TW200804426A - Fusion protein of porcine reproductive and respiratory syndrome virus as PRRS vaccine - Google Patents

Abstract

The present invention provides a fusion protein, PE-PQAB-K3, of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) for use as a vaccine for porcine reproductive and respiratory syndrome (PRRS), which comprises Pseudomonas exotoxin domain I (combination domain-transposition domain II), a PQAB structural peptide which comprises the N-terminals of ORF5 and ORF 6 of PRRSV and binds the cell receptors, and a signal code, KDEL-KDEL-KDEL (KDEL3), linking at the carboxyl terminal of the fusion protein. The serum samples obtained from the mice or pigs immunized with the PE-PQAB-K3 fusion protein of the present invention can inhibit the growth and reproduction of PRRSV in MAC-10A cells, and the neutralization titer can reach the range from 16 to 32.

Description

200804426 IX. Description of the Invention: [Technical Field of the Invention] The present invention relates to the construction of a fusion protein, and according to the development of a fusion protein sub-unit vaccine which induces a PRRSV neutralization valence reaction in pigs. [Prior Art] Porcine reproductive and respiratory syndrome (PRRS) is a kind of infection caused by porcine reproductive and respiratory syndrome virus (PRRSV) and is caused by sow reproductive disorders and respiratory infections of pigs of all ages. The pig is infected with the disease. PRRSV is considered to be a very tenacious virus based on the fact that it is not easy to induce antibodies with neutralizing power in the host, and PRRSV is an RNA virus that propagates in a host cell via a simplified gene replication system, and the probability of gene mutation is very high. In addition, PRRSV infection can be divided into two phases: A upper respiratory tract and lower respiratory epithelial cell infection system; and mononuclear spheres and macrophages in the peripheral tissues of B respiratory tract. Therefore, the host needs to have both a humoral or mucosal immune response mechanism and a cellular immune mechanism to remove the infected cells for defense. Because antibodies have little effect on PRRSV, they can even mutate the virus. In antibody-dependent potentiating mechanisms, the use of antibodies may result in more severe infections. Patent No. 1-2289933 of the Republic of China provides a fusion protein specific for a target cell, which utilizes the PRRSV ORF7 nuclear protein of the functional region of the Pseudomonas aeruginosa exotoxin site, and adds KDEL to the carboxyl terminus. code. The fusion protein can be produced in a large amount in E. coli, and after the immunization of the pig, it can effectively reduce and eliminate the viremia after the pig is challenged. The entire text of this patent is incorporated herein by reference. In the ORF5-6 heterodimerization of PRRSV, the Cys-34 epitope region of ORF5 and the Cys-8 anti-determination region of ORF6 are critical for viral infection and its envelope assembly. (Snijder Eric J., Jessica C. et al., Journal of Virology, January 2003, Vol. 77, No. 1:97-104) The same sequence of 〇PRRSV ORF5 (consensus sequence) YKNTHLDLIYNA, between amino acids 38 The epitope between 44 and 44, located in the N-terminal region of PRRSV ORF5, identified as a neutralizing epitope (Ostrowski M·, J. A. Galeota, et al.,

Journal of Virology, May 2002, Vol. 76, No. 9: 4241-4250) ° The traditional technique of ORF5 or ORF6 has been constructed in the middle of PE and KDEL. After immunization to pigs, the infected pigs were found. In the lungs, more serious inflammation occurs, and it is clear that ORF5 or ORF6 is more immunotoxic than ORF7. Therefore, in order to effectively prevent pigs from infecting PRRS, it is still necessary to develop vaccines with high and high prices and safe. SUMMARY OF THE INVENTION One object of the present invention is to provide a fusion protein, and a combination thereof. Another object of the present invention relates to the preparation of a subunit PRRS vaccine having the composition of a neutralizing force fusion protein using the fusion proteins. A further object of the invention is to provide a pharmaceutical composition comprising a fusion protein of the invention and a pharmaceutically acceptable carrier. The detailed description of the invention is set forth in the following description. Other features, objects, and advantages of the invention will be apparent from the description of the invention. [Embodiment] The present invention is characterized in that most of the antigens of ORF5 and ORF6 are removed, leaving only a few amino acids at the N-terminus of ORF5 and ORF6, thereby constructing a heterozygous PQAB and embedding it in PE. In the middle of KDEL3, the fusion protein PE-PQAB-KDEL3 was tested by immunoassay in mice and pigs to confirm the ability to neutralize the serum. The following examples are provided to illustrate the invention, but are not intended to limit the scope of the invention. Example 1: The PE-PQAB-K3 fusion protein is known to be transfected according to the above-mentioned viral infection. The region of the PRRSV virus with neutralizing power is the fusion protein region of the N-terminus of ORF6 and ORF5, which is the 0RF6 structural protein No. 2 to 26 Amino acid peptide region and amino acid peptide region of ORF5 structural protein Nos. 30 to 63. In this example, the fusion region is used to construct a segment of a key protein (i.e., an antigenic determinant) that induces neutralizing valency immunoprotection to achieve an in vivo immune effect. The flow chart of the PE-pQAB-K3 fusion protein and the flow chart of the PE(MII)-PQAB plastid are shown in Figures 1 and 2, respectively. The DNA sequence of PRRSV was obtained from the National Center for Biotechnology Data Center (NCBI) and the ORF5 and ORF6 structural protein amino acid sequences were sequenced into standards. The nucleotide sequence corresponding to the target amino acid sequence is first ligated to a target nucleic acid sequence, and the sequence of the fusion protein region is as follows: 107547.doc 200804426 GSSLDDFCYDSTAPQKVLLAFSITYASNDSSSHLQLIYNL TLCELNGTDWLANKFDWA (SEQ ID NO. 2) ie? 1^\^-0^^6-2~26-0^^5-31~63 fusion skin area, which consists of two segments (ORF6)-G2SSLDDFCYDSTAPQKVLLAFSITY26 and (ORF5)·A31SNDSSSHLQLIYNLTLCELNGTDWLANKFDWA63, among them, GSSLDDFC The fragment is called P, the YDSTAPQKVLLAFSITY fragment is called Q, the ASNDSSSHLQLIYNLTLC fragment is called A, and the ELNGTDWLANKFD WA fragment is called B. The PQ fragment belongs to ORF6, and the AB fragment belongs to the ORF5 protein fragment. The following describes the preparation of the nucleotide sequence. Since there are many pairs of nucleotide sequences corresponding to the amino acid sequence, it can be known in the academic literature (such as http:"www·kazusa.or.jp/codon) in Escherichia coli. (5 · a ") The host system is more suitable for the nucleotide corresponding sequence, avoiding the nucleotide corresponding sequence that E. coli is not easy to identify and express. Similarly, if the sequence is to be expressed in yeast, it is also necessary to select a sequence suitable for expression by the yeast system (Saee/zaromycw or P/c/n: spp.). The nucleotide sequence corresponding to the expression of the E. coli host system is designed as follows, and the restriction enzyme sequences are respectively attached to the 5' and 3*-termini of the nucleotide pair to facilitate the subsequent gene transfer step. In order to increase the efficiency of enzyme cleavage and facilitate the design of PCR primers, the gene can be added with three consecutive CCC, AAA, GGG or TTT °. Table 1. Genetic code of PQAB fusion peptide SEQ ID N0.1:

5!.CCC AAA CCC CAT ATG GAA TTC GGT TCC TCC CTG 107547.doc 200804426

G S S L

GAC GAC TTC TGC TAC GAC TCC ACC GCT CCC CAG DDFC YDS ΤΑ P Q

AAA GTT CTG CTG GCT TTC TCC ATC ACC TAC GCT K V L L A F S I T Y A

TCC AAC GAC TCC TCC TCC CAC CTG CAA CTG ATC S N D S S S HLQ LI

TAC AAC CTG ACC CTG TGC GAA CTG AAC GGT ACC YNLTLC EL NGT

GAC TGG CTG GCT AAC AAA TTC GAC TGG GCT CTC D WLAN K F D W A GAG AAA AAA-3, a total of 204 nucleotide pairs, at least 189 to 183 nucleotide pairs in length when entering into the plastid when splicing with an enzyme. The computer software (such as DNA strider) is used to analyze the restriction enzyme map of the target nucleic acid sequence, and according to the result of the map, the restriction enzyme cleavage sequence used for splicing after the end of the target nucleic acid sequence is installed, since the generated target substance is required For restriction enzyme splicing, it is best not to have the restriction enzyme cleavage required for splicing in the target DNA sequence. Therefore, it is necessary to use the commonly used software to analyze whether there are these restriction maps in the DNA sequence, if the target There is a restriction enzyme cleavage sequence that should not be contained in the nucleic acid sequence, and the amino acid signal must be searched again, and the nucleotide sequence corresponding to the same amino acid is used to change, so that the sequence no longer has a restriction enzyme cleavage position. Using the method disclosed in the aforementioned Patent No. 1-2289933, the nucleotide sequence encoded by the wild type amino acid is modified so that the protein of 107547.doc 200804426 can be expressed in large quantities by the Escherichia coli system; The focus of the reform is mainly on the nucleotide sequence of the wild-type virus strain, in the condition that it does not affect its originally displayed amino acid, and can be effectively expressed in the E. coli host system. The upgrade. The nucleotide sequence after the modification can be synthesized by a polymerase chain reaction using a plurality of pairs of primers respectively, and the corresponding numbers of all the primers are shown in Table 2. Table 2: Target antigen PQAB primer pair corresponding numbered antigen forward primer sequence number (Seq. ID No.) Reverse primer sequence number (Seq. ID No.) PQAB F1 3 R1 7 PQAB F2 4 R2 8 PQAB F3 5 The sequences of the R3 9 PQAB F4 6 forward and reverse primers are as follows: The forward primer F1 (SEQ ID Νο·3) is the amino acid sequence of Nos. 81 to 124 of SEQ ID Νο·1, ie, 5 '-GCT TTC TCC ATC ACC TAC GCT TCC AAC GAC TCC TCC TCC CAC CT-31 ; Forward primer F2 (SEQ ID Νο·4) is the amino acid sequence of Nos. 48 to 96 of SEQ ID No. 1, ie, 5f-C GAC TCC ACC GCT CCC CAG AAA GTT CTG CTG GCT TTC TCC ATC ACC TA-3f ; Forward primer F3 (SEQ ID Νο·5) is the amino acid sequence 22 to 65 of SEQ ID Νο·1 That is, 5 107547.doc -10- 200804426 5,-GGT TCC TCC CTG GAC GAC TTC TGC TAC GAC TCC ACC GCT CCC CA-3f ; Forward primer F4 (SEQ ID No. 6) is the first of SEQ ID No. To amino acid sequence No. 41, ie, 5f-CCC AAA CCC CAT ATG GAA TTC GGT TCC TCC CTG GAC GAC T-3,; reverse primer R1 (SEQ ID Νο·7) is the 148th of SEQ ID No. To the amino acid sequence of 106, ie 5f-A CAG GGT CAG GTT GTA GAT CAG TTG CAG GTG GGA GGA GGA GTC-3f ; Reverse primer R2 (SEQ ID Νο·8) is the amino acid sequence of Nos. 176 to 133 of SEQ ID No. 1, ie, 5*-GC CAG CCA GTC GGT ACC GTT CAG TTC GCA CAG GGT CAG GTT GTA-31 ; Reverse primer R3 (SEQ ID Νο·9) is the amino acid sequence of Nos. 204 to 164 of SEQ ID No. 1, ie , 5»-TTT TTT CTC GAG AGC CCA GTC GAA TTT GTT AGC CAG CCA GTC GG-3,; wherein R1, R2 and R3 are the reverse complement gene sequences of the first gene sequence code. First, the polymerization of nucleotide fragments is carried out by means of a polymerization method without a DNA template, using a forward primer F1 and a reverse primer R1, wherein each of the primers has 10-18 bases each of which is designed to be complementary to each other. In order to become a double-stranded DNA template polymerization product by reading and writing and complementing the polymerase. After the first PCR is completed, 0.01~4 μΐ of the polymerized product is taken as the template DNA of the second 107547.doc 200804426 PCR, and the second set of primers is added to the positive primer F2 and the reverse primer R2 each 0.01 to 4 μΐ. Along with the required dNTPs, reagents, and Pfu polymerase, the second PCR is started; then, in the same manner, different primers are added to perform PCR on F3 and R3, and then PCR steps of primers on F4 and R3 are performed. The 204 bp nucleotide sequence after PQAB modification can be synthesized. * After the electrophoresis test of the separately synthesized nucleotide fragments, it was confirmed that the fragment size of the product was as expected. PQAB-1 segment (204 bp), as shown in Figure 3; PQAB- produces a total of 4 DNA fragments of a, b, c and d (a segment 70 bp, b segment 129 bp, c segment 186 bp, d segment 204 bp). Example 2: plastid construction with the indicated sequence The above-mentioned 204 bp DNA fragment was synthesized by restriction enzymes EcoRl and Xhol, respectively, and the peptide sequence which has a binding and translocation function, and a carboxyl group terminal Shengtai's large intestine were inserted. In the bacilli, the resulting plastid is the pPE-PQAB-K3 plastid. Constructed in a pET15 plastid system containing a T7 promoter and an antibiotic (ampicilin) resistant fragment, which can express a fusion protein containing a PRRSV PQAB and a detoxified A exoioxh A without domain III), a plastid map system As shown in Figure 4. Finally, the above plastids are separately transfected into a strain or cell which can express the fusion protein. Example 3: Performance and analysis of the target protein

More than 90% of the bacteria containing the above plastids contained plastid and PQAB genes, which were stored in a 2 ml aliquot in a glycerol storage method at -70 ° C 107547.doc -12- 200804426. In a sterile room, 2 ml of the above preserved vaccine is inoculated into a sterilized 500 ml flask containing 200 ml LB (+500 pg/ml Amp) at a rotary incubator at 37 ° C at 150 rpm. The culture is carried out for 10 to 12 hours to complete the inoculum culture. The OD600 of the vaccine should reach 1.〇±〇.4. In a sterile room, 50 ml of the bacterial solution is inoculated separately into a sterilized 3000 ml triangle with a total of eight bottles containing 1250 ml LB (+500 pg/ml Amp + 50 ml 10°/. Glucose). At 37. (: Rotary incubator is incubated at 150 rpm for 2 to 3 hours, and when OD600 is detected at 0·3±0·1, the final concentration of 50 ppm of IPTG protein inducer is injected, and the rotary incubator is at 37 °C. The protein production step was completed by incubating at 150 rpm for 2 hours. Next, the PE-PQAB-K3 (Fig. 3) antigen protein fragments in the inclusion bodies were extracted and dissolved by the 8M urea method, each with ten liters of bacteria. Liquid (a small batch, lot) extraction, can get 300 ~ 400 mg antigen amount. Using Western-blotting method, coomasie blue staining, SDS-PAGE electrophoresis analysis, and density meter (densitometer) The density of the electrophoresis bands was measured to quantify the protein of each antigen solution. The above antigen protein was taken as 22±〇·〇2 mg as the main component of the high dose of the injection. Take 0·02±0·002 mg as the main component. The low-dose main component of the injection is used for the efficacy test of pig immunization and challenge. Example 4: Efficacy test of pig immunization and challenge. In SPF farm, pigs were randomly divided into three groups of five pigs each. And each group of pigs is raised separately In the isolation room with air conditioning and ventilation equipment. Pigs in the PE-PQAB-E3 vaccination group were injected intramuscularly with 1 ml of PE-PQAB-E3 (containing 200 gg protein/dose at 14 and 28 days of age). ), 107547.doc •13· 200804426 2 ml vaccine emulsified in 1 ml ISA206 (SEPPIC® France), immunized twice. GP5 & M immunized group with PE-ORF5-K3 and PE-ORF6-K3 (each containing 200 pg protein/dose was immunized. The control pigs were reared without immunization. Two weeks after the last vaccination, the pigs were intramuscularly administered with 100 mg of ketamine solution for sedation. Then, after intranasal drops of 1 ml of 2% lidocaine to inhibit cough-reflex, the pigs were challenged intranasally. Fresh 1 ml of MD-1 strain of PRRSV culture was used, approximately 1×l07 TCID50 A dose of /ml was used to challenge five pigs in each group. On the 14th day after inoculation, all sacrificed pigs were completely dissected. Liver samples were collected (from the two parts of the hood and each from the hepatic caudate lobe Middle and subsidiary parts) and secured with 10% neutral buffered fumarin For subsequent histopathological examination, the test was based on blind fashion and was assessed according to the severity of interstitial pneumonia (Opriessnig T, PG Halbur, et al., Journal of Virology, 76 (2002): 1 1837-11844 and Halbur, PG·, PS Paul, et al., 1996· J. Vet· Diagn· Investig. 8:11-20), distinguished by 0 to 6. The greater the number, the more severe the lesion. EXPERIMENTAL RESULTS Two weeks after the second immunization, pig blood leukocyte samples were tested for PRRSV by RT-PCR. The results showed that all pigs did not cause viremia before PRRSV challenge. However, after 3, 4, and 14 days after the challenge, the blood leukocyte samples of the pigs were again detected by RT-PCR, and the results are shown in Table 3. 107547.doc • 14· 200804426 Table 3 ·· PRRS V viremia proportion of pigs after PRRS V challenge control group PE-PQAB-K3 PE-ORF5-K3 PE-ORF6-K3 3 3/5 3/5 2/5 7 3/4 (dead 1*) 2/5 2/4 (dead 2*) 14 3/4 (dead 1) 2/5 2/4 (dead 2*) * Table of dead pigs before PRRS V viremia was detected by RT-PCR and all pigs (including pigs that had been sacrificed and pigs that survived at the end of the two-week study) were dissected. The giant examination showed that the pigs showed lung after challenge. In the control group and the ORF5& ORF6 vaccine group, a wide range of lesions and severe interstitial pulmonary inflammation were observed, while the PE-PQAB-K3 vaccine group of the present invention did not find such extensive lesions and interstitial pulmonary membranes. As shown in Table 4, the severity of the giant lung lesions exhibited by the PE-PQAB-K3 vaccine group of the present invention was significantly smaller than that of the control group and the ORF5& ORF6 vaccine group. Tables 4 and 5 respectively illustrate the PRRSV attack. Comparison of macroscopic lung lesions induced by PRRSV 14 days after poisoning, and macroscopic lung lesion index of PE-PQAB-K3 vaccine group 107547.doc 15 200804426 Table 4: 14 after PRRSV challenge Comparison of PRRSV-induced macroscopic lung lesions Pig number control group Lesion index PE-PQAB-K3 vaccine group lesion index PE-ORF5-K3, PE-ORF6-K3 vaccine group 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 3.80 5.80 Index ±0.50 ±0.84 ±0.45 *: Indicates the index of interstitial pneumonia. Table 5. Giant view of PE-PQAB-K3 vaccine group The lung lesion index was significantly different from the control group and the ORF5& ORF6 vaccine group (ρ<0·01). The total number of variances in the control group was 5 29 5.8 0.2 PE-PQAB-K3 group 5 19 3.8 0.7 PE- ORF5&ORF6-Κ3 group 5 29 5.8 0.2

ANOVA variable source SS degrees of freedom MS F P-value threshold group 13.33333 2 6.66666718.18182 0.000233 3.885294 group 4.4 12 0.366667 sum 17.73333 14 107547.doc •16- 200804426 comparison control group and ORF5& ORF6 vaccine group low (Ρ< 〇·〇1) According to the above experimental results, it is clear that the PE-pQAB-K3 of the present invention is effective not only to protect pigs from PRRSV infection and the vaccine according to the present invention, which is relative to other vaccines (for example, PE-ORF5-K3) , PE-ORF6-K3) showed milder interstitial pneumonia. Table 6 shows the change in antibody titer in immunized pigs. In group A, the IgG ELISA price of Liangzhu is shown, but the indirect fluorescent antibody analysis (IFA) and the neutralization (NT) price are both Lower than Group C. Furthermore, as shown in the results of Table 5, antigen-specific allergic effects were produced after immunization and challenge with pigs with PRRSV ORF5 or ORF6. Therefore, ORF5 and ORF6 are not suitable for use as PRRSV vaccine antigens. Table 6. Serum price group of pig immunization experiment coated antigen IFA Price price NT price * ΡΕ (ΔΙΙΙ) PQGAB IgG-ELISA price (S/BK) A PE-ABCF-K3 PE-PQGF-K3 12 80 8-16 8-16 B Negative CTL 1 1 <8 <8 C PE-PQG1AB-K3 17 30 32-64 16-64 : The neutralization price is obtained by adding serially diluted PRRSV samples to MAC_ 1 〇 In the A cell-assisted system, the degree of inhibition of growth and proliferation was measured and measured. BRIEF DESCRIPTION OF THE DRAWINGS 107547.doc -17- 200804426 Figure 1 shows a schematic diagram of the PE-PQAB-K3 fusion protein according to Example 1. 2 is a flow chart showing the construction of a PE(AIII)-PQAB plastid according to Example 1. Fig. 3 is a view showing the electrophoresis pattern of the nucleotide fragment synthesized according to Example 1, which has a total of four DNA fragments (a segment 70 bp, a b segment 129 bp, a c segment 186 bp, and a d segment 204 bp). Figure 4 shows the PE(MII)-PQAB plastid map. 107547.doc 18· 200804426 Sequence Listing <110> Taiwan Institute of Zoology, <120> As a Fusion Protein of PRRS Virus (PRRSV) for Porcine Reproductive and Respiratory Syndrome (PRRS) Vaccine<140>141>;<150><151><160> 9 <170>Word7.0 <210> 1 <211> 204 <212> DNA <213> Artificial sequence <400> 1 cccaaaccccatatcgaattc ggt Gly Tee Ser tee Ser ctg Leu gac Asp 5 ttc gac Asp ttc Phe tgc Cys tac Tyr gac tee acc get ccc cag aaa JL gtt ctg ctg get tee ate acc tac Asp 10 Ser Thr Ala Pro Gin 15 Lys Val Leu Leu Ala 20 Phe Ser Lie Thr Tyr 25 get tee aac gac tee tee tee cac ctg caa ctg ate tac aac ctg acc Ala Ser Asn Asp Ser 30 Ser Ser His Leu Gin 35 Leu He Tyr Asn Leu 40 Thr ctg tgc gaa ctg aac ggt acc gac tgg ctg get Aac aaa ttc gac tgg Leu Cys Glu Leu 45 Asn Gly Thr Asp Trp 50 Leu Ala Asn Lys Phe 55 Asp Trp 48 96 144 152 204 get ctcgagaaaaaa Ala 58 <210> 2 <211> 58 <212> PRT <213> Manual Sequence <400〉 2

Gly Ser Ser Leu Asp 5 Asp Phe Cys Tyr Asp 10 Ser Thr Ala Pro Gin 15 Lys Val Leu Leu Ala 20 Phe Ser lie Thr Tyr 25 Ala Ser Asn Asp Ser 30 Ser Ser His Leu Gin 35 Leu lie Tyr Asn Leu 40 Thy Leu Cys Glu Leu 45 Asn Gly Thr Asp Trp 50 Leu Ala Asn Lys Phe 55 Asp Trp Ala 58 107547.doc 200804426 <210> 3 <211> 44 <212> DNA <213> Artificial Sequence <400> 3 Getttctcca tcacctacgc ttccaacgac tcctcctccc acct <210> 4 <211> 48 <212> DNA <213> Artificial sequence <400> 4 cgactccacc gctccccaga aagttctgct ggctttctcc atcaccta <210> 5 <211> 44 <212> DNA <213> Artificial Sequence <400> 5 ggttcctccc tggacgacctt ctgctacgac tccaccgctc ccca <210> 6 <211> 40 <212> DNA <213>Artificial Sequence<400> 6 cccaaacccc atatggaatt cggttcctcc ctggacgact <210&gt 7 <211> 43 <212> DNA <213> Artificial sequence <400> 7 acagggtcag gttgtagatc agttgcaggt gggaggagga gtc <210> 8 <211> 44 <212> DNA <213>Artificialsequence<400> 8 gccagccagt cggtaccgtt cagttcgcac agggtcaggt tgta 44 48 44 40 43 44 107547.doc -2- 200804426 <210> 9 <211> 44 <212> DNA <213> Artificial sequence< 400> 9 ttttttctcg agagcccagt cgaatttgtt agcccagccag tcgg 107547.doc

Claims (1)

200804426 X. Patent application scope: 1. A PE-PQGAB-K3 fusion protein comprising: - ORF6 and ORF5 of the porcine reproductive and respiratory syndrome virus (PRRSV) - N-terminal fusion protein region; - Pseudomonas aeruginosa Toxin A shifts the functional site; and -carboxy terminal moiety KDEL-KDEL-KDEL (KDEL3). 2. The fusion protein of claim 1, wherein the ORF6 of the PRRSV is a 2nd to 26th amino acid sequence. 3. The fusion protein of claim 1, wherein the ORF5 of the PRRSV is a 30th to 63th amino acid sequence. 4. A pharmaceutical composition for use as a vaccine comprising the fusion protein of claim 1 and a pharmaceutically acceptable carrier. 107547.doc