strong cross-immune protection existed between the Propionibacterium acnes(PA)and APP,and their common antigen epitope,named Ba1,Bb5 and C1,could effectively prevent the infection of APP.Moreover,intensive study found that the epitope PH1 on exotoxin Apx? produced by APP and PH2 on high affinity cytoplasmic zinc transporter of APP had high homology with single-stranded DNA binding protein of PA,and brought a good immune protection effect against APP infection.In order to research the immune effect of epitope antigen and the impact of cytokines on the immune effect,this experiment predicted effective antigen epitopes of ADH by DNAstar and Bepipred 1.0,who was linked with the Ba1,Bb5,C1,PH1 and PH2 by means of flexible linker,after which multiple different sequences were compared to get the optimal combination sequence.The sequence gene was synthesized and used to construct the prokaryotic expression vector p ET28a-RTA,and then,the target protein RTA was expressed in E.coli BL21 and purified by nickel column affinity chromatography.The RTA protein was mixed with aluminum adhesive brine adjuvant at 0,14,28 d,which was used to subcutaneously immunize the ICR mice in the back.At the same time,the control groups were set,including the APP5 b viable bacteria,APP1 inactivated bacteria,APP5 b inactivated bacteria and PBS group.The blood was collected to detect the serum antibody titer through tail vein at 0,14,28,35 d.Two RTA immune groups were intraperitoneally treated with IL-2 and intranasally treated with GM-CSF at 24 h and 0 h before challenge with APP1 and APP5 b at 35 d,respectively.The clinical symptoms and weight loss of mice were observed after challenge,and three days later,mice were killed.The lung index,the number of T cells in the lung homogenate,survival rate and the level of cytokines in BALF and serum were detected to evaluate the immune protection effect,the impact of RTA protein on cytokines,and the effect of pretreatment with IL-2,GM-CSF on protection effect of RTA protein.Results showed that the RTA was a 20.6 k Da protein,and its optimal expression condition was induced for 6-8 h at 37?.Western blot result showed that RTA protein had good binding activity with anti-APP5 b hyperimmune serum.After the RTA protein was immunized,the serum antibody titers of ICR mice reached 103 orders of magnitude,and the antibodies could specifically bind to APP1 and APP5 b.RTA protein could effectively reduce the clinical symptoms and weight loss after challenge,and the lung index in IL-2 and GM-CSF pretreatment groups were slightly higher than that of the RTA group.In addition,the pretreatment of IL-2 reduced the number of T cells in the lung homogenate,and GM-CSF had no influence on the number of T cells.The immunization of RTA protein provided 40% survival rate for mice against APP1 challenge.It was indicated that RTA protein had certain protective effect on APP infection,but pretreatment with IL-2 and GM-CSF decreased the protection effect of RTA protein.Moreover,the levels of IL-17,IL-1?,Fas-Ligand,IL-21,G-CSF and CCL4 in the RTA immune group were decreased,which showed that RTA immunization could effectively reduce the degree of inflammatory response.Compared with the RTA group,IL-2 and GM-CSF pretreatment increased the levels of inflammatory factors,including TNF-?,IL-1?,IL-21,G-CSF,GM-CSF,CCL4,which increased the inflammatory response and decreased the immune protection effect of RTA protein.The epitope antigen RTA was designed in this study,and its immune protection effect was evaluated through clinical symptoms,weight loss,survival rate,cytokines change and other index,which provides a theoretical basis for a new APP epitope-based vaccine design.This is of great significance for the prevention of APP infection.