TWI228993B - Fusion antigen specific for a target cell - Google Patents

Abstract

The present invention mainly provides a fusion antigen specific for a target cell comprising a ligand moiety which is capable of reacting, recognizing or binding to the receptors on the target cell, a Pseudomonas exotoxin A translocation domain II, an antigenic moiety, and a carboxyl terminal moiety which permits combination of the fusion antigen to the endoplasmic reticulum (ER) membrane of the target cell. A pharmaceutical composition comprising the fusion antigen is also provided.

Description

1228993 (ii) Description of the invention: TECHNICAL FIELD The present invention relates mainly to fusion antigens. More specifically, the present invention relates mainly to the use of a fusion antigen as a τ-cell vaccine. Previous techniques In animals, the mechanisms of immunity include humoral immunity and cell-mediated immune responses. Humoral immunity is mainly related to the production of antibodies. Antibodies, by binding to them, provide protection from pathogens or their toxic products ' and thereby block them and enter cells that can be infected or destroyed. Antibodies can also trigger giant salamander cells, ingesting and destroying pathogens like bacteria. The third function of antibodies is to activate the system called complement plasma proteins, which can directly destroy bacteria. Cell-mediated immune response relies on direct interactions between τ_lymphocytes and cells that carry antigens recognized by τ 'cells. These slime packs can recognize virus-infected cells and use the synthetic mechanism of the cells themselves. Somatic cells that replicate in slime packs, but antigens derived from virus replication, appear on the surface of infected cells (MHC class I) There, they are recognized by the poisonous τ_cells (cd8 + t_cells), and infection can then be controlled by killing the cells before virus replication is complete. 'Vaccines used to prevent viral infections, usually live attenuated organisms, have reduced pathogenicity but still stimulate protective immunity. When the virus replicates, live virus foreign proteins used as live attenuated vaccines are identified and processed into endogenous processed peptides in the endoplasmic reticulum (ER) cavity of antigen presenting cells (APCs). This treatment includes antigen modification and appropriate decomposition. However, live attenuated

O V85 \ 85IIO DOC 1228993 vaccine has a strong tendency to restore toxicity. For example, the toxicity of infectious laryngotracheitis virus (ILTV) can be restored in both vaccine or drug-reduced lines. In addition, multiple generations of the virus are required; therefore, its ability to evoke an immune response is also questioned. Therefore, developing live attenuated vaccines is a time-consuming task. To prevent the recovery of live vaccines, the development of genetically defective vaccines, such as pseudorabies vaccines, gl vaccines, and PRV-labeled vaccines. Among other things, recombinant subunit vaccines and DNA vaccines have also been revealed. Use vaccinia or fowlpox virus or bacteria as a carrier ▲

Body W, the gene that carries the antigen. Via recombinant DNA technology: It reduces the time to develop a good vaccine and can complete vaccines of multiple serotypes at the same time. Examples of such vaccines are the fowl pox vaccine, the Salmonella vector system, and the Syntro Vet (US) genetic recombinant vaccine. On the other hand, when using microorganisms, especially RNA viruses as vectors, ‘these micro-organisms will generate new species or new strains, so the safety of such vaccines is challenged. In addition, recombinant sub-single immunization vaccines usually act on the immune response that causes cellular mediation. Because of them. It is an exogenous antiprotogen and is taken up into giant pheasant cells, dendritic cells, and B lymphocytes. Such a peptide derived from an exogenous antigen is produced after the antigen is internalized into APCs via a liquid phase cytosolic effect or a membrane-bound receptor. Peptides produced in the 'yi cystic compartment of APCs and classified by empty MHC class II sub-classes' are based on the affinity between MHC class II molecules and peptides to form the peptide-MHC-section Class II complexes, and then the peptide-MHC class II complexes were shifted to the surface of APCs for recognition by CD4 + T-cells. However, small subunit proteins recognized by CD8 + T-cells cannot be effectively used as vaccines, because once administered parenterally, they are internalized into the inner cystic compartment and

O: \ 85 \ 85l 10 DOC 1228993 may be extensively degraded or unable to interact with the MHC Class I pathway. In addition, CD4 + cells (Th cells) can activate humoral immunity and cell-mediated immune responses through Th1 and Th2 helper T-cells, respectively. Th1 and Th2 cells can regulate each other to maintain a balance of humoral immunity and cell-mediated immune response. Therefore, if only humoral antibodies are produced in all immune responses, it will be difficult to control virus infection due to excessive sensitization of the immune system. Fortunately, it is currently possible to prepare safe T-cell vaccines to defuse protective cells-mediated immunity against all viruses (Constantin A. | · Bona et al., 1998 · Immunology today 19th volume 126- 131) ^. Vaccines that infect immune cells, such as T-cells, B-cells, dendritic cells, monocytes, and macrophages, are yet to be developed. Examples of such viruses are porcine reproductive and respiratory syndrome virus, Circovirus type II, and human immunodeficiency virus. • This type of virus turns off the ability to recognize foreign proteins as antigens in antigen-presenting cells. Immune cells are not / capable of functioning and carry the virus at the same time. This type of infection II 'often causes acute injury to infected animals. Animals that are already infected are vulnerable to other pathogens. Recent | Recent reports 'show that killing T-cells (CTLs) is necessary to control HIV infection (Hanne GS et al., 2000, Journal of viro-logy, Volume 74, Issue' / 4, Issues 1694-1703 ). Unfortunately, radon still lacks effective vaccines against viruses that infect immune cells. In particular, porcine reproductive and respiratory syndrome virus (PRRSV), which causes significant losses in animal agriculture each year. The virus infects giant pheasant cells (in the alveoli and spleen), brain microglia, and monocytes and is present in the blood and organs of infected animals. Antibodies have little effect on the virus and may even stimulate the virus

〇 \ 85 \ 85IIO DOC 1228993 changes. In the mechanism of antibody-dependent enhancement (ADE), the use of antibodies leads to more severe infections. About 50 to 80% of pigs are infected with this virus. Generally, animals infected with the virus have no obvious symptoms, but the immunity of the infected animal is reduced. This leads to a reduction in weight gain and an increase in mortality. PRRS V is an RNA virus. Not only domestic animals, ducks can also be infected with PRRSV, although a live attenuated vaccine against PRRSV has been developed. However, mutations in viruses often occur in live vaccines, and safe vaccines still need to be developed. φ SUMMARY OF THE INVENTION _ The present invention provides a fusion antigen that can be used as a T-cell vaccine, and preferably a T-cell vaccine against a virus that infects immune cells. The present invention is characterized by the design of the carboxy terminal portion of the fusion antigen. An object of the present invention is to provide a fusion antigen specific to a target cell, including an antigenic portion; a ligand portion capable of reacting with, and recognizing or binding to, a known receptor on the target cell; a pseudomonad The bacteria are exotoxin. A shift work. Energetic site Π: and a carboxyl terminal moiety that allows retention of the fusion antigen in the endoplasmic reticulum (ER) of the φ target cell. : Another object of the present invention is to provide a pharmaceutical composition including the fusion antigen of the present invention and a pharmaceutically acceptable carrier. Embodiments The present invention provides a fusion antigen specific to a target cell, including an antigenic portion; a ligand portion capable of reacting with, or identifying, or binding to a receptor on the target cell; a false order Exotoxin A translocating functional site Π; and a carboxyl terminal moiety that allows retention of the fusion antigen in the target cell

0 \ S5 \ 85IIO DOC 1228993 Quality Network (ER). As used herein, the term "fusion antigen" refers to a recombinant protein capable of evoking an immune response in an animal. The fusion antigen preferably includes an epitope that directly evokes an immune response, and other parts that can enhance the lean response, such as parts that regulate delivery, delivery, processing, and performance, or multifunctional devices. Preferably, the 'target cell line is an antigen-presenting cell. More preferably, the target cell line is selected from the group consisting of T-cell, B-cell, dendritic cell, monocyte and macrophage. ru-As used herein, the term "antigenic portion" refers to a peptide fragment that elicits an immune response. In a specific embodiment of the invention: in this embodiment, the antigenic portion is an epitope. According to the present invention, the antigenic portion is a protein of a pathogenic species that can highly activate an immune response. Such proteins include, for example, but are not limited to, ectin, nucleoprotein, or cell membrane protein. The antigenic part is a peptide directly cloned from a pathogenic species, and a skilled artist, in order to raise the ability of immune response, to make it more conveniently, and to make it easier to 'deliver' Modified recombinant protein f. Preferably, the antigenic Shao line is derived from porcine reproductive and aspiration virus (pRRsv), circular disease mother type II, or human immunodeficiency virus. In one preferred embodiment of the present invention, the antigenic portion may be PRRSVORF1, 2, 3, 4, 5, 6, or 7. 'In a more preferred embodiment of the present invention, the antigenic portion PRRSV0RF7. In order to evoke a more severe immune response, the antigenicity unit has been incorporated into a primordial unit, and connected with the anti-resistance unit through the bridge region. According to the present invention, the bridge region may be a small fragment of a peptide, which is called;

O: \ 85 \ 85IIO DOC 1228993 A little immune response, preventing the immune system from recognizing it. As used herein, the term "ligand moiety" generally refers to all molecules capable of reacting, recognizing, or binding to a receptor on a target stem cell. Examples of such receptors include, but are not limited to, antibody receptors, growth receptors, lymphokine receptors, cytokinin receptors, hormone receptors, and the like. In some specific embodiments of the present invention, the receptor that binds to the ligand portion is selected from the group consisting of TGF receptor, IL2 receptor, IL4 receptor, IL6 receptor, IGF1 receptor, CD4 receptor, IL18 receptor, The group consisting of IL12 receptor, EGF receptor, LDL receptor and α2-macroglobulin receptor. The ligand part has the ability to bind to the cell membrane of the target cell in order to fix the fusion antigen on the target cell, and it activates the immune system by the binding effect of the fusion antigen and the receptor on the target stem cell. The ligand part is preferably a Pseudomonas exotoxin A binding functional site I. Pseudomonas exotoxin A (PE) is a single polypeptide chain of 613 amino acids. X-ray crystallographic study and mutation analysis of PE molecules revealed that PE is composed of three functional sites. Amine terminal cells, receptor-binding functional sites (functional sites, I.); intermediate shift functional sites (functional sites) Π); and a carboxyl terminal φ-terminal active functional site (functional site II) (see US Patent No. 5,705,163 :, which is incorporated herein by reference). ", ... β As used herein, the term" Pseudomonas exotoxin A binding functional site I "means a site having a binding function to an amine terminal cell receptor of Pseudomonas exotoxin A Peptide fragments of the same sequence, or functionally equivalent fragments. Pseudomonas is an amine terminal cell receptor binding functional site of exotoxin A, and includes two secondary functional sites, which are named functional site I a and functional site I b, respectively. The configuration of functional sites I a and I b can bind to LDL receptors or α2-macroglobulin receptors on the surface of cells OA85 \ 85IIODOC -10- 1228993. As used herein, the term "Pseudomonas exotoxin A binding functional site Π" refers to a peptide fragment having the same sequence as an intermediate translocating functional site of Pseudomonas exotoxin A, or functionally On equal fragments. Pseudomonas brevis exotoxin A has a function of translocating the functional site Π, and has the ability to displace the fusion antigen into the cytoplasm of the target cell. After the fusion antigen is attached to the stem cell membrane, it is translocated into the stem cell. f As used herein, the term "carboxyl terminal moiety, which allows the fusion antigen to remain in the endoplasmic reticulum (ER) membrane of the target cell", refers to the ability to bind the fusion antigen to the ER membrane and Peptide fragments retained in the ER cavity. In a specific embodiment of the present invention, the carboxyl terminal is divided from an amine terminal to a terminal terminal, and includes the following amino acid residues: R, R2-R3-R4- (R5) n wherein, R1 is a positively charged amino acid residue; R2 is a negatively charged amino acid residue; R3 is a negatively charged amino acid residue;

R4 is L γ, R5 is a positively charged amino acid residue; and η is 0 or 1. The carboxyl terminal portion is preferably a member of the KDEL family of proteins. As used herein, the term "KDEL family proteins" refers to a group of proteins that have a similar carboxyl terminus that can bind to cellular ER membranes and further have the ability to retain such proteins in the ER cavity. Generally, the length of this carboxyl terminus is 0, 35, 85, and 10 DOC -11-1228993 degrees, with 4 to 16 residues. As discussed in U.S. Patent No. 5,705,163 (incorporated by reference herein), amine residues on the KDEL family protein < end-terminal, especially the last 5 An amino acid is very important. Studies on similar sequences showing a specific biological function performed in different molecules have shown that the sequence that retains newly formed proteins in the endoplasmic reticulum is LysAspGluLeu. These findings suggest that the sequence at the carboxyl terminus of the fusion antigen according to the present invention acts as some type of recognition sequence to help the fusion primordia shift from the intracellular compartment to the ER and retain it in the cavity. In a preferred embodiment, the carboxy-terminal portion includes a sequence of kjdel. In a more specific embodiment, the carboxyl terminal portion includes the sequence 歹 ij of KKDL-RDEL-KDEL ·. The present invention is characterized by the design of the carboxyl terminal portion, which enables the fusion antigen to be processed in the ER of target cells to be complexed with MHC class I molecules, and recognized by T-cells. The fusion antigen according to the present invention can be used to trigger a bismuth cell-mediated immune response. \ According to the present. Ming 'fusion antigen system is used to immunize animals. It is an object of the present invention to provide a pharmaceutical composition comprising the fusion antigen of the present invention, and a pharmaceutically acceptable carrier. The pharmaceutical composition is preferably a T-cell vaccine. As used herein, the term "T-cell vaccine" refers to a vaccine that protects an individual from infection through immune responses mediated by living cells. Tau cells (also known as Tau lymphocytes, CD8 + T-cells, and CTLs), and memory T-cells (Tcm and Tem) are important roles for T-cell vaccines. The present invention also provides a method for immunizing animals, including the following steps: (a) a fusion antigen specific for a target stem cell, including an antigenic portion;

〇: \ 85 \ 85 丨 丨 O.DOC 1228993 A ligand part, which can recognize or bind to the receptor on target cells; a Pseudomonas exotoxin A translocation functional site Π; And a carboxyl terminal portion that allows the fusion antigen to be retained in the endoplasmic reticulum (ER) membrane of the target cell; and (b) the fusion antigen is put into the animal body. In step (b) of the method, the fusion antigen can be introduced into the animal body in any manner known to the artist. For example, the fusion antigen can be delivered to an animal by injection, f or in the form of an oral vaccine. It can be reinforced if necessary. Preferably, μ is inoculated before infection. Newly born animals, even embryos, can be vaccinated with fusion antigens to produce better immunity. According to the present invention, during the period of immune action, the following actions occur: (c) Standard stem cell membrane binds to the ligand part to fix the fusion antigen on the standard stem cell; '(d) Move by Pseudomonas exotoxin A Position the functional site Π to transfer the fusion antigen into the cytoplasm of target Huaba cells; (e) the ER membrane of the target cell binds to the carboxy terminal portion of the fusion antigen and retain the fusion antigen in the ER cavity; / (f) Processing the antigenic portion in the ER cavity; < (g) the processed antigenic portion is bound to the MHC class I molecule; (h) the MHC class I molecule carries the processed antigenic portion to the surface of the target cell, (i) CD8 + T-cells recognize the processed antigenic portion carried by MHC class I molecules and obtain immune information; and O: \ 85 \ 85 IIODOC -13-1228993 (j) Store immunization information to immunize animals. In action (C), the ligand portion of the fusion antigen directs the fusion antigen to bind to the receptor on the target cell membrane, and fixes the fusion antigen to the target cell. In action (d), the fusion antigen is translocated into the cytoplasm of the target cell by the Pseudomonas exotoxin a. Translocating functional site Π '. This shift causes fusion antibodies to enter the target cells.

In action (e), the carboxy terminal portion of the fusion antigen is bound to the ER membrane of the target cell 'to retain the fusion antigen in the ER cavity for processing and processing of the fusion antigen. -• From — In action (f), process the antigen portion in the ER cavity. This processing includes, but is not limited to, modification of the antigen in the ER cavity, such as glycosylation, and proper degradation by enzymes. In action (g), the processed fusion antigen can bind to MHC class I molecules. MHC class I molecules are themselves incompletely folded proteins and bind many chaperones. The processed 4 fusion antigen binds to the peptide-binding cleft, completes folding and stimulates the release of concomitant proteins. In action (h), the processed antigen is displayed on the surface of the target cell by the MHC class I molecule. The folded MHC class VII and processed antigen portions are delivered to the cell surface. "The processed antigen portion carried by the MHC class I molecule in" in action "is recognized by the CD8 T-cells to obtain immune information about toxin _ cell recognition" and stores the immune information in memory _ cells Inside. Examples of memory τ_ cells are Tem and Tem cells. In Action ⑴, immune information stored by memory T-cells can be used to protect animals from 0'85 \ 8511〇 dqc -14-1228993. When the animals immunized with the fusion antigen were re-infected with the same antigen, the memory D-cells evoked a stronger immune response in a shorter period of time. T-cell vaccines provide endogenously processed antigens that can be processed in the ER cavity of target cells. The invention also provides a fusion of porcine reproductive and respiratory syndrome virus (PRRSV) 〇RF 7 antigen, including a PRRS V ORF 7 part; a Pseudomonas exotoxin A binding functional site I; a Pseudomonas exogene Toxin A shifts the function f site Π; and a carboxyl terminal moiety, which allows the fusion antigen to be retained in the endoplasmic reticulum (ER) membrane of the target cell. -— Also provided is a pharmaceutical composition comprising a fusion antigen of the invention, and a pharmaceutically acceptable carrier. For the purpose of explaining the present invention, the following examples are provided, but they are not intended to limit the scope of the present invention. Example 1: PRRSV ORF 7 fusion antigen

犮 and hydroxyl # points: nucleoprotein PRRSV ORF 7 i, 'The sequence is as shown in the sequence identification number .: 1 (Genbank accession number AF035409, also shown in Figure 2'). The PRRSV ORF 7 gene was cloned with a specific primer, which is the forward primer 5, -GTC ACA TAT GCC AAA TAA CAA CGG s! T CA-3 '(sequence identification number: 2) and the reverse primer 5f-AAG AAT TCC AGC TCA TC_C ATG CTG-3f (sequence identification number: 3). The restriction sites were linked using the restriction enzyme II and inserted into the Pseudomonas exotoxin A translocation functional site Π. Pseudomonas exotoxin A binding functional site I and Pseudomonas exotoxin / swim in place and: using pJH4 as the starting plastid, which encodes a false 0 \ 85 \ 85IIO DOC -15-1228993 single Exotoxin A (PE) full-length gene, as described by Liao CW et al. (LiaCW. Et al., 1995, Applied Microbiol Biotechnol 43:49 & -507). In pET15-derived plastids, construct a 1-valent ORF DNA fragment of the full-length PE gene, including functional sites I, Π, and m, to form a pet-pe plastid with a RI and X at the 3 'end of the PE gene / zo I Restriction enzyme recognition inclusion. Fusion PEE with antigen gene (ΔΙΙΙ) 'borrowed from Wei 魏 \ 91, · / base pair of the II- 仏 0 RI fragment (that is, the D fragment), which is the c-terminal DNA of the PEESV 0RF7 nucleoprotein gene Fragment and digested with jw II and RI restriction enzymes. The DNA fragments were then purified by gel electrophoresis and electroelution. The T fragment ligase was used to ligate the IUco R I fragment of pET-PE plastid of 7.1 kb with this D fragment to form plastid pET15-Η6-PE (Δin) -PRRS7-D (7.31 kb). This plastid is at the end of the fusion gene, including R I and Zao I cognitive positions. In order to increase the antigenicity, a DNA fragment was created according to the recombinant technology round, with two vertically repeated D fragments (ie, DOD) connected by a bridge (g). Then by connecting the / I-DgD-Pw I fragment with a 6.0 kb / I-PW I fragment of ρΕΤ15-Η6-ΡΕ (ΔIII) -PRRS7-D, a coding PE (AlII) -PRRS7-DgD 7.7 kb plastid (as shown in Figure 3) (ie / PE-DGD) 〇 'Assault end skirt_min: Shows the gene sequence encoding KKDELRDELKDEL (sequence identification number: 4) in sequence identification number: 5 J. I also created an I restriction enzyme recognition site at the 5 'end, and a stop codon TAA-TGA (as shown in Figure 4) at the 31 end and inserted into the pET23a plastid digested with I and X / zo I Formation of pET23-3KDEL plastids (as shown in Figure O: \ 8S \ 85llO DOC -16-1228993 5). A polynucleotide encoding a Sa / I position-KKDELRDELKDEL-stop codon R I sequence was synthesized via a continuous polymerase chain reaction (PCR). The linear DNA of pET23 was cut with / I as a PCR DNA template, and the primers and reverse primers 5 ^ TTC ATC TCT CAG TTC GTC TTT TTT GAG GTA GTC GAC GGA GCT CGA ATT CGG-3f (sequence identification number: 6). A fragment of the first PCR product of 168 base pairs is generated. The DNA product contains a Sa / I recognition site. Then f

Then, using this 168 base pair DNA as the second PCR template, the gene primer and reverse primer were initiated by T7 5. A GAA TTC CTC GAG JTCA TTA CAG TTC GTC TTT CAG TTC ATC TCT CAG TTC GTC-3 '(Sequence identification number · 7), resulting in a second PCR product, a fragment of 206 base pairs. A final PCR DNA fragment containing positions at / I, especially / 20I and five co R I was obtained. The PCR-amplified DNA fragment was cut with I and penta-R I and purified by gel electrophoresis and electroelution. The purified ☆ / I- 仏 RI DNA fragment was ligated to a 3Jkb / I-pentaRRI DNA fragment obtained from pET23a. Finally, construct a plasmid pI23-6WI-3KDEL that encodes I-KKDELRDELKDEL-stop codon I-five a R I. The hairpin and PRRSV ORF 7 fusion antigen lines are shown in FIG. 1. ’Prepare two DNA fragments. A 6.4-kd I-X / ζο I fragment obtained from plastid pET15-H6-PE (AIII) -PRRS7-DgD (7.7kb), containing PE (ΔIII), and obtained by digestion with I and I Of the antigen. Another 1.345-kb I fragment, which contains a serotonin terminal fragment, was also obtained by digesting the plastid ET23-3KDEL with PWI and 仏 / 1 restriction enzymes. These two fragments, -17-1228993, were purified and then ligated by T4 ligase to form ρET23-Η6-PE (ΔIII) -DgD-3KDEL (ie, PE-DGDk; as shown in Figure 5). filing # 4 涊 and 趑 趑: at 37 ° C, in a Luda Bertani broth containing 100 to 500 ppm ampicillin, culture the plastids that contain PE-DGD and PE-DGDk molecules E. coli BL21 (DE3) pLys cells. When the culture reached the early log phase (A600-0.1 to 0.4), isopropyl-1-thio-β-D-t-galactopyranoside (IPTG) was added for induction at a final concentration of 0.5 mM. Cells were harvested after 2 hours of incubation and immediately stored at -70 ° C. The fusion antigen was partially purified by urea extraction as described previously (Liao et al., 1995, Appl. Microbiol Biotechnol. 43: 498-507). In order to improve the binding to Ni-NTA matrix (Ni-NTA agarose; Qiagen® Inc. CA) under denaturing conditions, the PE-DGD and PE-DGDk molecules containing the 6XHis tag were fully exposed. Therefore, by reducing the possibility of non-specific binding, the efficiency of purification is maximized. Batch purification of 6-His-labeled PE-DGD and PE from E. coli is performed under denaturing conditions as described below. -The role of DGDk:-Add 1 ml of 50% Ni-TNA slurry to 4 ml of lysate, and gently mix at room temperature by shaking (eg 200 rpm) for 60 minutes to form a lysate-resin Mixture;-Carefully pack the lysate-resin mixture into an empty column with a bottom cap still attached;-remove the bottom cap and collect the solution flowing through;-rinse the buffer solution with 4 ml ( 100 mM NaH2PO4, 10 mM Tris-HCl, 8 M urea, pH 8 · 0) Rinse twice; 〇 \ 85 \ 85110 DOC -18-1228993-elution buffer solution (0.5 mM NaH2PO4, 10 mM Tris-HCl, 8M) Urea, ρΗ5 · 9) eluted the protein 4 times, then eluted 4 times with 0.5ml ρΗ4.5 elution buffer solution 100mM NaH2P04, 10mM Tris-HCl, 8M urea, pH 4.5); and-collected The fractions were dissolved and subjected to SDS-PAGE. The results are shown in FIGS. 6 and 7. Quantitative analysis was performed using standard BSA proteins. Shows successful construction of fusion antigens. f Example 2: Use of a PRRSVORF7 fusion antigen as a T-cell vaccine. Example 1 describes the preparation of a PRRS V / ORF 7 fusion antigen used herein. ##: From PRRS V pigs regularly tested by RT-PCR and known to be virus-free, pigs were obtained and plasma lysates were collected. RNA was extracted using NucleoSpinRNA ΠTM kit (Macherey-Nagel GmbH & Co. KG, Germany). Add 3 50 microliters of RA1 solution and 3.5 microliters of β-mercaptoethanol to / 100 microliters of plasma dissolution fraction. After reducing the viscosity and clarifying the lysate by # 'filtration, the lysate was mixed with 350 microliters of 70 ° /. Ethanol mixed. The RNA was absorbed into a NucleospinTM RNA column by centrifugation and rinsed. 95 microliters of DNase solution was applied to the column for DNA digestion. After repeated rinsing and centrifugation several times, 60 μl of RNase-free elution was performed. By using Qiagen Onestep RT-PCR Kit '(Qiagen ^ Inc · CA)' for RT-PCR 〇 Provide 5 丨-(^ 八 GCC AGT CAA TCA GCT GTG-3 '(sequence identification number: 8) forward Primers, and 5, GCG GAT CAG GCG CAC-3 '(sequence identification number: 9) reverse primers, synthesized 293 O: \ 85 \ 85IIO.DOC -19-1228993 base pair fragment. Agarose coagulation Gel electrophoresis, determined that the detection limit of RT-PCR is about 10 PRRS (TCID50 / ml). Blood leukocyte samples derived from 5 sows in the SPF farm were subjected to RT-PCR. 5 sows were not PRRSV Infection. The results are not shown in Figures 8 to 10. Slimming: On the SPF farm, 6 newborn piglets were selected from 3 sows, respectively, and confirmed, weighed, and sex determined. Piglets were randomized F

Three groups were vaccinated with PE-DGD vaccine, PE-DGDK vaccine and control group, and were graded on the basis of body weight. Each group included 2 piglets from each sow. In the vaccinated group, intramuscular immunization was performed twice during lactation. During the weaning period (approximately 3 to 4 weeks of age), each group is removed and kept in an isolated room equipped with air conditioning and ventilation equipment. The vaccinated group, at the age of 4 and 18, was injected intramuscularly with 1 ml of PE-DGD; or PE-DGDK (containing 50 micrograms of protein / dose) in 1 liter ISA206 (SEPPIC®, France) An emulsified 2 ml · 'vaccine was immunized twice. The control group was raised without immunization. In the pound carbonyl formula Yin's attack #: Two weeks after the last vaccination, sedated with 1000 mg of ketamine solution intramuscularly, and then 1 ml of 2% lidocaine (Lidocaine) was dripped intranasally. ) After suppressing the cough and reflex, attack the pigs intranasally. A fresh 1 ml MDRS PRRS culture was used to challenge at a dose of approximately 1 × 107 TCID5G / ml. In each group, 5 piglets were challenged.

Two weeks after the second immunization, blood leukocyte samples of piglets were measured by RT-PCR, PRRSV was detected, and the results are shown in FIG. 12. Prior to PRRSV O: \ 85 \ 85IIO DOC -20-1228993, no viremia was present in any piglets. After 3, 4 and 14 days after challenge, the blood leukocyte samples of piglets were measured again by RT-PCR to detect PRRSV, and the results are shown in Figs. 13, 14 and 15, respectively. The results are also summarized in Table 1:

After using PRRSV challenge, the proportion of PRRSV viremia in piglets days Control group PE-DGDk PE-DGD 3 3/5 ”3/5 3/5 '7 3/4 (dead Γ) 2/5 2 / 4 (dead Γ) 14 3/3 (dead 2 *) 0/5 2/3 (dead 2 *) •• PRRS V viremia was detected by RT-PCR before death of all dead animals At the end of the 2-week study, autopsies were performed on all survivors. Macroscopic examination showed that 5 pigs in the control group, 4 pigs vaccinated with PE-DGD vaccine, and 2 in the PE-DGDK group Pleural pneumonia was present in the lungs of pigs. In the control group and the group vaccinated with PE-DGD, more extensive lesions were observed, but not in the group vaccinated with PE-DGDK. PRRS VORF 7 fusion protein PE-DGDK was shown It can protect pigs from infection. The specific embodiments of the present invention are used to explain and describe the content of the present invention, but the scope of the present invention can include various modifications and improvements that can be made by those skilled in the art. The present invention is not limited to these The specific form used to explain the content of the present invention, all modifications that do not depart from the spirit and scope of the present invention are within the scope defined in the scope of the attached patent application . Brief Description of the drawings

〇 \ 85 \ 85IIO DOC 1228993 Figure 1 illustrates the construction of a PRRS V ORF 7 fusion antigen according to Example 1. Figure 2 explains the gene sequence of PRRSVORF 7. Figure 3 illustrates the construction of PE-PRRSV-D in pET23a protein expression plastids. Figure 4 explains the construction of pET23-3KDEL. Figure 5 illustrates the construction of the PRRS V ORF 7 fusion antigen.

Figure 6 illustrates SDS-PAGE detection of protein purification and quantitative analysis of PE-DGD protein < samples. By IPTG induction, Table I f in BL21 (DE3) plys presents the PE-DGD protein gene of the PE (ΔIII) and PRRS V 0RF-7 chimeras in the pET plastid system. SDS-PAGE images of total bacterial proteins were shown for the samples after IPTG induction (i-channel A) and urea extraction samples (runway B or runway 4). A strong staining band at 80K Da is PE-DGD. Protein. Runways 1, 2 and 3 are samples of standard BSA proteins with 1000 nanograms, 300 nanograms, and 100 nanograms, respectively. Runways 4, 5 :, and 6 are samples of PE-DCfD. Urea-extracted protein with loadings of 1 μl, 0.1 μl, and 0.01 μl, respectively. Figure 7 explains. SDS-PAGE detection of protein purification and quantitative analysis of PE-DGDK protein φ quality samples. By IPTG induction, the PE-DGDK protein gene of the PE (ΔIII) and PRRSV ORF-7 chimeras, expressed in the pET plastid system in BL21 (DE3) plys. SDS-PAGE diagrams of total bacterial proteins for samples before (track A) and after (track B) run by IPTG and urea extraction samples (runway C or runway 4). A strong staining band at 80K Da is the PE-DGDK protein. Runways 1, 2 and 3 are samples of standard BS A protein with 1000 nanograms, 300 nanograms, and 100 nanograms, respectively. Runways 4, 5, and 6 are samples of 1 μl, 0.1 μl O: \ 85 \ 85llO DOC 22 1228993, and 0.01 μl of PE-DCDK urea-extracted protein samples, respectively. Figure 8 illustrates the detection limits of RT-PCR using BM-0RF7 in swine blood leukocyte samples. Total RNA was extracted from 100 μl of pig blood leukocyte samples with 1 μl serial dilution of PRRS virus (106TCID5G / ml). By performing RT-PCR using the BM-ORF7 primer and a 10/25 volume RNA template, the PRRSV detection limit was determined. RT-PCR products containing approximately 300, 100, 30, 10, 3, and 1 (TCID50 / ml) feed samples were loaded into runways 1, 2, 3, 4, 5, and 6, respectively, and Perform on a 2% agarose gel in TBE buffer solution. . '— Figure 9 explains the results of real-time PCR analysis of PRRSV by BM-primer. Figure 10 illustrates the results of comparing the detection limits of real-time PCR with conventional RT-PCR. Figure 11 explains the results of PRRSV detection by RT-PCR of 5 sows, where M represents the DNA marker and P represents the positive control group. Figure 12 illustrates the results of PliRSV detection by RT-PCR before challenge of 15 piglets, where M represents the DNA marker and P represents the positive control group. ‘Figure 13 explains the results of PRRS V detected by RT-PCR 3 days after challenge of 15 piglets, where M represents the DNA marker, and P represents the 1½ sex control group. Figure 14 illustrates the results of PRRSV detected by RT-PCR 7 days after challenge of 15 piglets, where M represents a DNA marker and P represents a positive control group.

Figure 15 illustrates the results of detecting PRRSV by RT-PCR O: \ 85 \ 85IIODOC -23-1228993 14 days after challenge with 15 piglets, where M represents the DNA marker and the sex control group. O: \ 85 \ 85llO.DOC -24-1228993 Sequence Listing < 110〉 Taiwan Institute of Animal Science & Technology &120; Fusion Antigen for Vaccine < 130 > A < 160〉 9 < 170〉 < 210〉 Patentln version 3.2 1 < 211〉 388 < 212 > DNA '' " < 213 > < 300 > Porcine Reproductive and Respiratory Syndrome Virus < 308 > Genbank / AF03 5409 < 309 > 1998-12 -29 < 313 > < 400 > (2898) .. (3269) '1 ,, catatgccaa ataacaacgg caagcagcag aagaaaaaga agggggacgg ccagccagtc 60 aatcagctgt gccaaatgct gggtaagatc atcgcccagc aaagtcagtc cagagttaag 120 ggaccgggaa.ggaaaaataa gaagaaaaac ccggagaagc cccattttcc tctggcgact 180 gaagatgacg tcagacacca ctttaccccc agtgagcggc aattgtgttt gtcgtcaatc 240 cagactgcct ttaatcaagg cgctggaact tgcatcctgt cagattctgg gaggataagt 300 tacactgtgg agtttagttt gcctacgcat catactgtgc gcctgatccg cgt t acagca; u 3 60 ccaccctcag cataatgggc tggaatt, < 212, < < 212 > < 212 O: \ 85 \ 85IIO DOC -25-26 1228993 < 223 > Forward primer for the synthesis of PRRSV〇RF 7: '< 400 > 2 gtcacatatg ccaaataaca acggca < 210 > 3 < 211> 24 < 212 > DNA < 213 > artificial < 220 > < 223 > Reverse primer for synthesis of PRRSV ORF 7 < 400 > 3 aagaattcca gctcatccat gctg < 210〉 4 < 211> 13 < 212 > PRT < 213 > Artificial < 220 > < 223 > Carboxy-terminal partial sequence in 1 < 400 > 4

Lys Lys Asp Glu Leu Arg Asp Glu Leu Lys Asp Glu Leu 15 10 2 4

< 210> 5 < 211 > 39 < 212> DNA < 213 > Artificial < 220 > O: \ 85 \ 85nODOC -26-1228993 < 223 > Gene of carboxy terminal portion in Example 1: ' < 400 > 5 aaaaaagacg aactgagaga tgaactgaaa gacgaactg < 210 > 6 < 211〉 51 < 212 > DNA < 213> Artificial < 220 > < 223 > In Example 1, the first polymerase chain reaction was prepared ≪ 400 > 6 ttcatctctc agttcgtctt ttttgaggta gtcgacggag ctcgaattcg g < 210 > 7 < 211 > 52 < 212> DNA < 213 > Artificial < 220 > '< 223 > Second polymerization in Example 1 Enzyme chain reaction reverse reaction < 400 > 7 agaattcctc gagtcattac agttcgtctt tcagttcatc tctcagttcg tc < 210 > 8 < 211 > 21 < 212 > DNA < 213 > Artificial < 220> < 223> for RT -PCR detects PRRSV forward primer O: \ 85 \ 85llO.DOC -27-1228993 < 400 > 8 ccagccagtc aatcagctgt < 210 > 9 < 211> 15 < 212 > DNA < 213 > < 220 > Artificial < 223 > Forward primers for RT-PCR detection of PRRSV < 400 > 9 gcg gatcagg cgcac O: \ 85 \ 85IIO.DOC -28-

Claims (1)

Yi: 丨 586 patent application Replacement of the patent scope (November 1993) Patent application park · A fusion antigen specific to the standard stem cells, the burden is: an antigenic part, which is not outside the genus Pseudomonas Active site of toxin 111; a ligand moiety that can react with the receptor on the target cell, recognize and bind to it; a Pseudomonas exotoxin A translocation functional site υ; and ~ The terminal is divided into directions from the amine terminal to the carboxy terminal, including the following amino acid residues: where R1 is a positively charged amino acid residue; r2 is a negatively charged amino acid residue; r3 is A negatively charged amino acid residue; R4 is L; R5 is a positively charged amino acid residue; and n is 0 or 1. 2. The fusion antigen according to item 1 of the application, wherein the target cell is an antigen-presenting cell. 3. The fusion antigen according to item 1 of the patent application scope, wherein the target cell line is selected from the group consisting of T-cells, B-cells, dendritic cells, monocytes and macrophages. 4. The fusion antigen according to item 1 of the scope of the patent application, wherein the antigenic portion is derived from porcine reproductive and respiratory syndrome virus, circular virus type II, or human immunodeficiency virus. 1228993 5. The fusion antigen according to item 1 of the scope of patent application, wherein the antigenic portion is selected from the group consisting of porcine reproductive and respiratory syndrome virus (PRRS V) ORFs 1, 2, 3, 4, 5, 6, and 7 group. 6. The fusion antigen according to item 1 of the patent application, wherein the antigenic portion includes at least one antigenic unit and is connected to an adjacent antigenic unit by a bridge region; wherein the bridge unit is a small fragment of a peptide. 7. The fusion antigen according to item 1 of the scope of the patent application, wherein the receptor to be bound to the ligand is selected from the group consisting of antibody receptors, growth factor receptors, lymphokine receptors, cytokinin receptors, and A group of hormone receptors. 8. The fusion antigen according to item 1 of the scope of patent application, wherein the receptor to be bound to the ligand is selected from the group consisting of TGF receptor, IL2 receptor, IL4 receptor, IL6 receptor, IGF1 receptor, and CD4 receptor. Body, IL18 receptor, IL12 receptor, EGF receptor, LDL receptor and α2-macroglobulin receptor. 9. The fusion antigen according to item 1 of the scope of patent application, wherein the ligand portion is a Pseudomonas exotoxin A binding functional site I. 10. The fusion antigen according to item 1 of the scope of patent application, wherein the carboxy terminal portion includes the sequence of KDEL. 11 · The fusion antigen according to item 1 of the scope of patent application, wherein the carboxyl terminal portion includes the sequence of KKDL-RDEL-KDEL. 12. The fusion antigen according to item 1 of the scope of the patent application, wherein the antigenic site is the fusion porcine reproductive and respiratory syndrome virus ORF 7 antigen, and the ligand part is a Pseudomonas exotoxin A binding functional site I. 13. —A pharmaceutical composition that can be used as a vaccine, which contains a fusion antigen specific to a standard stem cell according to any one of items 1 to 12 in accordance with the scope of the patent application: O: \ 85 \ 85110-931130.DOC 1228993 Acceptable vehicle. 14. The pharmaceutical composition according to claim 13 of the scope of patent application, which is used as a T-cell vaccine. 15. The pharmaceutical composition according to item 13 of the scope of patent application, wherein the fusion antigen specific to the target cell is the fusion antigen according to item 12 of the scope of patent application 0 O: \ 85 \ 85110-931130.DOC