Analysis On Characterization Of The Mutant Strain Of Aeromonas Hydrophila By The Tn916 Mutagenesis And The Insertion Sites Of Transposon

Aeromonas hydrophila is an important anaerobic freshwater bacterium that can inflict severe losses to aquaculture and can present a risk of infection not only for territrial and aquatic animals but for human handlers and consumers. The vivulence of the organism may involve several extracellular products including proteases, hemolysins, enterotoxins, acetylcholinesterase, and adhesion, i.e., pilus and a surface array protein layer (S layer). Transposon mutagenesis has been successfully used to study virulence genes in Aeromonas hydrophila.Mutant MJ-1 strain some virulence determinants-deficient was obtained by transposon Tn916 insertion into Aeromonas hydrophila J-l strain. By serially passaged for 20 times, Tn916 was still located in the chromosome of MJ-1 strain stably. Compared with the parent J-l strain, some genes of main virulence determinants, such as exoproteases (caseinase and elastese), amylase, DNase, and hemolysins and S layer, could not be expressed, some biochemical characters, outer membrane protein, extracellular products, cytotoxic effect on EPC cells and adherence to HEp-2 cells of the MJ-1 strain had changed, also, MJ-1 lost its pathogenicity for mice. MJ-1 was deficient in extracellular secretionary virulent factors and cellular surface proteins, which suggested that there might exist pathogenicity island in Aeromonas hydrophila J-l strain.PCR detection of transcriptional regular protein (ahyR) gene from MJ-1 genomic DNA showed that the full length could not be obtained. It is suspected that Tn916 might insert into ahyR gene. To confirm the assumption, the ahyR gene of J-l was inactivated by insertional mutagenesis. According to the relevant nucleotide sequence from GenBank, a pair of specific primers was designed to amplify the partial fragment of ahyR gene from Aeromonas hydrophila J-l strain by PCR. The PCR product was cloned into pMD18-T vector, and after sequencing, ligated into the suicide plasmid pJP5603 which contains a Kan resistant gene to get a recombinant plasmid pJP- ahyR. A mutant strain was obtained after the positive recombinant plasmid was transformed into the J-l strain. On the basis of PCR, an ahyR gene homologous recombinant mutant strain named J-lAahyR wasconfirmed. The main biological characters of J-lâ–³ahyR were studied. Compared with Ah J-l strain, some genes of main virulent determinants could not be expressed, such as proteases, amylase, DNase, hemolysins and S-layer; some biochemical characters, outer membrane protein, extracellular products had changed; also, it had no cytotoxic effect on EPC cells; the pathogenicity capability was greatly decreased and the 50% lethal dose for mice was more than 1.0X 109CFU. The main characters of MJ-1 and J-l â–³ ahyR were identical, which affirmed that Tn916 did insert into the ahyR gene. The other insertion sites of Tn916 will be furthered.Iphophophorus helleri Heckel were immunized with the live attenuated MJ-1. The experiment fish were divided into four groups. Three groups were i.p. infected with various doses of the MJ-1 ( 10⁷, 10⁵,10³CFU/fish ), and the control group were injected with 0.1 mL steril PBS. 30 days after immunization, the levels of immune response (the agglutinating antibody titres and the phagocytic activity) were detected, followed by the challenge test with the i.p. injection of the wild type Ah J-l strain (20LD₅0), The agglutinating antibody titres, phagocytic activity and relative percent survival (RPS) of vaccined fish infected with 10⁷CFU were higher than those in other groups. The results showed that avirulent MJ-1 strain remained good immunogenicity and could provide high protective ability for fish. This article will apply basis for development of attenuated vaccine against Aeromonas hydrophila.