Analysis The Major Adhesin Gene Sequences And It's Recombinant Gene Products Function Of Aeromonas Hydrophila

In order to deeply investigate on sequence difference of major adhesin genes among the different Aeromonas hyrophila strains, as well as to understand the function of the major adhesin and its role in invading and inducing host producing protective immunity, the major adhesin genes of 6 Aeromonas hydrophila strains were cloned and sequenced. At the time, the expression condition of the recombinant gene of the major adhesion from Aeromonas hydrophila ZN1 strain was optimized and some biological function of expression products were identified and analyzed. The results were as follows:The analytic results of nucleotide and amino acid sequence demonstrated that the homology range of nucleotide and amino acid sequence of 6 Aeromonas hydrophila strains were 35.6%~99.6% and 34.2%~99.5% respectively; the homology of nucleotide and amino acid sequence between BL①and BL④from the same type and the same source were 39.8%~99.6% and 34.2%~99.5% respectively; the homology of nucleotide and amino acid sequence between BL①and TPS-30 from the different type and the different source were 98.1% and 42.3% respectively; in addition, amino acid sequence of the major adhesion from strain ZN1 (serotype O: 10501, isolated from European eel) was quite different from other strains, the homology range was 35.6~63.6%. These results suggested that correlation of nucleotide and amino acid homology of the major adhesion gene among Aeromonas hyrophila strains to the source of strains and serotype was not strict, but showed some diversity.The optimizing results of recombinant expression of the major adhesion gene showed that: the recombinant Ecoli BL21 (named ZAPB) of the major adhesin gene (Mah) from Aeromonas hydrophila ZN1 strain enriched for 4hrs or 5hrs expressed better than enriched for 2hrs or 3hrs before induction; the expression results was mostly similar at different concentration of IPTG (0.1mmol/L,0.5mmol/L,1.0mmol/L,1.5mmol/L); the expression results of induced for 2hrs or 3hrs was better than induced for 4hrs or 5hrs; and the expression results of induced at 27℃were better than induced at 30℃,33℃or 37℃; Beside, it could not improve the effect of expression by increasing the oxygen partial pressure or regulating the medium pH (with 6.0 or 8.0) or inducing at low-temperature and for delay, and the expression products are mainly in the form of inclusion bodies. The results show that the optimal expression conditions of ZAPB were as follows: the rotational speed was 200r/min, enriching bacteria for 4hrs before inducing and the OD600 of the bacteria were between 0.6 and 0.8, the concentration of IPTG was 0.5mmol/L, and inducing expression was at 27℃for 3hrs.The analytic results of immunological characteristics and function of the expression product of the genes recombination showed that: (1) The enzyme-linked immunosorbent assay indicated that the antibody titers of rabbit anti-ZN1-OMP (outer membrane protein of Aeromonas hydrophila ZN1 strain) serum to expression products Mah-TrxA (the recombinant protein of thioredoxin and major adhesin) and ZN1-OMP were 1:3200 and 1:12800 respectively; the antibody titers of rabbit anti-Mah-TrxA serum to Mah-TrxA and ZN1-OMP were 1:12800 and 1:1600 respectively, this showed that it had similar immunological characteristics between the expression products and the natural OMP. (2) The relative adhetion rate of Aeromonas hydrophila from fish ZN1 strain and BL①strain (serotype O:Ah10501),TPS-30 strain and Ah9805 stain (serotype O:9),CQ-3 strain (serotype O:CQ-1) to epithelioma papillosum cells of carp (EPC) was up to 97%; and the adhetion rate of Aeromonas hydrophila ATCC7966 strain which wasn't from fish was 78.5%. (3) The results of adhesion inhibition and blocking tests show ,the average number of adhesive bacteria of the different Aeromonas hydrophila adhered to EPC (which was pre-incubated with Mah-TrxA )was between 0.3 and 3.9 (less than 10); the average number of adhesive bacteria of the different Aeromonas hydrophila (which was pre-incubated with rabbit anti-Mah-TrxA serum) adhered to EPC was between 0.3 and 3.7 (less than 10). These results showed that the Mah-TrxA had cross-inhibition effect on different Aeromonas hydrophila, and the different Aeromonas hydrophila adhered to EPC were decreased significantly by antigen competivive inhibition and antibody blocking, it was confirmed that the cloning and expression Mah-TrxA was really the major adhesin of ZN1 strain by forward and reverse side; and it was also proved that there were similar biological activity between Mah-TrxA fusion protein which was expressed by genetic engineering technology and the major adhesin which was expressed by wild Aeromonas hydrophila ZN1 strain.In summary, the study compared the nucleotide and amino acid sequence homology of major adhesin genes of 6 different Aeromonas hydrophila, elucidated that the major adhesion gene homology of Aeromonas hyrophila strains didn't have strict correlation with the source of strains and serotype, and verifid that Mah-TrxA was able to adhere to fish cell, and proved that the Mah was the major adhesin genes of Aeromonas hydrophila ZN1; the results of cross-inhibition test show that the Mah-TrxA fusion protein was able to inhibit the Aeromonas hydrophila from the different serotype and the different source to adhere to EPC, which suggested that the adherent sites of the different Aeromonas hydrophila had a certain conservation. Perhaps, this discovery would provide a new idea for prevention and control fish disease of the Aeromonas hydrophila.