TWI314559B - Fusion protein of porcine reproductive and respiratory syndrome virus as prrs vaccine - Google Patents

Description

1314559 IX. Description of the Invention: [Technical Field of the Invention] The present invention relates to the construction of a fusion protein and develops a fusion protein subunit vaccine having a PRRSV neutralization valence reaction in pigs. [Prior Art] Pig Porcine reproductive and respiratory syndrome (PRRS), a swine infectious disease that is infected by the Reproductive and Respiratory Syndrome Virus (PRRSV) and is a reproductive disorder in sows and respiratory infections of all ages. . 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 mutation rate is very high. In addition, PRRSV infection can be divided into two stages: A upper respiratory tract and lower respiratory tract epithelial cell infection system; and mononuclear spheres and macrophages in the peripheral tissues of B respiratory tract. Therefore, the host must 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 the PRRSV ORF5, the consensus sequence YKNTHLDLIYNA, between the amino acids 38 to The epitope between 44, which is located in the N-terminal region of PRRSV ORF5, has been identified as a neutralizing epitope (Ostrowski M., JA Galeota, et al.,

Journal of Virology, May 2002, Vol. 76, No. 9:4241-4250). Traditional techniques have also constructed the entire protein structure of ORF5 or ORF6 in the middle of PE and KDEL. After immunization to pigs, it was found that the infected pigs developed more serious inflammation in the lungs. It is obvious that ORF5 or ORF6 is more immune than ORF7. toxicity. 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 and appended claims. [Embodiment] The present invention is characterized in that most of the antigens of ORF5 and ORF6 are removed, leaving only a few amino acids of the N-terminal of 0RF5 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 neutralization power is the fusion protein region of the N-terminus of ORF6 and ORF5, which is the ORF6 structural protein No. 2 to 26 Amino acid peptide region and ORF5 structural protein No. 30 to 63 amino acid peptide region. 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. First, the nucleotide sequence corresponding to the target amino acid sequence is sequenced into a target nucleic acid sequence, and the sequence of the fusion protein region is as follows: 107547.doc 1314559 GSSLDDFCYDSTAPQKVLLAFSITYASNDSSSHLQLIYNL TLCELNGTDWLANKFDWA (SEQ ID NO. 2) That is, PRRSV-ORF6 -2-26-ORF5-31~63 fusion peptide region consisting of two fragments (ORF6)-G2SSLDDFCYDSTAPQKVLLAFSITY26 and (ORF5)-A31SNDSSSHLQLIYNLTLCELNGTDWLANKFDWA63, wherein the GSSLDDFC fragment is called P, YDSTAPQKVLLAFSITY fragment is called Q, ASNDSSSHLQLIYNLTLC fragment It is called A, and the ELNGTDWLANKFDWA 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 E. coli by academic materials (eg http://www.kazusa.or.jp/codon). (5. co/z') The nucleotide system corresponding sequence suitable for the host system avoids the nucleotide corresponding sequence which is not easily recognized and expressed by E. coli. 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 or the MM 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'-ends 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, some three consecutive CCC, AAA, GGG or TTT can be added to both ends of the gene. 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 1314559

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 KFDWA GAG AAA AAA-3' A total of 204 nucleotide pairs, when using enzyme splicing, there will be at least 189 to 183 nucleotide pairs of length into the plastid. The computer soft Zhao (such as DNA strider) analyzes 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 1314559 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 virus strain, in the condition that it does not affect the original amino acid, and can be effectively expressed in the E. coli host system. The upgrade. The modified nucleotide sequence can be synthesized by polymerase chain reaction using multiple pairs of primers respectively, and the corresponding numbers of all primers are shown in Table 2. Table 2: Target antigen PQAB primer pair corresponding numbered antigen forward primer sequence number (Seq.IDNo.) Reverse primer sequence number (Seq.IDNo.) PQAB F1 3 R1 7 PQAB F2 4 R2 8 PQAB F3 5 R3 9 The sequences of the PQAB F4 6 forward and reverse primers are as follows: Forward primer F1 (SEQ ID No. 3) is the amino acid sequence of Nos. 81 to 124 of SEQ ID No. 1, ie, 5'- GCT TTC TCC ATC ACC TAC GCT TCC AAC GAC TCC TCC TCC CAC CT-3'; Forward primer F2 (SEQ ID Νο.4) is the amino acid sequence No. 48 to 96 of SEQ ID No. 1, ie, 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. 5) is amino acid sequence No. 22 to 65 of SEQ ID No. 1. , ie, 107547.doc -10· 1314559 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 SEQ ID No. 1 Amino acid sequence No. 1 to 41, ie, 5'-CCC AAA CCC CAT ATG GAA TTC GGT TCC TCC CTG GAC GAC T-3'; reverse primer R1 (SEQ ID No. 7) is SEQ ID No. Amino acid sequence Nos. 148 to 106, ie, 5'-A CAG GGT CAG G TT GTA GAT CAG TTG CAG GTG GGA GGA GGA GTC-3'; Reverse primer R2 (SEQ ID No. 8) is the amino acid sequence 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-3'; Reverse primer R3 (SEQ ID No_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 primer 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 •11 - 1314559 PCR, and the second set of primers is added to the positive primer F2 and the reverse primer R2 each 0.01~ 4 μΐ, together with the required dNTPs, reagents, and Pfu polymerase, start the second PCR; then, according to the same method, add different primers to PCR for F3 and R3, and then introduce the primers to F4 and R3. In the PCR step, a 204 bp nucleotide sequence modified by PQAB can be synthesized. After the gel fractions synthesized by the respective electrophoresis tests, 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 PRRSV PQAB and a detoxified Pseudomonas aeruginosa exotoxin A (A without domain III), plastid map The system is 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· 1314559. 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 1.0±0.4. In a sterile room, inoculate 50 ml of the bacterial solution into a total of eight bottles of sterilized 3000 ml flask containing 1250 ml LB (+500 pg/ml Amp + 50 ml 10% Glucose) at 37 The °C rotary incubator is incubated at 150 rpm for 2 to 3 hours. When the OD600 is detected at 0.3±0.1, the final concentration of 50 ppm IPTG protein inducer is injected, and the rotary incubator is rotated at 150 rpm at 37 °C. The protein production step was completed by incubating for 2 hours. Next, extract and dissolve the PE-PQAB-K3 (Fig. 3) antigen protein fragments in the inclusion bodies by 8M urea method, and extract them in 10 liters of bacterial solution (a small batch, lot) to obtain 300~400. The amount of mg antigen. The Western blotting method, the coomasie blue staining method, the SDS-PAGE electrophoresis method, and the density of the electrophoresis bands were measured by a densitometer to quantify the protein of each antigen solution. The above antigenic protein was taken as 0.2±0.02 mg as a main component of a high dose of an injection. Take 0·02±0.002 mg as the main component of the low dose of the injection, and carry out the efficacy tests of each immunization and challenge. Example 4: Efficacy test for pig immunization and challenge In pig farms, pigs were randomly divided into three groups of five pigs each, and each group was housed separately in isolation with air conditioning and ventilation equipment. indoor. Pigs in the PE-PQAB-E3 vaccinated group received 1 ml of PE-PQAB-E3 (containing 2〇〇pg protein/dose) by intramuscular injection at 14 and 28 days of age, 107547.doc •13- 1314559 2 ml of vaccine emulsified in 1 ml ISA206 (SEPPIC® France), immunized twice. The GP5 & M immunized group was immunized with PE-ORF5-K3 and PE-ORF6-K3 (each containing 200 μ8 protein/dose). The pigs in the control group were raised without immunization. Two weeks after the last vaccination, pigs were intramuscularly administered with 100 mg of ketamine solution for sedation, followed by intranasal drip of 1 ml of 2% lidocaine to inhibit cough. - After the reflex, the pigs were challenged intranasally. Five pigs in each group were challenged with fresh 1 ml of MD-1 strain of PRRSV culture at a dose of approximately 1 x 107 TCID 5 〇 / ml. On the 14th day after inoculation, all sacrificed pigs were completely dissected. Liver samples were collected (from the two parts of the cephalic lobes and from the middle and accessory parts of the hepatic caudate lobe) and fixed with 10% neutral buffered formalin 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 〇 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 white blood cell samples of the pigs were again detected by RT-PCR, and the results are shown in Table 3. 107547.doc • 14- 1314559 Table 3: Days of PRRS V viremia in pigs after PRRSV 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*) * The dead pigs were borrowed before PRRS V viremia was detected by RT-PCR for all pigs (including pigs that had been sacrificed and pigs that survived at the end of the two-week study). Giant examinations showed that pigs showed lungs after challenge. A broader range of lesions and severe interstitial pleuropitis were observed in the control group and the ORF5 & ORF6 vaccine group, whereas these extensive lesions and interstitial pneumonia were not found in the PE-PQAB-K3 vaccine group of the present invention. . 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 show a comparison of the giant lung lesions induced by PRRSV 14 days after PRRSV challenge, and a comparison of the giant lung lesion index exhibited by the PE-PQAB-K3 vaccine group. 107547.doc 15- 1314559 Table 4: Comparison of PRRSV-induced macroscopic lung lesions 14 days after PRRSV challenge. 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 interstitial pneumonia lesion index . Table 5, the giant lung lesions index of the PE-PQAB-K3 vaccine group showed a very significant difference in biostatistics compared with the control group and the ORF5& ORF6 vaccine group (ρ < 0.01)

Group number average variability control group 5 29 5.8 0.2 PE-PQAB-K3 group 5 19 3.8 0.7 PE- ORF5 & ORF6-K3 group 5 29 5.8 0.2 ANOVA variable source SS DOF MS F P-value threshold between groups 13.33333 2 6.66666718.18182 0.000233 3.885294 Within the group 4.4 12 0.366667 Total 17.73333 14 107547.doc -16- 1314559 The control group and the ORF5& ORF6 vaccine group were low (p<〇〇1). According to the above experimental results, it is clearly shown that the present invention I> E_pQAB-K3 not only effectively protects against PRRSV infection, but the vaccine according to the invention is more resistant to interstitial pneumonia than other vaccines (eg PE-ORF5-K3, PE-ORF6-K3). slight. Table 6 shows the changes in the antibody valence of the immunized antibodies. The A group showed good IgG ELISA, but the indirect fluorescent antibody analysis (IFA) and the neutral (NT) valence were compared. Group C is low. 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-10A In the cell-like system, the degree of inhibition of growth and proliferation was measured and measured. [Simple description of the schema] 107547.doc 17 1314559

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- 1314559 Sequence 袅<110> Taiwan Institute of Zoological Sciences<120> fusion protein pRRS virus as a vaccine for porcine reproductive and respiratory syndrome<140><141><150>;151><160> 9 <170〉 Word7.0

<211> 204 <212> DNA <213> Artificial sequence <400>

1 gac tee acc get ccc cag aaa gtt Asp Ser Thr Ala Pro Gin Lys Val 10 15 get tee aac gac tee tee tee cac Ala Ser Asn Asp Ser Ser Ser His 30 ctg tgc gaa ctg aac ggt acc gac Leu Cys Glu Leu Asn Gly Thr Asp 45 get ctcgagaaaaaa Ala 58 cccaaaccccatatcgaattc ggt Gly tee tee ctg gac gac ttc tgc tac 48 Ser Ser Leu Asp 5 ttc Asp Phe Cys Tyr ctg ctg get tee ate acc tac 96 Leu Leu Ala 20 Phe Ser lie Thr Tyr 25 ctg caa ctg Ate tac aac ctg acc 144 Leu Gin 35 Leu lie Tyr Asn Leu 40 Thr tgg ctg get aac aaa ttc gac tgg 152 Trp Leu Ala Asn Lys Phe Asp Trp 50 55 204 <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 He 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 44 1314559 <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 48 cgactccacc gctccccaga aagttctgct ggctttctcc atcaccta

<210> 5 <211> 44 <212> DNA <213> Artificial sequence <400> 5 44 ggttcctccc tggacgacctt ctgctacgac tccaccgctc ccca <210> 6 <211> 40 <212> DNA <213>; manual sequence <400> 6

Ccacaacccc atatggaatt cggttcctcc ctggacgact <210> 7 <211> 43 <212> DNA <213> Artificial sequence <400> 7 40 43 acagggtcag gttgtagatc agttgcaggt gggaggagga gtc <210> 8 <211> 44 <212&gt DNA <213> Artificial sequence <400> 8 44 gccagccagt cggtaccgtt cagttcgcac agggtcaggt tgta 107547.doc -2- 1314559 <210> 9 <211> 44 <212> DNA <213> Artificial sequence <400 〉 9 ttttttctcg agagcccagt cgaatttgtt agcccagccag tcgg

107547.doc

Claims (1)

1314559 X. Patent application scope: 1. A PE-PQGAB-K3 fusion protein comprising: - ORF6 of porcine reproductive and respiratory syndrome virus (PRRSV) and N-terminal fusion protein region of ORF5; Pseudomonas aeruginosa exotoxin A The functional site; and the KDEL-KDEL-KDEL (KDEL3) of the carboxyl terminal moiety. 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. A pharmaceutical composition for use as a vaccine comprising the fusion protein of claim 1 and a pharmaceutically acceptable carrier. 107547.doc