Construction And Characterization Of AopB~- AopD~- AroA~- Triple Deleted Mutant Of Aeromonas Hydrophila

Aeromonas hydrophila is a zoonotic pathogens,is also a food-bome pathogens.Aquatic bacterial septicemia is caused by Aeromonas hydrophila which induces high incidence of a disease and mortality in aquatic animals each year.It endangers the most aquatic species and scopes and different stage of fish and breed aquatic area.As an acute infectious disease,aquatic bacterial septicemia brings huge economic loss.At present,the extensive use of antibiotics and immunization are main methods to prevent and control it in acquculture.The current dominant commercial vaccines are still killed whole cell bacterins and subunit vaccines,which generally reduce the mortality of A.hydrophila infection.However those vaccines frequently failed to prevent severer morbidity and economic losses.Above all,it's very urgent to develop a safe,effective,inexpensive and convenient vaccine to control A.hydrophila.In contrast,the attenuated live vaccines could re-present protective antigens and stimulate a lasting immune response that may be more efficacious in preventing the disease.Therefore,studies of A.hydrophila vaccines tend to focus on live attenuated vaccines constructed by inactivating virulence-associated genes.Based on above considerations,this study was aimed to develop a safer,effective vaccine strain and exploit A.hydrophila as a live vaccine.Ah2056 strain was used as the parent strain.The aopB/aopD(encoding A.hydrophila out membrane protein B,D) gene was deleted in the Ah2056 genome to decrease virulence and remove the risk of virulence return.Then the aroA gene(encoding 5-enolpyruvyl shikimate 3-phosphate synthase) was deleted in the aopB^-/aopD^-to construct the aopB^-/aopD^-/aroA^- strain for removing the risk of virulence return again.This aopB^-/aopD^-/aroA^-strain was lower virulence and aromatic amino acids dependent for removing the risk of virulence diffusion.The main research was described as follows:1.Construction of aopB^-/aopD^- mutantAccording to the GenBank sequences of A.hydrophila,the upstream and downstream of aopB/aopD gene were respectively amplified from Ah2056 genome,subcloned into suicide plasmid pRE112.The recombination suicide plasmids were designated as pREaopB /aopD which contained 369bp-deleted aopB/aopD and sucrose sensitive(sacB) gene. The aopB^-/aopD^-mutant,from which aopB/aopD gene was deleted,was constructed first. The E.coil donor strain X7213 transformed with the suicide plasmid pREaopB/aopD was conjugated with the recipient strain,the wide-type Ah2056 strain.After transconjugation,Chloramphenicol-resistant(Cm^R) and sucrose-sensitivity(Suc^S) transconjugants in which the whole plasmid had been incorporated into the recipient chromosome were analyzed by PCR.Colonies with the correct PCR profile were incubated in LB.Aliquots were then plated on to NA plates containing 10%(w/v) sucrose. Sucrose-resistant(Suc^R) colonies were tested for the chloramphenicol-sensitivity(Cm^S) phenotype,which was indicative of loss of plasmid vector sequences.Following this, Sue^R and Cm^S colonies were identified using PCR to determine the presence of the second crossover.2.Construction of aopB^-/aopD^-/aroA^- mutantThe upstream and downstream of aroA gene were respectively amplified from Ah2056 genome according to the GenBank sequences of A.hydrophila,subcloned into suicide plasmid pSUP202.The recombination suicide plasmids were designated as pSUParoA which contained 1290bp-deleted aroA and gentamycin resistance(aacCl) gene.The aopB^-/aopD^-/aroA^- mutant,from which aroA gene was deleted,was constructed first.The E.coil donor strain S17-1 transformed with the suicide plasmid pSUParoA was conjugated with the recipient,the aopB^-/aopD^- mutant.After transconjugation,Chloramphenicolresistant (Cm^R) and gentamycin-resistance(Gm^R) transconjugants were analyzed for the presence of a first crossover event.Colonies were then plated on to NA plates containing gentamycin.Gentamycin-resistance(Gm^R) colonies were tested for the chloramphenicol-sensitivity(Cm^S) phenotype,which was indicative of loss of plasmid vector sequences.Following this,Gm^R and Cm^S colonies were identified using PCR to determine the presence of the second crossover.3.Biological characteristic of aopB^-/aopD^-,aopB^-/aopD^-/aroA^- mutantBiochemical assays of aopB^-/aopD^- mutant was the same as those of Ah2056,while AhaopB^-/aopD^-/aroA^- mutant failed to ferment maltose,sucrose,xylose and produce urea, arginnine decarboxylase,lysine decarboxylase.The exoenzyme activities analysis of revealed that aopB^-/aopD^- and aopB^-/aopD^-/aroA^-mutant could not produce the detectable exoproteases,haemolysin,amylase and Dnase, while Ah2056 had a high level of exoenzyme activities.The cultural supematant of aopB^-/aopD^-,aopB^-/aopD^-/aroA^- were inoculated in EPC cell culture,and the result indicating that lost the toxigenicity to EPC cell.The cultural supematant of Ah2056 had highly the toxigenicity to EPC cell,which 10^-3-10^-4μl cultural supernatant could restrain the growth of 50%EPC cell.The growth curve in LB of aopB^-/aopD^-/aroA^- showed that the mutant grew significantly more slowly than aopB^-/aopD^- and Ah2056 did,and didn't grow after incubation for 8-9h.The growth curve in M9 of aopB^-/aopD^-/aroA^- showed that the mutant didn't grow,while aopB^-/aopD^- and Ah2056 did.The results suggest aopB^-/aopD^-/aroA^- was lower virulence and aromatic amino acids dependent. The i.p LD₅₀ value of Ah2506 and aopB^-/aopD^- mutant were determined to be approximately 1.0×10⁵ and 9.7×10⁷ CFU,respectively.The virulence of aopB^-/aopD^-mutant in Carassius auratus gibelio reduced about 1000 times than parent strain. The i.p LD₅₀ value of AhaopB^-/aopD^-/aroA^- mutant was higher than 1.0×10¹⁰,which lose its virulence.4.The immune and protective assay of the intraperitoneally immunized with aopB^-/aopD^-,aopB^-/aopD^-/aroA^- mutants in Carassius auratus gibelio100-150g Carassius auratus gibelio were intraperitoneally immunized with the aopB^-/aopD^-,the aopB^-/aopD^-/aroA^- mutant and the killed cell vaccine at the same dose of 1.0×10⁸CFU.ELISA was used to assay serum antibodies tirtre to A.hydrophila by coating with A.hydrophila sonicated antigen.The results showed that the serum antibodies tirtre of all groups were detect at week after intraperitoneally immunization. The antibody level for killed Ah2056 group was the highest at week 3 after intraperitoneally immunization,and then to descent gradually.The antibody level for the aopB^-/aopD^-,aopB^-/aopD^-/aroA^- mutant groups were both higher than those of the killed cell vaccine group at week after oral immunization,and then to increase gradually,which were keeping a highly serum antibodies tirtre.Thirty-five days later,the serum antibodies tirtre of aopB^-/aopD^-mutant group was 2¹².Carassius auratus gibelio were intraperitoneally immunized with the aopB^-/aopD^-, aopB^-/aopD^-/aroA^- mutant and the killed cell vaccine at the same dose of 1.0×10⁸CFU. Thirty-five days later,Carassius auratus gibelio were challenged i.p.with 100×LD₅₀ of Ah2056(O:9) and AhJ-1(O:5).Relative percent survivals(RPS) of intraperitoneally immunized mutants were higher than those of the killed cell vaccine,and were 66.7%, 63.3%respectively.With the hige dose challeng,still offered Carassius auratus gibelio 63.3%-66.7%protection against homologous serovar and 60%protection against heterogenous serovar.The results suggest that intraperitoneally immunized with aopB^-/aopD^- or aopB^-/aopD^-/aroA^- is better than the killed cell vaccine.5.The immune and protective assay of the orally immunized with aopB^-/aopD^-, aopB^-/aopD^-/aroA^- mutants in Carassius auratus gibelio100-150g Carassius auratus gibelio were orally immunized with the aopB^-/aopD^-,the aopB^-/aopD^-/aroA^- mutant and the killed cell vaccine at the same dose of 1.0×10¹⁰CFU. ELISA was used to assay serum antibodies tirtre to A.hydrophila by coating with A. hydrophila sonicated antigen.The results showed that the serum antibodies tirtre of all groups were detect at week after oral immunization.The antibody level for killed Ah2056 group was the highest at week 2 after oral immunization,and then to descent gradually. The antibody level for the aopB^-/aopD^-,aopB^-/aopD^-/aroA^- mutant groups were both higher than those of the killed cell vaccine group at week after oral immunization,and then to increase gradually,which were keeping a highly serum antibodies tirtre. Thirty-five days later,the serum antibodies tirtre of aopB^-/aopD^- mutant group was 2⁷.Carassius auratus gibelio were orally immunized with the aopB^-/aopD^-,the aopB^-/aopD^-/aroA^- mutant and the killed cell vaccine at the same dose of 1.0×10¹⁰CFU. Thirty-five days later,Carassius auratus gibelio were challenged i.p.with 100×LD₅₀ of Ah2056(O:9) and AhJ-1(O:5).Relative percent survivals(RPS) of orally immunized groups were lower than those of intraperitoneally immunized groups respectively.Both aopB^-/aopD^- and aopB^-/aopD^-/aroA^- group had the same of RPS of orally immunized groups.With the hige dose challeng,offered Carassius auratus gibelio only 46.7% protections against homologous serovar and 50%protection against heterogenous serovar. The results suggest that orally immunized with aopB^-/aopD^- or aopB^-/aopD^-/aroA^- is inferior to the killed cell vaccine.6.Expression of A.hydrophila out membrane protein in Bacillus subtilisWith the technology of PCR,the promoter P43 was amplified from B.subtilis total DNA,the outer membrane protein gene ompTS was amplified from A.hydrophila total DNA without its signal peptide-encoding sequence.The PCR productions were sequenced, digested and cloned into the corresponding site of an E.coli-B.subtilis shuttle vector pNW33N to generate the shuttle expression vector pNWP43omp.The recombinant vector was transformed into B.subtilis BS01.BS01 cells harboring pNWP43omp expressed the outer membrane protein,which were confirmed by SDS-PAGE and western blot.In conclusion,the shuttle expression vector pNWP43omp were successful constructed. Under the control of the promoter P43,the ompTS gene were expressed in B. subtilis.There is a good prospect for the application of B.subtilis expression system in aquaculture.