Abstract:
A fusion protein of porcine reproductive and respiratory syndrome virus (PRRSV) for PRRSV vaccine. The fusion protein includes: (a) a Pseudomoitas Exoloxin A (PE) peptide that comprises a binding domain and a translocating domain; (b) a peptide fragment that contains a N-terminal portion of PRRSV ORF6 protein; (c) a peptide fragment that has a N-terminal portion of PRRSV ORF5 protein; and (d) a carboxyl terminal domain that comprises an amino acid seciuence KDEL. The PE peptide is located at the N-terminus of the fusion protein, and the peptide fraament containinC the N-terminal portion of PRRSV ORF5 protein is located between the peptide fragment containing the N-terminal portion of PRRSV ORF6 protein and the carboxyl terminal domain.

Description:
BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     The present invention relates to a fusion protein of PRRS subunit vaccine inducing PRRSV neutralization titers in pigs. 
     2. Description of Related Art 
     Porcine reproductive and respiratory syndrome is a porcine infectious disease that primarily strikes respiratory tracts of pigs at various ages and results in sows having reproductive dysfunction. PRRSV is a tough and resistant virus not prone to evoke antibodies having neutralization titers in the infectious pig. Besides, PRRSV is a RNA virus and reproduces easily on the basis of a simplified genetic system, so the probability of genetic mutations is very high. Furthermore, infections and pathogenesis pathway of PRRS virus can be sorted into two stages: (A) infections of epithelium tissues of upper and lower reproductive tracts; and (B) infections of monocytes and macrophages in tissues surrounding reproductive tracts. Thus, the host must have body fluid immunity as well as mucosal immunity with neutralization titers and also a cell-mediated immune response to facilitate removal of the infected viruses and strengthen the host protection mechanisms. However, it is not very easy for PRRSV infectious pig to have neutralization titers in natural conditions, hence typical antibodies basically have little effect on PRRSV, and they even induce mutations in viruses. Furthermore, in antibody-dependent enhancement of phagocytosis, the antibodies could only cause more severe PRRSV infections. 
     Taiwan Patent No.1-2289933 (also as U.S. patent publication no. 2004/02147617) discloses a target-cell-specific fusion protein, which utilizes a moiety and a functional domain of  Pseudomonas aeruginosa  exotoxin to fuse a PRRSV ORF7 nuclear protein fragment, with a KDEL signal peptide added on the carboxyl terminus. The fusion protein can be mass-produced in  E. coli.  When immunized the fusion protein to pigs, it is possible to decrease or to eliminate viremia after being PRRSV-challenged in the immunized pigs. The full text of the patent is incorporated herein. 
     In heterodimerization between ORF5 and ORF6 of PRRSV, epitope Cys-34 of ORF5 and epitope Cys-8 of ORF6 play a critical role in viral infection and the envelope assembly thereof. (Snijder Eric J., Jessica C. et al., Journal of Virology, January 2003, Vol. 77, No. 1:97-104). Besides, the consensus sequence of PRRSV ORF5 (YKNTHLDLIYNA) is an epitope between amino acid 38 th  and amino acid 44 th , which is located at N-terminal extracellular domain of PRRSV ORF5 and had been identified as a neutralization epitope (Ostrowski M., J. A. Galeota, et al., Journal of Virology, May 2002, Vol. 76, No. 9:4241-4250). 
     Prior arts disclose constructing whole PRRSV ORF5 or ORF6 antigens between PE and KDEL. After immunization of these fusion proteins, the pigs suffered from severe inflammation in their lungs post being PRRSV-challenged, indicating that PRRSV ORF5 or ORF6 have an antigen-specific allergy effect. Manifestly, it is difficult to use them as PRRS vaccine antigens. Thus, to develop a vaccine and effectively protect pigs from PRRS infections, there are a lot of difficulties that have to be overcome. It should need to be designed such as a lower immunotoxicity and having a high neutralization titer. 
     SUMMARY OF THE INVENTION 
     An object of the present invention is to provide a fusion protein, and the method to construct thereof. 
     Anther object of the present invention relates to using the fusion protein to prepare a subunit vaccine composed of proteins having neutralization titers. 
     Still another object of the present invention is to provide a pharmaceutical composition, comprising the fusion protein of the present invention and pharmaceutically acceptable adjuvants. 
     PE-PQGAB-K3 fusion protein of the present invention comprises: a chimeric polypeptide containing N-terminal portions of PRRSV ORF5 and ORF6 structure proteins; a portion of  Pseudomonas exotoxin  A binding and translocation domains; and a carboxyl terminal domain containing fragment. 
     The present invention further comprises a pharmaceutical composition that can serve as a vaccine, comprising: a chimeric polypeptide, which contains N-terminal portions of PRRSV ORF5 and ORF6 structural proteins; a portion of  Pseudomonas exotoxin  A binding and translocation domain; a carboxyl terminal domain containing fragment; and a pharmaceutically acceptable adjuvant. 
     The strain of PRRSV in the present invention is not particularly limited. It can be an American strain, European strain, or Australian strain. There is no particular limitation to the fragments contained in the N-terminal domain of the fusion protein, but they are preferably any PRRSV fragment that is antigenic, such as PRRSV ORF5, ORF6. Taking the American strain as an example, the portion of PRRSV ORF6 sequence is preferably as SEQ ID NO.13. The amino acid sequence of the N-terminal portion of PRRSV ORF5 structural protein can be as SEQ ID NO.12. For the European strain, the amino acid sequence of the N-terminal portion of PRRSV ORF6 structural protein is preferably as SEQ ID NO.14, and that of the N-terminal portion of PRRSV ORF5 can be as SEQ ID NO.15. 
     In the present invention, the nucleic acid sequence of the fusion protein containing a portion of PRRSV ORF5 and a portion of PRRSV ORF6 is modified, and there is no particular limitation to the sequence, but it is preferably a nucleic acid sequence that can be expressed in large amounts in  E. coli  host-vector system, and the expressed proteins are identical to wild type ones. Taking the American strain of PRRSV as an example, preferably the modified nucleic acid sequence is as seen in SEQ ID NO.1. For the European strain, preferably the modified nucleic sequence is as SEQ ID NO.11. 
     A preferred embodiment of the portion of  Pseudomonas exotoxin  A binding and translocation domain of the fusion protein in the present invention is a detoxified  Pseudomonas exotoxin,  which is a fragment from  Pseudomonas exotoxin  A without domain III. Preferably, the fragment of  Pseudomonas exotoxin  A binding and translocation domain acts as a ligand moiety which is capable of reacting, recognizing or binding to a receptor on the target cell. 
     The pharmaceutical composition of the present invention can comprise a suitable adjuvant known in the art: dispersant, humectant (such as Tween 80), or sterile injections prepared with suspensions (such as sterile injection solutions or oily solutions). Sterile injection preparations can also be used in diluents or solvents of sterile injections or suspensions during innocuous injections, for example, in solutions of 1,3-butanediol. Acceptable carriers or solvents include mannitol, water, ringer solution, and isotonic sodium chloride solution. Besides, sterilized and fixed oils are used in prior arts as solvents or suspension media(for example, synthesized monoglycerides or diglycerides). Fatty acids (such as oleic acids or glyceride derivatives) and natural pharmaceutically acceptable oils (such as olive oil or castor oil, especially polyoxyethylated derivatives thereof) can be used in injectable preparations. The oil solutions or suspensions can also comprise long-chain alcohol diluents, dispersants, caboxylmethyl cellulose, or similar dispersants. Other commonly used surfactants like Tweens and Spans, or emulsifiers and bioavailability enhancers (usually used in manufacturing pharmaceutically acceptable alum solids, liquids or other dosage forms) can also be used for preparing purposes. 
     Compositions for oral administration can be any dosage form acceptable for oral administration, comprising, but not limited to, capsules, tablets, emulsions, water suspensions, dispersants, and solutions. In cases of tablets for oral administration purposes, the typical carriers include lactose and corn starch. A lubricant is often added, such as magnesium stearate. For oral administration with capsules, suitable diluents include lactose and corn starch. In cases of oral administration of water dispersants or emulsions, active ingredients could associate with emulsions or suspensions to suspend or disperse in the oil phase. Depending on needs, certain sweeteners, flavoring agents, or coloring agents can be added. 
     A nasal aerosol or inhalation composition can be prepared according to techniques well-known in the art of pharmaceutical formulation. For example, such a composition can be prepared as a solution in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other solubilizing or dispersing agents known in the art. Compositions containing fusion proteins can also be administered in the form of suppositories for rectal administration. 
     The carriers of the pharmaceutical composition must be “acceptable”, i.e. compatible to active ingredients in the formulation (preferably, capable of stabilizing the active ingredient) and not deleterious to the subject to be treated. Other examples of the carriers include colloidal silicon dioxide, magnesium stearate, cellose, sodium lauryl sulfate, and D&amp;C yellow No.10. 
     The pharmaceutical composition of the fusion protein in the present invention preferably comprises an immune adjuvant. The immune adjuvant used is not limited and can be any conventional one used in vaccines known in the art, comprising Alumigel and oil emulsion, such as Freund&#39;s FCA or FIA or mannide mono-oleate emulsifier (ISA720 or ISA206, SEPPIC®, France), preferably ISA206. 
     Other objects, advantages, and novel features of the invention will become more apparent from the following detailed description when taken in conjunction with the accompanying drawings. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  is a schematic illustration of PE-PQGAB fusion protein of Example 1; 
         FIG. 2  is a flowchart of plasmid construction of PE(ΔIII)-PQGAB of Example 1; 
         FIG. 3  is the electrophoresis diagram of the nucleic acid fragments synthesized according to Example 1, with four DNA fragments (a:70 bp, b:129 bp, c:186 bp, d:204 bp); and 
         FIG. 4  is the plasmid map of PE(ΔIII)-PQGAB. 
         FIG. 5  is the result of proteins induced in  E. coli  Host-vector system and extracted from inclusion bodies by 8M urea extraction. lane 0h, 2h: the total lysis samples at 0 hr and 2 hr after IPTG induction of  E. coli  with pPE-PQGAB-K3; and lane 8M: 8M urea protein extraction of PE-PQGAB-K3. 
     
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT 
     The feature of the present invention is based on a finding that, when most structure proteins in ORF5 and ORF6 were removed, leaving dozens of N-terminal amino acids of ORF5 and ORF6, by which constructing a fusion peptide chain PQGAB, and then inserting the peptide chain between PE and K3 sequence was possible, it was confirmed that the fusion protein PE-PQGAB-K3 had serum neutralization titers by mice and porcine immunization tests. 
     The following examples are proposed to explain the present invention, but not set forth as to limit the scope thereof. 
     EXAMPLE 1  
     PQGAB Fusion Peptide of PRRSV American Strain 
     The protein sequences of ORF5 and ORF6 of PRRSV were obtained from the National Center for Biotechnology Information (NCBI, USA) database. Based on aforementioned mechanisms of viral infections, the regions of PRRSV that have neutralization titers are located at the N-terminus of ORF5 and ORF6. That is, amino acid residues 2 to 26 of ORF6(SEQ ID NO.13) and amino acid resiues 31 to 63 of ORF5 structural proteins (SEQ ID NO.12). The amino acid sequence resulting from the fusion of the two peptides is illustrated below: 
     
       
         
               
               
             
           
               
                     GSSLDDFCYDSTAPQKVLLAFSITYASNDSSSHLQLIYNLTLC 
                   
               
               
                 ELNGTDWLANKFDWA 
               
             
          
         
       
     
     The sequence of PRRSV-ORF6-2˜26-ORF5-31˜63 fusion peptide was the combination of (ORF6)-G 2 SSLDDFCYDSTAPQKVLLAFSITY 26  (SEQ ID NO.13) and (ORF5)-A 31 SNDSSSHLQLIYNLTLCELNGTDWL ANKFDWA 63  (SEQ ID NO.12) peptides, wherein the fragment GSSLDDFC is designated “P”, fragment YDSTAPQKVLLAFSITY “Q”, fragment ASNDSSSHLQLIYNLTLC “A”, and ELNGTDWLANKFDWA “B”. Fragment PQ is a portion of ORF6, and Fragment AB is a portion of ORF5. G is the gap or bridge of PQ and AB ploypeptides. G can be the 27 th  animo acid of ORF6 or any polypeptide fragment of ORF6 from 27 th  to any linked codons. The position G can also be not added any amino acid within the polypeptides of PQ and AB. 
     The example employs the PQGAB fusion peptide region to construct a key protein (epitope) capable of inducing neutralization titers and immune protection in order to obtain effects of inducing immune protection in vivo. The schematic illustration of PE-PQGAB-K3 fusion protein and the flowcharts of plasmids construction of PE(ΔIII)-PQGAB and PE(ΔIII)-PQGAB-K3 are shown in  FIG. 1  and  FIG. 2 , respectively. 
     EXAMPLE 2 
     The preparation of nucleic sequence encoding PQGAB peptide is illustrated below. An amino acid corresponds to various sets of nucleotide triplets, so it is preferable to obtain corresponding nucleotide triplets from literature (such as http://www.kazusa.orjp/codon) that is suitable to be expressed in the  E. coli  system instead of the corresponding nucleotide triplets not easy to be recognized and expressed by  E. coli.  Likewise, if the sequence encoding PQGAB peptide is to be expressed in yeast systems, the appropriate nucleotide triplets for expression in yeast systems (such as  Saccharomycesor Pichia  spp.) are preferred. 
     A corresponding sequence with nucleotide triplets suitable to be expressed in  E. Coli  system was designated according to the amino acid sequence of PQGAB fusion protein. The 5′ and 3′ ends of the corresponding sequence were added by restriction sites for subsequent cloning. To improve efficiency of digestion and facilitate designing PCR primers, both ends of the sequence could be added to with nucleotide triplets with replicating bases, such as CCC, AAA, GGG, or TTT. The nucleic acid sequence encoding PQGAB fusion protein (SEQ ID NO:9) of the American strain of PRRSV is illustrated in SEQ ID NO:1. 
     There are totally 207 nucleotides in SEQ ID NO:1, in which the coding region is flanked by the restriction sites CATATG (NdeI), GAATTC (EcoR I) at 5′ end, and CTCGAG (XhoI) at 3′. When it was cloned into a plasmid restriction enzyme sites, some of the nucleotide triplets were cut off, leaving 180-186 nucleotides ligated to the plasmid. 
     When the target nucleic acid sequence encoding PQGAB fusion protein was identified, the restriction map of the nucleic acid sequence was analyzed by DNA Strider before synthesis, and then each end of the target sequence was linked to restriction site sequences for subsequent cloning, in accordance with the restriction map. The synthesized product of the target sequence must be digested by certain restriction enzymes before cloning, so it is preferable that any restriction site susceptible to the enzymes used be avoided in the structural region of the sequence. If the restriction sites subjected to cloning enzymes exist in the structural region of the target sequence, the target sequence must be re-designated such that different codons of the same amino acids were used, to eliminate restriction sites that were identical for cloning in the structural region of the target sequence. 
     Subsequently, the method disclosed in Taiwan Patent No. 1-2289933 (also as U.S. patent publication no. 2004/02147617) is used to modify the corresponding nucleotides codons of wild type amino acids sequence such that the wild type protein was mass expressed in by  E. coli  system. The essence of modification is to modify wild type nucleic acid sequence such that the normally expressed amino acids were not affected, and expression in  E. coli  was kept effective. The modified nucleic acid sequence can be synthesized by PCR using a variety of primer pairs. The primers are numbered as shown in Table 1. 
     
       
         
               
             
               
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 the corresponding numbers of primers for PQGAB antigens 
               
               
                 of PRRSV American Strain 
               
             
          
           
               
                   
                 Forward 
                   
                 Reverse 
                   
               
               
                 Target antigen 
                 primer 
                 Seq. ID No. 
                 primer 
                 Seq. ID No. 
               
               
                   
               
               
                 PQGAB-US 
                 F1 
                 2 
                 R1 
                 6 
               
               
                 PQGAB-US 
                 F2 
                 3 
                 R2 
                 7 
               
               
                 PQGAB-US 
                 F3 
                 4 
                 R3 
                 8 
               
               
                 PQGAB-US 
                 F4 
                 5 
               
               
                   
               
             
          
         
       
     
     The sequences of forward and reverse primers are shown as follows: 
     Forward primer F1 (SEQ ID No.2) corresponds to the nucleotides 79 -122 of SEQ ID NO: 1, namely 
     
       
         
               
               
             
           
               
                 5′-GCT TTC TCC ATC ACC TAC GCT TCC AAC GAC TCC TCC 
                   
               
               
                 TCC CAC CT-3′; 
               
             
          
         
       
     
     Forward primer F2 (SEQ ID No:3) corresponds to the nucleotides 48-95 of SEQ ID No:1, namely 
     
       
         
               
               
             
           
               
                 5′-C GAC TCC ACC GCT CCC CAG AAA GTT CTG CTG GCT 
                   
               
               
                 TTC TCC ATC ACC TA-3′; 
               
             
          
         
       
     
     Forward primer F3 (SEQ ID No:4) corresponds to the nucleotides 22-65 of SEQ ID No:1, namely 
     
       
         
               
               
             
           
               
                 5′-GGT TCC TCC CTG GAC GAC TTC TGC TAC GAC TCC 
                   
               
               
                 ACC GCT CCC CA-3′; 
               
             
          
         
       
     
     Forward primer F4(SEQ ID No.5) corresponds to the nucleotides 1-40 of SEQ ID No:1, namely 
     
       
         
               
               
             
           
               
                 5′-CCC AAA CCC CAT ATG GAA TTC GGT TCC TCC CTG 
                   
               
               
                 GAC GAC T-3′; 
               
             
          
         
       
     
     Reverse primer R1(SEQ ID No.6) corresponds to the nucleotides 148-106 of SEQ ID No.1, namely 
     
       
         
               
               
             
           
               
                 5′-A CAG GGT CAG GTT GTA GAT CAG TTG CAG GTG GGA 
                   
               
               
                 GGA GGA GTC-3′; 
               
             
          
         
       
     
     Reverse primer R2(SEQ ID No.7) corresponds to the nucleotides 176-133 of SEQ ID No:1, namely 
     
       
         
               
               
             
           
               
                 5′-GC CAG CCA GTC GGT ACC GTT CAG TTC GCA CAG GGT 
                   
               
               
                 CAG GTT GTA-3′; 
               
             
          
         
       
     
     Reverse primer R3(SEQ ID No.8) corresponds to the nucleotides 207-164 of SEQ ID No:1, namely 
     
       
         
               
               
             
           
               
                 5′-TTT TTT CTC GAG AGC CCA GTC GAA TTT GTT AGC 
                   
               
               
                 CAG CCA GTC GG-3′; 
               
             
          
         
       
     
     wherein R1, R2 and R3 were reversely complementary sequences of a gene sequence. 
     The fragment synthesized with no DNA template was performed firstly. Forward primer F1 and reverse primer R1 were hybridized to each other, wherein 10-18 bases at 3′ ends of each primer were designed complementary to each other, and the resultant complex was read and complemented by polymerase so as to obtain a double-stranded DNA template product. 
     After the first round of PCR, 0.01˜4 μl of the PCR product was taken as the template DNA of the second round of PCR, adding therein the second primer pair, i.e. forward primer F2 and reverse primer R2, 0.01˜4 μl each, in conjunction with needed dNTPs, reagents and Pfu polymerse, and the second round PCR was performed. Likewise, primer pair F3 and R3 were added therein and PCR was performed again; the procedures were repeated with primer pair F4 and R3, and thereby a modified PQGAB nucleic acid sequence having 207 bp was obtained. 
     The synthesized nucleic acid fragments were subjected to electrophoresis and confirmed that they had the expected sizes. PQGAB-1(207 bp), as shown in  FIG. 3 ; PQGAB generated 4 DNA fragments a, b, c, and d (a: 70 bp b: 129 bp c: 186 bp, d: 207 bp). 
     EXAMPLE 3  
     PQGAB Fragment of PRRSV European Strains 
     The design of the fusion protein in example 1 and 2 aimed at American strain PRRSV, but apart from American strain PRRSV, European strain and Australian strain is also very prevalent globally. Similarity of structural amino acids is not high, only 60-80%, so designs of other ORF5&amp;ORF6 fusion proteins can be done in the same manner as example 1 and 2 to design and synthesize primers. 
     Taking PQGAB of the European strain of PRRSV as an example, the amino acid sequence of the fusion protein is shown in SEQ ID NO.10. It contains the amino acid residues from 1to 28of the ORF 6 (i.e., SEQ ID NO.15) structual proteins of the European strain of PRRSV. 
     After the sequence is confirmed, preparation of PRRSV European strain fusion proteins can be performed in the same manner as examples 1-2. The modified nucleic acid sequence can be synthesized by PCR using a variety of primer pairs. The primers are numbered as shown in Table 2. 
     
       
         
               
             
               
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 the corresponding numbers of primers for PQGAB antigens of 
               
               
                 PRRSV European Strain 
               
             
          
           
               
                   
                 Forward 
                   
                 Reverse 
                   
               
               
                 Target antigen 
                 primer 
                 Seq. ID No. 
                 primer 
                 Seq. ID No. 
               
               
                   
               
               
                 PQGAB-EP 
                 F1 
                 16 
                 R1 
                 20 
               
               
                 PQGAB-EP 
                 F2 
                 17 
                 R2 
                 21 
               
               
                 PQGAB-EP 
                 F3 
                 18 
                 R3 
                 22 
               
               
                 PQGAB-EP 
                 F4 
                 19 
                 R4 
                 23 
               
               
                   
               
             
          
         
       
     
     The target nucleic acid sequence encoding PQGAB-EP fusion protein can be synthesized with those primers shown above in vitro, by following the procedure described in example 2. To improve efficiency of digestion and facilitate designing PCR primers, both ends of the sequence could be added to with nucleotide triplets with replicating bases, such as CCC, AAA, GGG, or TTT. The nucleic acid sequence encoding PQGAB-EP fusion protein is illustrated in SEQ ID NO.11: in which the coding region is franked by the restrictions site GAATTC (EcoR I), CATATG (Nde I) and GACGTC (Sal I) at 5′ end, and CTCGAG (Xho I) at 3′ . 
     EXAMPLE 4  
     Construction of Plasmids Containing the Target Sequence 
     Taking the product from example 2 as illustration. The synthesized 207-bp DNA fragment was digested with EcoR1 and Xho1, linked to a  E.coli  plasmid containing a peptide sequence having functions of binding and translocation, and a carboxyl terminus peptide, and the resultant plasmid was pPE-PQGAB-K3. 
     The pET15 plasmid having a T7 promoter and an antibiotic resistance(ampicillin) marker constructed therein can express the fusion protein of PRRSV PQGAB fragment and detoxified  Pseudomonas exotoxin  ( Pseudomonas exotoxin  A without domain III). The vector map is shown in  FIG. 4 . 
     Finally, the above-mentioned plasmid was transformed into bacterial strains or cells capable of expressing the fusion proteins. 
     EXAMPLE 5  
     Expression and Analysis of the Target Protein 
     Bacterial strains confirmed having the above mentioned plasmid contained both the plasmid and PQGAB gene in 90% of the population. The strains were prepared as glycerol stocks in 2-ml aliquots and stored at −70° C. In a sterile environment, 2 ml of the stored stocks was inoculated in an autoclaved 500 ml flask containing 200 ml LB (+500 μg/ml Amp), shaken in a rotary incubator at 37° C., 150 rpm for 10˜12 hours, and a culture was obtained. The liquid was cultured until OD600 reached 1.0±0.4. 
     In a sterile environment, 50 ml culture liquid was inoculated in each of eight 3000-ml flasks containing 1250 ml LB (+500 μg/ml Amp +50 ml 10% Glucose), shaken at 37° C., 150 rpm for 2˜3 hours until OD 600 reached 0.3±0.1, 50 ppm IPTG was added, the culture was shaken again at 37° C., 150 rpm for 2 hours such that protein production was accomplished. 
     Then PE-PQGAB-K3 contained in inclusion bodies was dissolved by 8M urea extraction method, the extracted PE-PQGAB-K3 proteins are shown in  FIG. 5 . 300˜400 mg antigen could be obtained from a 10-liter lot of the culture liquid. Obtained antigen was analyzed with Western blot, coomasie blue staining and SDS-PAGE electrophoresis, the density of the bands was measured by densitometer to quantify proteins contained in antigen solutions. 0.2±0.02 mg of the proteins were used as the main ingredient of a low-dosage injection in order to proceed immunization and virus-challenging. 
     EXAMPLE 6  
     Immunization and Virus-Challenging in Pigs 
     In an SPF farm, pigs were grouped randomly into 3 groups, each having five pigs. Each group was bred in an isolation room equipped with air conditioning and circulation instruments. For pigs of PE-PQGAB-K3 immunization group aged 14 to 28 days, 2 ml vaccine containing 1 ml PE-PQGAB-K3 (containing 200 μg proteins/injection) and emulsified in 1 ml ISA206 (SEPPIC®, France) was injected intramuscularly, and the procedure of immunization was performed twice. The immunization group GP5&amp;M was immunized with PE-ORF5-K3             PE-ORF6-K3(containing 200 μg proteins/injection), respectively. The control group was bred without immunization.
     Two weeks after the last inoculation, 100 mg ketamine solution was administered intramuscularly to tranquilize the pigs, then 1 ml 2% Lidocaine was dropped in the nasal cavities of the pigs to inhibit coughing reflex actions, and then the virus was inoculated in pigs nasally. Five pigs of each group were inoculated with 1 ml MD-1 strain PRRSV cultures having a 1×10 7  TCID 50 /ml dosage. 
     14 days after inoculation, the pigs were sacrificed to proceed with complete autopsy. Lung or liver samples were collected (from both parts of the head lobe central part and auxiliary part of caudate lobe) and fixed by 10% neutral buffered formaldehyde for subsequent tissue pathology examination. The examination was conducted in a blind fashion and evaluated on the basis of interstitial pneumonitis severity (Opriessnig T, P. G. Halbur, et al., Journal of Virology, 76(2002):11837-11844, and Halbur, P. G., P. S. Paul, et al., 1996. J. Vet. Diagn. Investig. 8:11-20) in a scale from 0 to 6, wherein the severity increases with the number. 
     Experimental Results 
     Two weeks post second immunization, leukocytes from porcine blood were tested for PRRSV. The results indicated that viremia did not occur in any pig before PRRSV inoculating. The leukocyte samples were tested with RT-PCR at 3, 7, and 14 days post virus inoculating, respectively, and the results are shown in Table 3. 
     
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 PRRSV viremia occurrence in pigs post PRRSV 
               
               
                 inoculating 
               
             
          
           
               
                   
                   
                   
                   
                 PE-ORF5-K3 
               
               
                   
                 Day 
                 Control 
                 PE-PQGAB-K3 
                 PE-ORF6-K3 
               
               
                   
                   
               
             
          
           
               
                   
                 3 
                 3/5 
                 3/5 
                 2/5 
               
               
                   
                 7 
                 3/4 (death1*) 
                 2/5 
                 2/4 (death2*) 
               
               
                   
                 14 
                 3/4 (death1*) 
                 2/5 
                 2/4 (death2*) 
               
               
                   
                   
               
               
                   
                 *identified with PRRSV viremia by RT-PCR before death 
               
             
          
         
       
     
     All pigs, including those that had been sacrificed and the surviving after the two-week study, were dissected. Macroscopic examinations indicated that the lungs from virus-inoculated pigs of ORF5&amp;ORF6 vaccine group and the control group showed more extensive lesions and severe interstitial pneumonitis, whereas the PE-PQGAB-K3 vaccine group of the present invention did not show as extensive lesions and severe interstitial pneumonitis. As shown in Table 4, the PE-PQGAB-K3 vaccine group of the present invention showed less severity in terms of interstitial pneumonitis than the control group and ORF5&amp;ORF6 vaccine group. 
     
       
         
               
             
               
               
               
               
             
           
               
                 TABLE 4 
               
             
             
               
                   
               
               
                 comparisons of macroscopic lung lesions induced by 
               
               
                 PRRSV, 14 days post PRRSV inoculating 
               
             
          
           
               
                   
                   
                 PE-PQGAB-K3 
                 PE-ORF5-K3, 
               
               
                   
                   
                 vaccine 
                 PE-ORF6-K3 vaccine 
               
               
                   
                 Control group 
                 group 
                 group 
               
               
                 Pig No. 
                 Lesion index 
                 Lesion index 
                 Lesion index 
               
               
                   
               
               
                 1 
                  6* 
                 5 
                 6 
               
               
                 2 
                 6 
                 3 
                 5 
               
               
                 3 
                 6 
                 4 
                 6 
               
               
                 4 
                 6 
                 4 
                 6 
               
               
                 5 
                 5 
                 3 
                 6 
               
               
                 Average 
                 5.75 ± 0.50 
                 3.80 ± 0.84 
                 5.80 ± 0.45 
               
               
                   
               
               
                 *interstitial pneumonitis lesion index 
               
             
          
         
       
     
     
       
         
               
             
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 5 
               
               
                   
               
               
                 macroscopic lung lesion indexes exhibited by the 
               
               
                 PE-PQGAB-K3 vaccine group are significantly lower than control group 
               
               
                 and ORF5&amp;ORF6 vaccine group in view of biostatistics. 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                   
                   
                 Number of 
                   
                   
                   
               
               
                   
                 Group 
                 individuals 
                 Total 
                 Average 
                 Variance 
               
               
                   
                   
               
               
                   
                 Control group 
                 5 
                 29 
                 5.8 
                 0.2 
               
               
                   
                 PE-PQGAB-K3 group 
                 5 
                 19 
                 3.8 
                 0.7 
               
               
                   
                 PE-ORF5&amp;ORF6-K3 group 
                 5 
                 29 
                 5.8 
                 0.2 
               
               
                   
                   
               
             
          
           
               
                 ANOVA 
               
             
          
           
               
                 Variation 
                   
                 Degree of 
                   
                   
                   
                 Critical 
               
               
                 source 
                 SS 
                 freedom 
                 MS 
                 F 
                 P-value 
                 value 
               
               
                   
               
               
                 Inter-group 
                 13.33333 
                 2 
                 6.666667 
                 18.18182 
                 0.000233 
                 3.885294 
               
               
                 Intra-group 
                 4.4 
                 12 
                 0.366667 
               
               
                 total 
                 17.73333 
                 14 
               
               
                   
               
             
          
         
       
     
     The above experiments clearly indicate that PE-PQGAB-K3 of the present invention not only can effectively protect pigs from PRRSV infections, but also cause slighter interstitial pneumonitis than other vaccines (such as PE-ORF5-K3, PE-ORF6-K3). 
     The antibody titers variation in immunized pigs are shown in table 6. The A group has good IgG ELISA titers, but the IFA and NT titers are less than that of C group. Also, from table 5, it indicates that PRRSV ORF5 or ORF6 have an antigen-specific allergy effect after immunization and virus challenged. Manifestly, it is difficult to use them as PRRS vaccine antigens. 
     
       
         
               
             
               
               
               
             
               
               
               
               
               
             
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 6 
               
             
             
               
                   
               
               
                 Serum titers 
               
             
          
           
               
                   
                 coating antigen 
                   
               
             
          
           
               
                   
                 PE(Δ III) 
                 PQGAB 
                   
                   
               
             
          
           
               
                 Group 
                 IgG-ELISA titers (S/BK) 
                 IFA titers 
                 NT titers* 
               
               
                   
               
             
          
           
               
                 A 
                 12 
                 80 
                  8-16 
                  8-16 
               
               
                 PE-ABCF-K3 
               
               
                 PE-PQGF-K3 
               
               
                 B 
                 1 
                 1 
                 &lt;8 
                 &lt;8 
               
               
                 Negative CTL 
               
               
                 C 
                 17 
                 30 
                 32-64 
                 16-64 
               
               
                 PE-PQG1AB-K3 
               
               
                   
               
               
                 *The neutralization titer is determined by the inhibition growth and proliferation of PRRSV under serial dilution sample added in MAC-10A cells culture system. 
               
             
          
         
       
     
     Although the present invention has been explained in relation to its preferred embodiment, it is to be understood that many other possible modifications and variations can be made without departing from the scope of the invention as hereinafter claimed.