Cloning, Expression, Immunogenicity Of Major Outer Membrane Protein From Aeromonas Hydrophlia

In this paper, the cloning of major outer membrane protein gene of Aeromonas hydrophila and the immunogenicity of the gene production were discussed. The major outer membrane proteins (MOMPs) of an Aeromonas hydrophila named AHL316 which isolated from diseased eel and the referential strain AhTPS-30 were purified and used for preparing Immunostimulating Complexs (MOMP-ISCOMs). The immunotrial was carried out by injecting European eel peritoneally with 20ug of MOMP-ISCOMs per eel. The immunized eels were challenged with 3 108 cfu and 3 107cfu of TPS-30 forty days post-injection respectively, and the sera from tolerant fishes were collected and analyzed. The results of the experiments showed that immunized European eel could resist to a lethal dose challenge(3 107cfu of TPS-30). The relative protection rate of the fishes immunized with the MOMP-ISCOMs of TPS-30 and L316 were 80% and 100% respectively. And the serum from immune fish could strongly bind to the outer membrane protein of Aeromonas hydrophila on Western-blot. The results demonstrated that the MOMPs were protective antigens and the MOMP-ISCOMs of Aeromonas hydrophila could induce the host to mount satisfied immunity.A pair of primers were designed according to the published nucleotide sequence of a putative outer membrane protein gene (omp) of Aeromonas hydrophila. With the specific primers, a target fragment about 1.1kb was amplified from Aeromonas hydrophila L316 via PCR .The target fragment was inserted into the linearized pGEM-T easy Vector. After enzyme restriction and sequencing analysis, The nucleotide data had been further analyzed by Antheprot 5.0 and ClutalW softwares. The analysis results showed that the cloned DNA fragment had a longest open reading frame (ORF) of 1035nt, it predicted to be encoded a 344-aa protein with the molecular weight of 36kDa. Hydrophobicity analysis suggested that the protein was highly hydrophilic, especialy at the first 24 amino-acid, this region could be function as signal peptide. The homologious comparison proved the cloned gene had 96% homology to the sequence of the omp gene, and the alignment of the amino acid sequence was 98% .the recombinant plasmid was constructed with the target gene and the expressing vector pGEX-4T-l and then was transformed into E.coli BL21 (DE3) The fusion protein was expressed under the IPTG inducing condition, and exhibited about 62kDa in size, very close to the predicted molecular weight of GST-MOMP. furthermore, the fusion protein was specifically recognized by anti-serum which raised against the major outer membrane protein of AhL316. Make all these together, it proved that the cloned gene represented the major outer membrane protein gene of AhL316, and the expressed gene products sharedidentical antigenicity with the natural main outer membrane protein.The studies on preparation and application of MOMP-ISCOMs of Ah L316 provided a new approach to fish vaccinology. The successfully cloning and expressing the major outer membrane protein gene of Ah L316 made it possible to describe this gene's function under a single factor level, and also provided technical support for developing an advanced gene engineering vaccine and subunit vaccine against Aeromonas hydrophila.