Construction Of AopB~-/aopD~-Ah2056 Strain Of Aeromonas Hydrophila

Aeromonas hydrophila is a pathogenic bacterium of anthropo zoonosis and also a common Food-borne pathogen.The bacterial septicemia of Aquatic animales caused by Aeromonas hydrophila had high incidence rate and mortality,and brought huge economic loss in aquaculture each year.At present,the main methods to prevent and control is extensive use of antibiotics and immunization with Inactivated vaccine. The immunization with inactivated vaccine is generally reduced the mortality,however failed to prevent severer morbidity.Thus,it's urgent to develop a safe,effective,inexpensive and convenient vaccine to control the disease by A. hydrophila.In contrast,the attenuated live vaccines could re-present protective antigens and stimulate a lasting immune response that may be more efficacious. Therefore,it is necessary that the live attenuated vaccines of A.hydrophila was constructed by inactivating virulence-associated genes.In this study,Ah2056 strain was used as the parent strain and aopB~-/aopD~-Ah2056 strain was constructed.The results as following:1.Identification of Ah2056 parent strainIn accordance with 16SrRNA sequences of A.hydrophila in GenBank,16 SrRNA fragment from the genome of Ah2056 was amplified.It was insected to the vector pMD18T.After screened positive cloning and sequencied,the analysis of their.species classification was made.2.Construction of recombination suicide plasmidsThe upstream and downstream sequences of aopB/aopD gene were respectively amplified from Ah2056 genome according to the GenBank sequences of A. hydrophila,subcloned into suicide plasmid pRE112 with pBluescriptâ…¡SK as transfer vector.The recombination suicide plasmids were designated as pREaopB~-/aopD~-which contained 369bp-deleted of aopB/aopD gene and sucrose sensitive(sacB) gene.3.Screening and identification of aopB/aopD gene deletionThe pREaopB~-/aopD-was conducted into the E.coil X7213 first.The positive E.coil X7213 was conjugated with the Ah2056 strain.It was designated as pREaopB~-/aopD~- Ah2056.The chloramphenicol-resistant(Cm~R) and sucrose-sensitivity(Suc~s) were used to selected positive clone and PCR was used toanalyze insected fragment into the recipient chromosome and loss of plasmid vector sequences.