Abstract:
An aquatic subunit vaccine comprises an antigenic fusion protein and suitable carrier or adjuvant. The antigenic fusion protein sequence consists from its amino terminus to carboxyl terminus of a receptor binding motif and a translocation domain of  Pseudomonas aeruginosa  exotoxin A and has the amino acid sequence of SEQ ID No: 8; a viral antigenic protein affecting fish disease; and a signal peptide having the amino acid sequence of SEQ ID No: 10.

Description:
BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     The invent relates to aquatic subunit vaccine and in particular, to an aquatic subunit vaccine prepared based upon recombinant DNA technology by fusing a fish antigenic protein with the receptor binding motif and the translocation domain of an exotoxin A, as well as with the signal peptide of KDEL. 
     2. Description of the Prior Art 
     Infectious Pancreatic Necrosis (IPN) is a worldwide highly contagious fish disease. Infectious Pancreatic Necrosis Virus (IPNV), the pathogen of IPN, belongs to the family of Birnaviridae. The genome of IPNV consists of two segments of double-stranded RNA (dsRNA), segments A and B. The virion is a non-enveloped icosahedron particle. The length of the A segment gene is 3.2 Kb and codes predominantly for primary virus binding protein (VP2), secondary virus binding protein (VP3), and enzyme protein (VP4). 
     The major hosts of Infectious Pancreatic Necrosis Virus are salmon and trout. Other economical fishes and shells like eels, barracudas, cods, tilapias, pond loaches, milkfish, and clams, had be found to be infected by IPNV. IPNV affects primarily the young fish of salmon and trout in the initial feeding stage. The young fish in the weeks of initial feeding stage and later in the freshwater stage are vulnerable to IPNV infection. Nevertheless, in the mid 1980&#39;s, indications of IPNV disease outbreaks in post-smolt salmons held in seawater had also been reported. IPNV can be traced in the primary Atlantic salmon aquaculture countries such as Chile, America, and Norway. In Norway, IPN is one of the most serious contagious fish diseases in the salmon aquaculture industry. IPNV can cause a mortality of near 100% in freshwater stage of salmon. In post-smolt salmons, the mortality of IPNV may range from 10-20% up to 70%. Therefore, IPNV is a severe threat, ecologically and economically, to the aquaculture and sea-farming industries. 
     Vaccination against IPNV is the major control approach used in the salmon aquaculture industry. Conventional aquatic vaccines against IPNV includes mainly two types, the pathogen killed vaccine and the subunit vaccine. The development of pathogen-killed vaccine requires cell lines non-infected by other viruses. However, the fish cell lines are uncommon and the options are rare in addition to its difficulty in development as well as the high cost therefore. Subunit vaccine can be manipulated by genetic engineering technology. VP2 protein of IPNV expressed by  E. coli  or structure proteins of IPNV expressed by Insect Baculovirus are two common types of IPNV subunit vaccines that can induce the specific antibody responses in salmons. However, protective immunological effects offered by those approaches described above can not satisfy the standard required in the aquaculture industry. 
     There are several theories as to why a subunit vaccine against viruses can not induce a protective immunity. Concepts that have several proponents are as follow:
         1) the antigen can not properly targeted to the cell that plays the key role in inducing immunity;   2) the antigen may be trapped in exogenous routes and not transported into cytoplasm of a target cell to associate with its endoplasmic reticulum; and   3) as a consequence of the foregoing, the antigen may not be presented to the effector cell in the correct context.       

     Accordingly, there are a lot of drawbacks associated with conventional aquatic vaccines and improvements thereof have to be improved urgently. 
     In recent years, as rapid development of biotechnologies, genetic engineering approaches are utilized to modify and alter genes, thereby microorganisms originally harmful to human being or animals can be transformed into beneficial tools in medical and agricultural application. For example,  Pseudomonas aeruginosa  is one of typical examples among these organisms. When a patient with cystic fibrosis is infected by  Pseudomonas aeruginosa  may develop often chronic respiratory tract infection followed with chronic pneumonia. As cancer patients with immunodeficiency or patients severely burned are infected by  Pseudomonas aeruginosa  tend to turn into acute pneumonia or sepsis. The main factor in causing diseases to infected patients is the exotoxin A produced by  Pseudomonas aeruginosa.    
     The protein structure of exotoxin A consists of three major functional domains. Domain I of exotoxin A is a receptor-binding domain that is responsible for binding with α2-macroglobulin/LDL receptor on the plasma membrane of a mammalian cell to form an internalized ligand-receptor complex and then enters into the endosome of the cell through endocytosis. Where it will be localized and processed by proteases within the endosome. After enzymatic cleavage of exotoxin A in endosome, truncated protein fragments containing domain II (the translocation domain) and the toxic domain III are released and further translocated under the action of the translocation domain II from endosome into cytoplasm where it will bind with Golgi body and endoplasmic reticulum (ER) to form reticulum-Golgi network. Then, the third domain of truncated exotoxin A enables to inhibit protein synthesis in the cell, resulting in cell death. 
     Currently, the exotoxin A of  P. aeruginosa  is used for developing anti-cancer medicines. Immunotoxin produced by fusing specific antibodies with exotoxin A can be targeted to specific types of cells, such as cancer cells, by taking the advantages of specificity associated with the conjugated antibodies. It is expected that the protein synthesis pathway will be blocked in the targeted cells, thereby achieves the purpose of destroying the growth of the particular cell. 
     In view of various disadvantages derived from conventional aquatic subunit vaccine and the application of  P. aeruginosa  on the genetic engineering, the inventor of this application has devoted to improve it and after studying intensively for many years, provides a novel aquatic subunit vaccine and thus accomplish the invention. 
     SUMMARY OF THE INVENTION 
     This invention provides an aquatic subunit vaccine prepared by using genetic engineering techniques to fuse an antigenic protein against fish disease with a receptor binding protein, a translocation protein and a signal peptide such that the antigen can be targeted efficiently and precisely to the effector cell as well as bind and then penetrate the plasma membrane barriers. The antigen from the aquatic subunit vaccine can be presented correctly onto the effector cell and then induce fishes an immunity sufficient to resist viral infection. A number of advantages of vaccines created by recombinant DNA technology include simpler process for mass-production, lower cost of manufacturing, higher purity of produced vaccine, and safety of utilizing the vaccine. 
     The inventor utilizes the first two functional domains of exotoxin A of  P. aeruginosa , i.e. the receptor-binding domain (Domain I) and the translocation domain (Domain II), as the PE protein of transporting protein of the antigenic protein of the fish vaccine. In addition, an ER retention signal (i.e. a KDEL signal peptide, whose C terminus has a KDEL signal peptide, and which is recyclable to endoplasmic reticulum) is also conjugated to the designed fish vaccine in order to target specifically the vaccine protein to ER inside the effector cells. 
     The inventor conjugates the PE protein (containing Domains I and II of the exotoxin A protein, whose amino acid sequence were shown as SEQ ID No: 8) to the N terminus of the fish antigenic protein and the KDEL signal peptide (shown as SEQ ID No: 10) to the C terminus of fish antigenic protein. Therefore, the fish disease-related antigenic protein (thereafter antigenic fusion protein) prepared by fusing PE protein and KDEL signal peptide can be targeted under the action of the PE protein to appropriate cell (i.e. CD8+ T cell) through specific protein-protein interaction between domain I of exotoxin A in the vaccine and receptors on the surface of CD8+ T cell. After the antigenic fusion protein entering the endosome of the CD8+ T cell via endocytosis, it will be processed by protease in the endosome and released into cytoplasm. Then, the KDEL signal peptide will target properly the vaccine protein to bind ER inside the CD8+ T cell for antigen processing and hence induce the effective immunity in fish. 
     Accordingly, the invention provides an aquatic subunit vaccine comprising an antigenic fusion protein and suitable carrier or adjuvant. The antigenic fusion protein comprises from its N terminus to C terminus sequentially: (1) a receptor binding motif and translocation domain of exotoxin A protein (PE protein); (2) a viral antigenic protein affecting fish disease; and (3) a signal peptide KDEL. 
     In a preferred embodiment, the PE protein has an amino acid sequence as shown in SEQ ID No: 8, and the KDEL signal peptide has an amino acid sequence as shown in SEQ ID No: 10. 
     The viral antigenic proteins affecting fish diseases include Infectious Hematopoietic Necrosis Virus&#39;s (IHNV) G protein (SEQ ID No: 22) and/or N protein (SEQ ID No: 24), Nervous Necrosis Virus&#39;s (NNV) RNA2 protein (SEQ ID No: 26), Grouper Iridovirus&#39;s (GIV) Major Capsid Protein (MCP, SEQ ID No: 28) and/or Myristylated Membrane Protein (MMP, SEQ ID No: 30), Spring Viremia of Carp Virus&#39;s (SVCV) G protein (SEQ ID No: 32) and/or N protein (SEQ ID No: 34), Viral Hemorrhagic Septicemia Virus&#39;s (VHSV) G protein (SEQ ID No: 36) and/or N protein (SEQ ID No: 38), Salmon pancreas disease virus&#39;s (PDV) E1 protein (SEQ ID No: 40), E2 protein (SEQ ID No: 42), E3 protein (SEQ ID No: 44), capside protein (CP, SEQ ID No: 46), and/or structural polyprotein (SEQ ID No: 48), and IPNV&#39;s VP2 (SEQ ID No: lor SEQ ID No: 4). 
     In a preferred embodiment, the viral antigenic protein is IPNV&#39;s VP2 that has an amino acid sequence encoded in SEQ ID No: 1 or SEQ ID No: 4. In another preferred embodiment, the viral antigenic protein is the antigenic domain of the viral protein VP2 of IPNV, that is the amino acid sequence of amino acids 173 to 431 in the viral protein VP2 of IPNV as shown in SEQ ID No: 3 or SEQ ID No: 6. 
     The antigenic fusion protein in this invention is created by a gene cloning process as used in usual recombinant DNA technology. The DNA sequence encoding PE protein (as shown in SEQ ID No: 7), the DNA sequence encoding the viral antigenic protein affecting fish disease and DNA sequence encoding the KDEL signal peptide (as shown in SEQ ID No: 9) are cloned in an expression vector system to create an expression plasmid containing DNA sequence encoding antigenic fusion protein. Then, the plasmid is transformed into an expression host cells where protein expression is induced and then the antigenic fusion protein is extracted therefrom. 
     Viruses affecting fish diseases include Hematopoietic Necrosis Virus&#39;s (IHNV) with its G protein (the DNA sequence is shown as SEQ ID No: 21) and/or N protein (the DNA sequence is shown as SEQ ID No: 23), Nervous Necrosis Virus&#39;s (NNV) with its RNA2 protein (the DNA sequence is shown as SEQ ID No: 25), Grouper Iridovirus&#39;s (GIV) with its Major Capsid Protein (MCP, the DNA sequence is shown as SEQ ID No: 27) and/or Myristylated Membrane Protein (MMP, the DNA sequence is shown as SEQ ID No: 39), Spring Viremia of Carp Virus with its (SVCV) G protein (the DNA sequence is shown as SEQ ID No: 31) and/or N protein (the DNA sequence is shown as SEQ ID No: 33), Viral Hemorrhagic Septicemia Virus&#39;s (VHSV) with its G protein (the DNA sequence is shown as SEQ ID No: 35) and/or N protein (the DNA sequence is shown as SEQ ID No: 37), Salmon pancreas disease virus&#39;s (PDV) E1 protein (the DNA sequence is shown as SEQ ID No: 39), E2 protein (the DNA sequence is shown as SEQ ID No: 41), E3 protein (the DNA sequence is shown as SEQ ID No: 43), capside protein (CP, the DNA sequence is shown as SEQ ID No: 45), and/or structural polyprotein (the DNA sequence is shown as SEQ ID No: 47), and IPNV with its VP2 (the DNA sequence is shown as SEQ ID No: 2 or SEQ ID No: 5). 
     In a preferred embodiment, the IPNV antigenic fusion protein has an amino acid sequence of SEQ ID No: 12 or SEQ ID No: 14. The DNA sequence encoding the antigenic fusion protein has a nucleotide sequence of SEQ ID No: 11 or SEQ ID No: 13. 
     The expression vector system includes but not limited to pET and pGEX vector systems. In a preferred embodiment, the expression vector is pET24a. The expression host may be but not limited to  Escherichia coli . In a preferred embodiment, the expression host is  E. coli  BL21. 
     The adjuvant includes but not limited to waterborne adjuvant aluminum hydroxide gel, Alum, Freund&#39;s incomplete adjuvant, oil-based adjuvant, water-based adjuvant, or water-in-oil-in-water (W/O/W) adjuvant. In one preferred embodiment, the oil-based adjuvant was used as the adjuvant. 
     These features and advantages of the present invention will be fully understood and appreciated from the following detailed description of the accompanying drawings. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1   a  to  1   d  are schematic view showing the strategy for constructing the expression vector (pET24-PE-VP2-KDEL) used for the antigenic fusion protein in the aquatic subunit vaccine according to the invention. 
         FIG. 2  shows amounts of proteins expressed in  E. coli  BL21 by separating with SDS-PAGE electrophoresis and then staining bands with coomassie blue. M: protein marker.  FIG. 2   a  Lane 1:  E. coli  BL21 protein extract containing vector pET24a;  FIG. 2   a  Lane 2-3:  E. coli  BL21 protein extract containing vector pET24a-VP2-015 (Lane 2, not purified; Lane 3, purified); Lane 4-5:  E. coli  BL21 protein extract containing vector pET24a-VP2-016 (Lane 4: not purified; Lane 5: purified);  FIG. 2   b , Lane 6-7:  E. coli  BL21 protein extract containing plasmid pET24a-PE-VP2-015-KDEL (Lane 6: unpurified; Lane 7: purified); Lane 8-9:  E. coli  BL21 protein extract containing plasmid pET24a-PE-VP2-016-KDEL (Lane 8: unpurified; Lane 9: purified). 
         FIG. 3  is a graph showing relative percentage of survival (R.P.S.) of each group in Example 3. 
     
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT 
     The aquatic subunit vaccine according to the invention will be illustrated in more detail by reference with an example using a fragment of IPNV viral protein VP2. 
     EXAMPLE 1 
     The Construction of pET24-PE-VP2-KDEL Expression Vector for Antigenic Fusion Protein 
     In this example, a fragment of IPNV VP2 was used as the antigen of the aquatic subunit vaccine. A receptor binding motif and the a translocation protein (PE protein) of exotoxin A were conjugated to the N terminus of VP2, as well as a KDEL signal peptide was conjugated to its C terminus, thereby formed the antigenic fusion protein (PE-VP2-KDEL). 
     The antigenic fusion protein (PE-VP2-KDEL) was created by a gene cloning technology comprising cloning DNA sequences encoding respective proteins into an expression vector to form an expression vector pET24a-PE-VP2-KDEL, and then inducing protein expression. 
     The strategy for constructing the antigenic fusion protein expression vector (24a-PE-VP2-KDEL) is shown in  FIG. 1 . First, the DNA sequence encoding KDEL signal peptide is cloned into a plasmid pET24a to form plasmid pET24a-KDEL (as shown in  FIGS. 1   a  and  1   b ). Next, the DNA sequence encoding a sequence of amino acid 173 to amino acid 431 in IPNV VP2 protein is cloned into plasmid pET24a-KDEL to form a construct pET24a-VP2-KDEL (as shown in  FIGS. 1   b  and  1   c ). Finally, the DNA sequence encoding PE protein is cloned into plasmid pET24a-VP2-KDEL to form a construct pET24a-PE-VP2-KDEL (as shown in  FIGS. 1   c  and  1   d ). 
     1. Construction of Plasmid pET24a-KEDL 
     The DNA sequence encoding KDEL signal peptide (SEQ ID No: 10) is shown as SEQ ID No: 9. The DNA sequence encoding KDEL signal peptide is amplified by a polymerase chain reaction (PCR). The sequence of KEDL-specific primers was shown below: 
     Forward primer (with Hind III restriction enzyme cleavage site): 
                     (SEQ ID No: 15)       5′-CCC          CTCAAAAAAGACGAACTGAGAGATGAACTGAAAGA-3′             Hind III            
Reverse primer (with Xho I restriction enzyme cleavage site):
 
     
       
         
               
               
             
           
               
                   
                 (SEQ ID No: 16) 
               
               
                   
                 5′-GTG          TCATTACAGTTCGTCTTTCAGTTCATCT-3′ 
               
               
                   
                        Xho I 
               
             
          
         
       
     
     Conditions for PCR reaction comprised 5 cycles of: 94° C. 3 minutes, 95° C. 1 minute, 55° C. 1 minute, 72° C. 20 seconds; and finally, with 72° C. 1 minute for elongation. PCR product and pET24a vector were subjected to double restriction enzymes digestion with Hind III and Xho I. Thereafter, the digested PCR product and plasmid pET24a were purified, respectively, followed by ligation to clone the PCR product into plasmid pET24a to form a plasmid pET24a-KEDL. Then, the construct pET24a-KEDL was transformed into host cells,  E. coli  RR1, to carry out mass replication. The replicated PCR products were further confirmed by sequencing. 
     2. Construction of Plasmid pET24a-VP2-KDEL 
     Two IPNV VP2 proteins, VP2-015 and VP2-016, were used in this example. The full-length sequences of VP2-015 protein and VP2-016 protein were shown as SEQ ID No: 1 and SEQ ID No: 4, respectively. The difference between them was that amino acid residues 217, 221, and 247, were TAT in VP2-015 but PTA in VP2-016. 
     In this example, the amino acid sequences from amino acid 173 to 431 of VP2-015 and VP2-016 were used as antigens. DNA sequence encoding amino acid 173 to 431 of VP2-015 protein (shown as SEQ ID No: 3) was shown as SEQ ID No: 2 while DNA sequence encoding amino acid 173 to 431 of VP2-016 protein (shown as SEQ ID No: 6) was shown as SEQ ID No: 5. Both DNA sequences were separately amplified by polymerase chain reaction (PCR). VP2 specificity primers were: 
     Forward primer (with EcoR I restriction enzyme cleavage site): 
                             (SEQ ID No: 17)           5′-CG          CCATACGTCCGCCTAGAGGAC-3′                 EcoR I            
Reverse primer (with Hind III restriction enzyme cleavage site):
 
     
       
         
               
               
             
           
               
                   
                 (SEQ ID No: 18) 
               
               
                   
                 5′-CCC          CGTGATTTCGTTGAAGA-3′ 
               
               
                   
                       Hind III 
               
             
          
         
       
     
     Conditions of PCR reaction comprised 30 cycles of: 94° C. 5 minutes, 94° C. 1 minute, 55° C. 1 minute, and 72° C. 1 minute and finally, 72° C. 7 minutes for an elongation. PCR products and pET24a-KEDL plasmid were subjected to double restriction enzyme digestion (with EcoR I and Hind III). The digested PCR products and pET24a-KEDL plasmid were purified separately followed by ligation to form construct pET24a-VP2-015-KEDL and construct pET24a-VP2-016-KEDL. Both constructs pET24a-VP2-KEDL described above were transformed into host cells ( E. coli  RR1) to carry out mass replication. The replicated PCR products were further confirmed by sequencing. 
     3. Construction of Plasmid pET24a-PE-VP2-KDEL 
     DNA sequence encoding PE protein (SEQ ID No: 8) was shown as SEQ ID No: 7. The DNA sequence encoding PE protein was amplified by polymerase chain reaction (PCR). The PE-specific primers were: 
     Forward primer (with BamH I restriction enzyme cleavage site): 
                     (SEQ ID No: 19)       5′-CG          GCCGAAGAAGCTTTCGACCTCTGGAACGAATGC-3′             BamH I            
Reverse primer (with EcoR I restriction enzyme cleavage site):
 
     
       
         
               
               
             
           
               
                   
                 (SEQ ID No: 20) 
               
               
                   
                 5′-CG          GCAGGTCAGGCTCACCAC-3′ 
               
               
                   
                       EcoR I 
               
             
          
         
       
     
     Conditions for PCR reaction comprised 30 cycles of: 94° C. 5 minutes, 95° C. 1 minute, 55° C. 1 minute, and 72° C. 1 and half minutes as well as 72° C. 7 minutes for elongation. PCR products and plasmid pET24a-VP2-015-KEDL or plasmid pET24a-VP2-016-KEDL were subjected to double restriction enzyme digestion (with BamH I and EcoR I). The digested PCR products and plasmids (pET24a-VP2-015-KEDL or pET24a-VP2-016-KEDL) were purified, respectively, followed by ligation to cloning PCR products into plasmid pET24a-VP2-015-KEDL or plasmid pET24a-VP2-016-KEDL to form construct pET24a-PE-VP2-015-KEDL (containing the DNA sequence of PE-VP2-015-KEDL antigenic fusion protein, as indicated in SEQ ID No: 11) and a construct pET24a-PE-VP2-016-KEDL (containing the DNA sequence of PE-VP2-016-KEDL antigenic fusion protein, as shown in SEQ ID No: 13). Constructs pET24a-PE-VP2-015-KEDL or pET24a-PE-VP2-016-KEDL was transformed into host cells ( E. coli  BL21) to carry out mass replication. The replicated PCR products were further confirmed by sequencing. 
     4. Construction of Plasmid pET24a-VP2 
     An IPNV&#39;s VP2 antigenic protein fragment (absence of PE protein and KDEL signal peptide) was used as a control. The amino acid sequences of amino acid 173 to 431 in VP2-015 and VP2-016, respectively, were used as antigens. The DNA sequence encoding amino acid 173 to amino acid 431 in VP2-015 protein (its amino acid sequence was shown as SEQ ID No: 3) was shown as SEQ ID No: 2. The DNA sequence encoding amino acid 173 to amino acid 431 in VP2-016 protein (its amino acid sequence was shown as SEQ ID No: 6) was shown as SEQ ID No: 5. DNA sequences encoding these VP2 proteins were amplified by polymerase chain reaction (PCR). The VP2-specific primers were: 
     Forward primer (with EcoR I restriction enzyme cleavage site): 
                             (SEQ ID No: 17)           5′-CG          CCATACGTCCGCCTAGAGGAC-3′                EcoR I            
Reverse primer (with Hind III restriction enzyme cleavage site):
 
     
       
         
               
               
             
           
               
                   
                 (SEQ ID No: 18) 
               
               
                   
                 5′-CCC          CGTGATTTCGTTGAAGA-3′ 
               
               
                   
                      Hind III 
               
             
          
         
       
     
     Conditions for PCR reaction comprised 30 cycles of: 94° C. 5 minutes, 94° C. 1 minute, 55° C. 1 minute, and 72° C. 1 minute as well as final 72° C. 7 minutes for elongation. PCR products and vector pET24a-KEDL were subjected to double restriction enzyme digestion (with EcoR I and Hind III). The enzyme digested PCR products and vector pET24a were purified separately followed by ligation to clone PCR product into vector pET24a and form constructs pET24a-VP2-015 and pET24a-VP2-016. Both two constructs pET24a-VP2 were transformed into host cells ( E. coli  BL21) to carry out mass replication. The replicated PCR products were further confirmed by sequencing. 
     EXAMPLE 2 
     Vaccine Preparation 
     1. The Expression of Antigenic Fusion Protein 
       E. coli  BL21 cells transformed as described above with plasmid pET24a-PE-VP2-015-KEDL or plasmid pET24a-PE-VP2-016-KEDL, or  E. coli  BL21 cells containing plasmid pET24a-PE-VP2-016 or plasmid pET24a-PE-VP2-016 were cultured, respectively, at 37° C. in LB broth containing 50 μg/ml kanamycin overnight. Then, 10 ml of the culture was added into 990 ml fresh LB broth containing 50 μg/ml kanamycin. The diluted cell culture was incubated further at 37° C. by shaking at 250 rpm for 3 hours. Then, 1 ml 1M IPTG (final concentration 1 mM) as an inducer, was added to the culture to induce the expression of the antigenic fusion protein (PE-VP2-015-KEDL whose amino acid sequence was shown as SEQ ID No: 12; or PE-VP2-016-KEDL whose amino acid sequence was shown as SEQ ID No: 14). After IPTG induction, cells were cultured for another three hours and then, harvested by centrifugation at 6,5000×g at 4° C. for 30 minutes. The supernatant was discarded, the cell pellet was recovered and stored at −70° C. till used. 
     2. Extraction of Antigenic Fusion Protein 
     The cell pellet described above was resuspended in 200 ml phosphate buffered saline (PBS) and mixed homogeneously. The suspension was centrifuged at 6,5000×g 4° C. for 15 minutes and the supernatant was discarded. The above procedure was repeated at least twice. Thereafter, the cell pellet was resuspended in 100 ml of 1×IB wash Buffer (20 mM Tris-HCl, pH 7.5, 10 mM EDTA, 1% Triton X-100) and placed the cell suspension on ice or at 4° C. to prevent high heat generation during cell breakage. The cells was ruptured then by adding 100 μg/ml of lysozyme into the suspension and incubated at 30° C. for 30 min in combination with sonication or a high speed homogenizer (Polytron) as follows. The lysates were divided into aliquots in 50 ml tubes, mixed by swirling and sonicated on ice with an appropriate tip. Repeat sonication until the solution is no longer viscous. Then, inclusion bodies were collected by centrifugation at 10,000×g for more than 10 min. The supernatant was removed and the pellet was thoroughly resuspended in 0.1 culture volume of 1×IB Wash Buffer. Repeated centrifugation and saved the pellet, until the pellet became white. Again, the pellet was resuspended thoroughly in 0.1 culture volume. Transfer the suspension to a clean centrifuge tube with a known tare weight. The inclusion bodies were collected by centrifugation at 10,000×g for more than 10 min. The supernatant was decanted off and the last trace of liquid was removed by tapping the inverted tube on a paper tower. After thoroughly removing the supernatant, the weight of the inclusion body was measured. 
     The inclusion body was examined by SDS-PAGE. The result was shown in  FIG. 2   b , where Land 6 and 7 were antigenic fusion protein PE-VP2-015-KEDL; and Land 8 and 9 were antigenic fusion protein PE-VP2-016-KEDL. The molecular weigh of these antigenic fusion proteins was 71 kDa. The VP2-015 protein fragments as negative control (absence of PE protein and KDEL signal peptide) were shown in  FIG. 2   a , Lane 2 and 3, while VP2-016 protein fragments as negative control (absence of PE protein and KDEL signal peptide) were shown in  FIG. 2   a , Lane 4 and 5. The molecular weights of these protein fragments were 28 kDa. 
     3. The Preparation of the Vaccine 
     Antigenic fusion protein PE-VP2-015-KDEL (SEQ ID No: 12), antigenic fusion protein PE-VP2-016-KDEL (SEQ ID No: 14), VP2-015 antigenic protein (SEQ ID No: 3), and VP2-016 antigenic protein (SEQ ID No: 6) were used to prepare monovalent vaccines, respectively. The inclusion bodies containing respective antigenic fusion protein or antigenic protein were dispersed directly in PBS to prepare vaccines each containing 10 μg of respective antigenic fusion protein or antigenic protein. Next, the inclusion bodies were embedded in water-in-oil emulsion and then stored in 4° C. 
     EXAMPLE 3 
     Anti-Viral Test in Salmons 
     The basic information about salmons tested was as followed: 
     
       
         
               
               
             
           
               
                   
               
             
             
               
                 Species 
                 Atlantic salmon ( Salmo salar  L.) 
               
               
                 Strain 
                 Aquagen AS, LR (Low resistance) 06AGHe01 
               
               
                 Size 
                 Average 60-90 g 
               
               
                 Vaccination history 
                 Not previously vaccinated 
               
               
                 Diseases history 
                 None 
               
               
                   
               
             
          
         
       
     
     Culture conditions of salmons in the stage of smoltification: 
     
       
         
               
               
               
               
               
               
               
             
           
               
                   
               
               
                   
                   
                 Water 
                   
                   
                   
                   
               
               
                 Salinity 
                 Temp. 
                 Quality 
                 Time 
                 Photoperiod 
                 Flow 
                 Density 
               
               
                   
               
             
             
               
                 0‰ 
                 8-12° C. 
                 Fresh 
                 5 weeks 
                 12 hours daylight 
                 0.8 l/kg fish 
                 Max. 40 
               
               
                   
                   
                 water 
                   
                 12 hours darkness 
                 pr. min. 
                 kg/m 3   
               
               
                 0‰ 
                 8-12° C. 
                 Fresh 
                 From week 5 
                 24 hour daylight 
                 0.8 l/kg fish 
                 Max. 40 
               
               
                   
                   
                 water 
                 and onwards 
                   
                 pr. min. 
                 kg/m 3   
               
               
                   
               
             
          
         
       
     
     There were four groups plus one control group of salmons used in virus resistant test. Each group had thirty salmons. The prepared vaccines were administrated by intraperitoneal injection (i.p.) into the fishes. The dose of the vaccine was 0.1 ml per fish, which corresponded to 0.1 μg of antigenic fusion protein or VP2 viral protein per fish. The virus resistant test was designed as described below:
     Group 1: inoculated with the vaccine containing VP2-015 viral protein   Group 2: inoculated with the vaccine containing PE-VP2-015-KDEL antigenic fusion protein   Group 3: inoculated with the vaccine containing VP2-016 viral protein   Group 4: inoculated with the vaccine containing PE-VP2-016-KDEL antigenic fusion protein   Control: with inoculated PBS solution.   

     Tests of both experimental and control groups were duplicated. The vaccination was performed at a condition of 900 degree days (temperature times days). For example, if the temperature was 20° C., it should take for 45 days; whereas if the temperature was 10° C., it should take 90 days. Then, salmons tested were transferred to salt water environment for further investigation. 
     
       
         
               
               
               
               
               
               
             
           
               
                   
               
               
                   
                   
                 Water 
                   
                   
                   
               
               
                 Salinity 
                 Temp. 
                 Quality 
                 Photoperiod 
                 Flow 
                 Density 
               
               
                   
               
             
             
               
                 34‰ 
                 10-12° C. 
                 Salt 
                 12 hours daylight 
                 0.5-0.61 l/kg 
                 Max. 40 
               
               
                   
                   
                 water 
                 12 hours darkness 
                 fish pr. min. 
                 kg/m 3   
               
               
                   
               
             
          
         
       
     
     All groups of fish were fed according to standard protocols. Prior to challenge, fishes were fasted for one day. Naïve fish (20% of fishes tested) were injected intraperitoneally with 1.0×10 5  TCID 50 /ml of a virulent strain of IPN virus (serotype Sp and genotype TAT) as the challenge carriers to undergo cohabitation challenge. Fishes of other test groups were moved to seawater tanks. Then, six challenge carriers (20% of the fishes tested) were added to each challenge tank the day after seawater transfer. Thereafter, the mortality of the control and test groups were observed and recorded. The statistic analysis was done when the mortality of the control group was over 60%. Relative percent survival (R.P.S.) was calculated and chi-square test was used for statistic analysis. R.P.S. was calculated according to the formula:
 
R.P.S.=[1−(vaccine mortality/control mortality)]×100%
 
     Next, fishes of test groups were sacrificed and their blood and head kidney samples were collected. The serum samples were collected and frozen for ELISA test. The head kidney samples were used for the virus culture test to reconfirm the death of the fish was caused by IPNV. 
     Relative percent survival (R.P.S.) of each group was indicated as  FIG. 3 . It indicated that:
     1. The level of survival in the VP2-015-only group (Group 1) was 6%, which is not different significantly from that of the control group (non-vaccinated controls; its R.P.S. was not shown in  FIG. 3 , but was 0% on calculation based on the above R.P.S. calculation formula).   2. The PE-VP2-015-KDEL immunised fish (Group 2) had an RPS of 23% and indicated a significantly increased level of protection (RPS value) compared to the VP-015-only group (Group 1) (p&lt;0.01).   3. For the VP2-016-only (Group 3) immunised fish, there was a significant protection effect (RPS value=12%; p&lt;0.05) in the vaccinated fish compared to non-vaccinated controls.   4. For the PE-VP2-016-KDEL (Group 4) immunised fish, the obtained RPS value was 39%, and was different significantly from the VP2-016 only group (p&lt;0.01).   

     Compared with other traditional technologies, advantages of the inventive aquatic subunit vaccine are listed in the following:
     1. The inventive subunit vaccine is prepared based upon recombinant DNA technology by fusing the receptor binding motif and the translocation domain of  Pseudomonas aeruginosa  exotoxin A, and the KDEL signal peptide, thereby it can enhance the expression of vaccine protein within host cells, and result in better immunological effect and protection of immunized fish.   2. In addition, the novel vaccine according to the invention is a subunit vaccine and its manufacturing process by gene cloning has advantages of being a simple process for mass-production, lower production cost, higher purity of vaccine products, and safety of using the vaccine.   

     Many changes and modifications in the above described embodiment of the invention can, of course, be carried out without departing from the scope thereof. Accordingly, to promote the progress in science and the useful arts, the invention is disclosed and is intended to be limited only by the scope of the appended claims.