This study was focused on the utilization of a recombinant expression system to produce a unique modified subunit vaccine possessing a self-contained delivery system which could potentially improve the uptake and delivery of vaccine products as well their immunogenic potential. For this purpose the A-layer protein (At-R) associated with the fish pathogen atypical Aeromonas salmonicida was cloned and modified by the genetic fusion of the protein transduction domain (MTS) derived from Kaposi fibroblast growth factor (At-MTS). The potential for these proteins to be employed as antigens for oral immunization of goldfish was examined by encapsulation of At-R, At-MTS and the control, BSA, into biodegradable alginate gel macrospheres which were fed to goldfish in place of standard pellet fish feed. The bead physical properties were modified only in the presence of At-R and the temporal release of proteins was significantly less when At-MTS was employed. Western blot analysis of serum samples collected from fish following intubation with the recombinant proteins determined that the rate of protein uptake from the digestive tract into the blood system improved considerably when MTS was fused to At-R. Experimental fish were fed one of three protein–alginate formulae on a schedule of 3 days/week or 5 days/month for a period of 2 months. After 1 month, animals fed on the 5-day protocol demonstrated increased serum antibody titers while following an additional month of feeding this level decreased and titers were found to be higher in fish maintained on the 3-day regime. Fish fed At-MTS maintained the highest titer at the end of 2-month period. To determine whether the diminished antibody titers were a result of oral tolerance fish were injected intraperitoneally with the At-R antigen. Only experimental groups which had been fed At-R or At-MTS demonstrated increased antibody titers which paralleled a typical secondary humoral response. In spite of the presence of an increased titer to A-protein, vaccinated fish did not demonstrate resistance to infection with atypical A. salmonicida.

A recombinant form of the outer membrane protein (A-layer protein) associated with atypical Aeromonas salmonicida was expressed, fused to a cellulose binding domain (CBD) isolated from Clostridium cellulovorans. The resultant chimerical protein was bound to either Sigmacell 20((R)) or Orbicell cellulose particles. Common goldfish were injected intraperitoneally with the cellulose-protein complex and blood serum antibody levels produced against A-protein were examined weekly by means of ELISA. These titers were compared to those induced by immunization of goldfish with the same protein, with or without Freund's incomplete adjuvant, as well as to a standard bacterin-adjuvant system. Small Orbicell beads (1-10 microM) induced antibody levels that were equal to the titers produced by the adjuvanted protein and bacterin formulae. In comparison, the larger Sigmacell particles (10-20 microM) proved to be poor immunopotentiators. The long-term titer elicited from a single injection of A-protein bound to Orbicell beads was equivalent to that induced by two injections. All the vaccinated fish demonstrated memory to the A-layer protein after exposure to a pathogenic load of atypical A. salmonicida with Orbicell treated fish displaying the highest titer. No direct correlation was found between the presence of anti-A-protein antibodies and protection against infection. The paper describes a simple and safe method to increase the potential immunogenicity of soluble recombinant proteins by employing relatively inexpensive cellulose particles.

The 1x myc-tagged cDNA encoding for human CIS2 protein was subcloned into a pET-29a+ vector in order to express and produce a recombinant S-peptide tagged and 1x myc-tagged protein in Escherichia coli BL21(DE3). The constitutively expressed protein was isolated from inclusion bodies by a simple solubilization–renaturation procedure and purified by anion-exchange chromatography on Q-Sepharose. The recombinant form was found to be pure and monomeric as judged by both SDS–PAGE and gel-filtration chromatography and its biological activity was proven by its ability to bind to the tyrosine-phosphorylated cytosolic fragment of human growth hormone receptor fused to glutathione-S-transferase. Recombinant CIS2 was compared by biochemical, immunological, and molecular methods to the CIS2 protein expressed in eukaryotic cells. This report describes the first substantial production of biologically active recombinant human CIS2.

The majority of Israeli fish farms that breed goldfish of the species Carassius auratus have dealt with goldfish ulcer disease since early 1980. During the spring of 1997 the first case of carp erythrodermatitis was observed within the Israeli leather common carp population, Cyprinus carpio (line Dor 70 x Yugoslavia). The disease spread quickly throughout the central and northern regions of the country, with the number of affected farms reaching 37 by summer 1999. Coomassie-positive, slow-growing bacteria were cultured from the wounds of affected fish. The isolates were compared with a strain of atypical Aeromonas salmonicida cultured from Israeli goldfish. This was accomplished by phenotypic and biochemical criteria as well as PCR and SDS-PAGE. Most of the isolates demonstrated an outer membrane protein profile seen in the atypical strain as well as a major 50 kDa outer membrane protein comparable to virulent A-protein of A. salmonicida. The 1400 bp yap gene was also cloned by PCR. Blood serum chemistry demonstrated an acute disease status.

Gilthead seabream (Sparus aurata) growth hormone (gsGH) cDNA coding for the mature protein was cloned in a pGEM-T vector and then transferred into prokaryotic expression vector pET-8 and expressed inE. coliBL21 (DE3) cells upon induction with IPTG. The expressed protein, contained within the inclusion-body pellet, was solubilized in 4.5 M urea, refolded at pH 11.3 in the presence of catalytic amounts of cysteine, and purified to over 98% purity, as evidenced by SDS-PAGE. Gel-filtration on a Superdex column under nondenaturing conditions and partial amino acid N-terminal sequence showed the purified protein to be a monomeric alanyl-gsGH. Over 90% pure bacterial β-lactamase was copurified as a by-product. Binding assays of the [125I]gsGH to gs liver microsomal fraction resulted in high specific binding characterized by aKd= 1.93 nM. Recombinant gsGH, like ovine placental lactogen, exhibited growth-stimulating activity when applied orally toS. auratalarvae or intraperitoneally to juvenile fish.

Achromogenic atypical Aeromonas salmonicida is the causative agent of goldfish ulcer disease. Virulence of this bacterium is associated with the production of a paracrystalline outer membrane A-layer protein. The species-specific structural gene for the monomeric form of A-protein was cloned into a pET-3d plasmid in order to express and produce a recombinant form of the protein in Escherichia coli BL21(DE3). The induced protein was isolated from inclusion bodies by a simple solubilization-renaturation procedure and purified by ion exchange chromatography on Q-Sepharose to over 95% pure monomeric protein. Recombinant A-protein was compared by biochemical, immunological, and molecular methods with the A-protein isolated from atypical A. salmonicida bacterial cells by the glycine and the membrane extraction methods. The recombinant form was found to be undistinguishable from the wild type when examined by SDS-PAGE and gel filtration chromatography. The immunological similarity of the protein samples was demonstrated by employing polyclonal and monoclonal antibodies in ELISA and Western blot techniques. All forms of A-protein were found to activate the secretion of tumor necrosis factor alpha from murine macrophage. To date, this represents the first large-scale production of biologically active recombinant A-protein.