Cloning, Expression And Characterization Of The Major Adhesin Gene Of Aeromonas Hydrophila

Abstract: A major adhesin gene (Mah) was amplified from Aeromonas hydrophila ZNl strain which was isolated from diseased European eel.The DNA sequence analysis showed that the Mah had a longest open reading frame (ORF) of 1074 nt, encoding a 357-aa protein with the molecular weight of 39．606kDa, with a twenty two highly hydrophobic amino-acids as signal peptide located on its' N-terminal. The homologous comparison proved that the Mah had about 70% homology with the other Mah sequences of Aeromonas hydrophila stains . The cloned gene was assignated to a new sub-group of Mah gene in phylogenetic tree. The recombinant plasmid was constructed with Mah and the expressing vector pET-32a, which was then transformed into E.coliDE3(BL21) . The Mah-TrxA fusion protein was efficiently expressed in E.coliDE3(BL21) under the IPTG inducing. Further identifying and analysing the expressed gene products, the results showed that: (1) ELISA indicated that the major outer membrane protein of Aeromonas hydrophila ZN1 strain (ZN1-MOMP) and Mah-TrxA fusion protein were both recognized by mouse anti-ZN1-MOMP serum, the antibodies titers were up to 1:12800 and 1:3200 respectively; Moreover, the mouse anti-Mah-TrxA fusion protein serum could also bind to ZN1-MOMP and Mah-TrxA, and the antibodies titers reached to 1:1600 and 1:12800 respectively. (2) Western-bloting probed with mouse anti-ZN1-MOMP serum showed the predicted positive bands both on Mah-TrxA fusion protein and ZN1-MOMP samples. Furthermore, major adhesin of Aeromonas hydrophila ZN1 strain and other hetero-serotype Aeromonas hydrophila strains were all recognized by mouse anti-Mah-TrxA serum. (3) Adhesive assay indicated that ZN1-MOMP and Mah-TrxA fusion protein were able to inhibit the adhesive fuction of the bacteria and the rabbit anti-Mah-TrxA serum could significantly block the ZN1 to adhere to EPC. (4) Immunotrial demonstrated that European eels intraperitoneally injected with Mah-TrxA could induce protective immunity. When challenged the immunized fish with virulent Aeromonas hydrophila (l×10~7 cfu per fish) at day 30 post-injection, the relative protection rate was as high as 87．5%. Based on the above results, the conclusion could be made as following: (1) Mah-TrxA fusion protein shared thesimilar biological characteristics and function with native major adhesion; (2) themajor adhesin could be the common antigens among the different Aeromonashydrophila stains;(3) Mah-TrxA fusion protein could stimulated European eels toproduce protective immunity which indicated that the major adhesin could be theprotective antigen in Aeromonas hydrophila.Therefore, the successful cloning and expression the major adhesin of Aeromonas hydrophila, and systematically analyzing the gene products' biological characteristics, makes us more understanding to major adhesin and its role in invading and inducing host producing protective immunity, the research data would provide a valuable information for more efficient control of the disease caused by Aeromonas hydrophila.