TWI328039B - - Google Patents

Description

1328, 039,, IX. Description of the Invention: [Technical Field] The present invention utilizes a baculovirus expression system to produce a recombinant E2 glycoprotein derived from swine fever virus from insect cells, and tests the recombinant E2 sugar produced. The immunity of the protein to pigs to assess its availability as a secondary unit vaccine and for the detection of V-infected pigs infected with wild virus strains. [Prior Art] • [Background of the Invention] The porcine (hog cholera; HC) is currently officially named and widely used internationally for the name of the disease swine fever (CSF). It is a highly contagious and highly lethal pig. Only viral diseases. Infected pigs are clinically characterized by fever and bleeding from all organs of the body (van Oirschot, in: Straw, BE et al., Pig Diseases, 8th Edition, Iowa State University Publishing, Amis, Iowa, 1 59-172, 1999), and the outbreak of this disease often causes significant economic losses in the pig industry. Therefore, the International Organization for Disease Control (0IE) and China's Animal Infectious Disease Classification Table respectively list it as List A and Class A major animal φ diseases. For the prevention and treatment of swine fever, many countries use the live attenuated vaccine of swine fever as a safe and effective preventive measure, or carry out strict culling policies to achieve the goal of eliminating gambling sores (Edwards et al., VetMicrobiol 73:103). -119, 2000). In Taiwan, there have been cases of swine fever since the Japanese occupation era, and it has caused a great threat to the pig industry* in the province. From the field, the LPC organ or tissue culture vaccine was fully applied as a preventive measure for swine fever. After that, the pig plague situation in the province gradually improved. For the prevention of swine fever, only live-attenuated vaccines were available in the early stage, including the LPC vaccine, the Japanese guinea pig ascending and negative (GPE) strain and the Thiverval strain. Where GPE - with 1328,039,

The Thiverval strain was domesticated by ALD and Alfort in successive passages of cells. The currently widely used live attenuated vaccine is the LPC swine fever vaccine, which provides very good immunoprotective efficacy in pigs. However, when immunized with a live attenuated vaccine, it is often difficult to control the appropriate timing of immunization due to interference with the migration antibody (van Oirschot, JT, Vet Microbiol 73:1 03- 1 19, 2003), and induced The antibody response is also indistinguishable from antibodies raised by wild-type viruses, thus increasing the difficulty of eradicating the disease. Therefore, although the LPC vaccine is highly safe and protective, many scholars are still working on the development of marker vaccines. The old names of the three envelope proteins of the swine fever virus were E2 (gp44/4 8), E3 (gp3 3) and El (gp5 5) before 1994, and they were officially renamed in the order of the genome. Erns (E0, gp44/48), El (gp33) and E2 (gp55) (Riimenapf et al., J. Virol. 67: 3288-3294) 1 993; and Stark et al., Virology 174: 286-289, 1990) . Among the glycoproteins of the three classical swine fever viruses, the E2 glycoprotein was the first major envelope protein to be discovered and studied more (Hulst et al., J Virol 67: 5435-5442, 1993; Moormann et al., Virology, 1 77: 184-198, 1990). Its importance is due to the fact that E2 protein is the most important component of the surface of swine fever virus, and it is also the main antigen that causes swine fever virus infection in pigs (Lin et al., J Virol 74: 11619-11625, 2000). At present, many studies have shown that the immune response elicited by pigs alone after immunization with E2 glycoprotein can produce sufficient immunoprotective effects (Hulst et al., supra: K&nig et al, J Virol 69: 6479-6486 , 1995). Since the E2 glycoprotein of classical swine fever virus has long been regarded as a viral envelope protein that mainly causes pigs to produce neutralizing antibodies, the E2 subunit vaccine has become one of the main targets of the swine fever marker vaccine. It is pointed out by the literature that the E2 glycoprotein is a highly conformation-dependent antigen and must form the correct conformation to have good immunogenicity (Andrew et al.' Vaccine 18: 1932-1983). , 2000). Insect cells have similar post-translational modification capabilities to eukaryotic cells such as mammals, thus providing a true b 1328039.. nuclear expression environment (0reilly et al., in: Baculovirus Expression Vectors, Laboratory Manual, Oxford University Press, New York, 27-; 2, 19, 94 94). Since the successful establishment of the baculovirus expression system, it has been widely used for the expression of many viruses and eukaryotic genes, including the envelope protein of hepatitis C virus (HCV) (Htissy - et al, Virus Res 45: 45- 57' 1996), nucleocapsid protein of porcine reproductive and respiratory syndrome ν (PRRSV) (Denac et al, J Virol Methods 65: 169-181, 1997), structural and non-structural proteins of classical swine fever virus (Hulst et al. , as mentioned above; Kiinig Temple people's heads (J., Steffens et al., Gen Virol 80: 2583-2 590, 1999), human matrix metalloproteinase-9 (MMP-9). (Sadatmansoori et al., Protein Express Purif 23: 447-452, 2001) with triglyceride hydrolyzing enzyme (hTGH) (Alam et al, Protein Express Purif 24: 33-42, 2002), etc., and further analysis by analyzing the characteristics of recombinant proteins The function of virus or human protein, or the development of vaccines and ELIS A detection reagents. This experiment attempts to select the E2 protein of classical swine fever virus strains using the baculovirus expression system, and hopes to develop pigs with protective effects.瘟 logo Vaccines. Pigs infected with wild-type infections can be detected by enzyme-binding immunosorbent assay (ELISA) for the detection of swine fever E2 or E1-8 antibodies ( Floegel-Niesmann » G., Vet Microbiol 83 : 121-136 , 2001;

Moormann et al., Vet Microbiol 73: 209-2 1 9,2000), thus facilitating the clearance of swine fever. However, in the current immunization group with E2 subunit vaccine, the timing of E"18 antibody production is difficult to control due to the pigs infected with wild virus, and • The Eins ELIS A test kit detects pigs infected with wild virus. On the one hand, it is not possible to provide sufficient sensitivity and specificity for C van Oirschot, as described above. Therefore, there is still much room for improvement in the detection kits that are compatible with the E2 marker vaccine. The single-source antibody to pig marks was first published by Wensvoort et al. in 1986. At the time, 13 single-source antibodies were prepared (Wensvoort et al., Vet Microbiol 12: 101-108, 1986). The successful preparation of single-source antibodies against swine fever has greatly contributed to the study of swine fever virus. For example, the structural region of the swine fever virus glycoprotein E2, 7 1328, 039., was confirmed by analysis using these 13 single-source antibodies (van Rijn et al., Vet Microbiol 33: 221-230, 1992; van Rijn et al., J Gen Virol, 74: 2053-2060, 1993; and Wensvoort, J Gen Virol 70: 2865-2876, 1989). After that, the single-source antibody to swine fever has been continuously prepared by scholars from various countries and used to identify the antigenicity analysis of the glycoprotein E0 and E2 of swine fever virus (Weiland et al., J Virol 64: 3563-3569, 19) 90 · Weiland et al, J Virol 66: 3677-3682, 1992) and antigen typing of swine fever virus isolates (Kosmidou et al, Vet Microbiol 47: 111-118, 1995; Sisen et al, J Vet Med Sci 58: 707-710, 1996) ° φ In clinical applications, swine fever single-source antibodies can be further used to develop antigen capture assays to detect swine fever virus antigens (Colijn et al., Vet Microbiol 59: 15-25, 1997). Or develop a complex capture-block enzyme-binding immunosorbent assay (CTB ELISA) to detect specific antibodies to swine fever with greater sensitivity and specificity (Clavijo et al., Vet Microbiol 60: 1 55- 168,1998;

Wensvoort et al., JGen Virol 70: 2865-2876, 1988), provide many useful tools for the diagnosis and monitoring of swine fever. [Description of the Invention] φ [Inventive Description] The present invention relates to the production of a classical swine fever virus (CSFV) type I swine fever virus strain and a second aspect thereof using an baculovirus expression system for insect cells. The recombinant swine fever virus E2 glycoprotein of the wild type isolate isolate, and the recombinant E2 glycoprotein produced is characterized in that the 'immune response' produced in the pig is similar to that of the wild swine fever virus infection. Accordingly, the present invention is also directed to the preparation of a porcine subunit identification vaccine using the recombinant E2 glycoprotein prepared according to the method of the present invention. The vaccine is characterized by a broad-spectrum protection of pigs that are resistant to different molecular typing. In one embodiment, the recombinant E2 glycoprotein 132, 39 CSFV-G1E2, produced by the method of the present invention, can be secreted into a culture medium by a transfected insect cell host having a molecular weight of about 56 kDa and non-reduced. Under conditions, a homodimer protein structure having a molecular weight of about 115 kDa can be formed. In another embodiment, the recombinant E2 glycoproteins CSFV-G2E2A and CSFV-G2E2B derived from the second type (2a) isolate are prepared, having molecular weights of about 55 kDa and 27 kDa, respectively, and in non-reducing conditions. The homodimer protein structure can also be formed. In one aspect, the invention also relates to a single-source antibody TY1 25 that specifically recognizes an E2 glycoprotein in the structure of a homodimer protein. In a specific aspect, the single-source antibody TY125 can be identified such as LPC, S-59, TD/9 6/TWN, 0406/CH/01/TWN, 38/KS/93/TWN, and 94.4/IL/94 Different molecular typing types of swine fever virus strains such as /TWN. Thus, the present invention also relates to a diagnostic reagent for detecting the presence or absence of swine fever virus in a sample, characterized in that it comprises a single-source antibody TY125. And a diagnostic reagent for detecting whether a pig is infected with classical swine fever virus, characterized by comprising a recombinant classical swine fever virus E2 glycoprotein prepared according to the method of the present invention. [details of the invention] The sore was first thought to be caused by ·βα£Γ///Μ·ϊ cfto/eraswiar (hog cholera bacillus), and in 1904 by de Schweinitz and Dorset confirmed by the viral virus cause. Classical swine fever virus (CSFV) was classified as a genus in the family of viruses. In recent years, due to molecular biology research, the genetic composition and viral replication of the genus of the virus are similar to those of the WavivzYWae virus family. Therefore, at the 5th International Virus Conference in 1991, the genus was classified as F/cfWv/ rWae virus family. The same genus is bovine viral diarrhea virus (BVDV) and sheep border disease virus (BDV). In addition to being structurally similar to antigenicity, the three viruses are also considered to have varying degrees of cross-reactivity. 1328039 The viral typing method can show the genetic correlation between different swine fever virus strains, and then analyze the genetic relationship among different strains through molecular epidemiology to trace the origin of the virus, the evolution and propagation path of the virus, and then Set a suitable prevention strategy. Choosing the appropriate genotype position as the ratio of the gene sequence - can't build the phylogenetic trees of the swine mad virus, 1 further explain the mutation and world distribution of the virus. Generally, the region for gene sequence comparison includes both the conservative regions and the variable regions» The early comparisons are performed with the E2 and NS5B gene sequences, and the φ nucleic acid sequences of different virus isolates are individually performed. The comparison found that the ratio of the two is most similar to the analysis results. Where the E2 gene sequence is aligned, the swine fever virus can be divided into two groups. The first type (group I) is attributed to non-European strains, represented by Brescia strain, including some older isolates, American and Asian isolates, and the second type (group Π) belongs to Europe. The system from which the isolate is derived is represented by Alfort strain. According to the infrastructure of this classification system, pat〇n et al. respectively added a 5' non-translated region (5'-NTR) for comparison, and with some genes of E2 and NS5B, After further analysis of the three-segment sequence, the swine fever virus can be mainly divided into three types. The first type is a long-established isolate, which is represented by ALD, Alfort 187 and Brescia strain, and can be subdivided into three subtypes: 1.1, 1.2 and 1.3. The second type is a strain that is popular in countries such as Europe, and is also divided into three subtypes: 2.1, 2.2 and 2.3. However, the third type of swine fever virus has only appeared in South Korea, Thailand, Japan and Taiwan, and can also be divided into four subtypes such as 3.1, 3·2, 3·3 and 3.4. Most research scholars use the classification criteria of Paton et al. as the main basis for studying the molecular epidemiology of the current swine fever virus. According to recent studies, there were only a few isolates of classical swine fever virus in Europe between 1920 and 1970, and they all belonged to the first type, but since the 1980s to the 1990s, the second type of molecular classification of swine fever Virus-based outbreaks have emerged in other countries such as Europe, and since 19 years, the 2.1 subtype has even been in European countries! 〇1328,039.. Become the mainstream of viral genotypes in cases of swine fever infections. As for the isolates of Taiwan before the 1990s, their genotypes can be located and classified as the third type (or native type) and should have existed in Taiwan before the Japanese era. However, according to the molecular epidemiological analysis of swine fever virus in Taiwan from 1993 to 2001, the genotype of the swine fever virus isolate from Taiwan after 1996 was completely replaced by the 2.1 subtype of swine fever virus that was invaded by the country. . The three envelope proteins of the swine fever virus were E2 (gp44/4 8), E3 (gp33) and El (gp55) before 1994, and were officially renamed Erns (in the order of the genome). E0; gp44/48), El (gp33) and E2 • (gp55)° The protein precursor of the swine fever virus is translated in the cytoplasm, after which the glycoprotein portion enters the endoplasmic reticulum lumen. At the same time, the cell's signalase will cleave the end of the endogenous signal sequence at the C-terminus of the nuclear protein and the E2 glycoprotein to form a protein complex of E0-E1-E2; The end of the signal sequence is cut off to produce a mature E2 glycoprotein; finally, the proteinase cleaves the E0-E1 protein complex to form the glycoprotein E0 and E. Because the C-terminus of the glycoproteins E1 and E2 has a hydrophobicity (hydrophobic) The amino acid sequence is thought to form a transmembrane region (TMR) of the protein, thereby immobilizing the protein on the envelope of the virus; while the glycoprotein E0 does not have a TMR, it may be covalently bonded. Connected to disease On the envelope. In addition, the TMR 糖 of glycoprotein E1 acts as a signal sequence for the E2 protein and has the function of transferring the E2 protein to the endoplasmic reticulum. The three envelope glycoproteins are present in purified viral particles or infected host cells, and even vinegar proteins expressed in cells by recombinant vacciniavirus, mostly in the form of dimer-bonded dimers. In addition to E0 and E2, in addition to forming Ε0-Ε0, E2-E2 homopolymers of about 100 kDa, E2 also forms an E1-E2 heterogeneous polymer with an size of about 75 kDa with E1 ( Heterodimer). 1328039 Among the glycoproteins of the three swine fever viruses, E2 is the first major envelope protein that was first discovered and studied. The importance of this is because E2 protein is the most important component of the surface of swine fever virus, and it is also the main antigen that causes swine fever virus infection in pigs. At present, many studies have shown that the immune response elicited by pigs after immunizing E2 glycoprotein alone can produce sufficient immunoprotective effects. In addition, glycoprotein E2 is also thought to be involved in the ability of the swine fever virus to infect host cells. Early research scholars used 13 swine fever single-antibody antibodies to classify the antigenic epitope (antigenic determinant or epitope) of E2 glycoprotein into four domains of A, B, C and D; among them, A and B C is a structural region with neutralization ability, and A structural region is considered to be highly reserved between different classical swine fever virus strains. These structural regions are located at the N-terminus of glycoprotein E2 and constitute two independent The unit of antigenic structure; one of the antigenic structural units consists of the B and C structural regions (uiiti B/C), and the other contains the structural region of the A site. The B/C structure unit is connected to the signal signal sequence, and the A structure unit is connected to the C terminal of the B/C. In the A structural unit, hydrazine has a highly hydrophobic region, and this segment also has a high degree of retention between different /Jew/Wrws. Immunization of the B/C or A structural units of the E2 glycoprotein alone will allow the pig to produce sufficient neutralizing antibodies to combat the lethal dose of the swine fever virus. When a pig is infected with a virulent strain of swine fever virus, the pig has died in the absence of an immune response in the clinical form of acute swine fever, but if it is resistant to pigs, it will produce a relatively high Humoral immune response" Generally speaking, in the acute swine fever period, the presence of neutralizing antibodies should not be detected in the blood of pigs. This may be related to the number of B lymphocytes in blood and lymphoid tissues caused by infection with swine fever. Rapidly reduced, and most of the virus in the lymphoid tissue center proliferation and replication, and damage surrounding lymphocytes. As for the medium and weak virulence of swine fever virus, when the course of disease is long, it can induce different degrees of antibody response in the host. However, if the sow is infected with the attenuated strain of swine fever virus before the 85th day of pregnancy, most of the piglets that are born are not immune to development and are susceptible to immune tolerance. Therefore, most of the piglets they produce are not IZ. There will be antibodies against swine fever virus. Infected animals produce antibodies against the structural proteins E^S, E2 and the non-structural protein NS3. Among them, the NS3 protein has a similar high degree of retention between different, and can cause cross-reactivity of antibodies between CSFV, BVDV and BDV. As for the E2 glycoprotein of the swine fever virus, the viral envelope protein which produces the neutralizing antibody is mainly caused by the pig, and the antibodies induced by Vns and NS3 have low neutralizing ability to the virus. In addition, due to the large variability between glycoprotein E"18 and E2, most of the swine fever antibody test kits were mainly based on the detection of *^^^ and £2 antibodies. Between 1950 and 1960, microbiologists used electron microscopy to observe baculovirus and investigate its pathogenic sequence. From the late 1960s to the early 1970s, a fine cell line of insect cells was developed, allowing the baculovirus to be subdivided by cell lines. The Environmental Protection Agency first applied baculovirus to biological insecticides in 1975. Since then, many scientists have studied the baculovirus and further developed the baculovirus into a eukaryotic expression vector. In the early stage of the establishment of the baculovirus expression system, the proportion of recombinant virus in the progeny virus is very low, which makes it extremely difficult to screen. In the early stage, most of the genes are substituted by foreign genes to regulate the recombinant protein gene by using its promoter. Performance. And because the wild type baculovirus forms a nuclear polyhedron when the cells are subcultured, it exhibits an occlusion-positive (occ + ) phenotype; but the recombinant baculovirus lacking the ρο//ι gene cannot form a nuclear polyhedron. Therefore, in cell culture, 'the phenotype of occlusion-negative (occ·) can be used as an excerpt marker. Another additional advantage of Occ_ virus is that it cannot cause natural infection among insect larvae, so the environment can be excluded. Security concerns. However, the occ phenotype is not easy to observe in cell culture, so it can be transformed into the gene of the recombinant virus by adding the /acZ gene to the foreign vector when the gene is transferred to the vector, and then the X-gal is selected to form a blue virus. Plaque recombinant virus to increase the sensitivity of recombinant virus screening. 1328,039 In recent years, researchers have used baculoviruses to produce virus-like particles (VPLs), which are used in the development of vaccines and have been shown to induce protective immune responses. The virus assembly process can also be further explored. In addition, VPLs have the potential to evolve as a gene delivery system tool. In addition, the baculovirus expression system is also used in the application of eukaryotic virion di_s play. The principle is similar to the mechanism of phage display, mainly the protein and rod shape to be expressed. The fusion of the envelope protein gp67 of the virus enables the foreign protein to be correctly expressed on the surface of the baculovirus, and in this way, the function of the unknown protein in the cell and the cell can be explored, in order to investigate the protein structure and its actual regulatory function. One of the important methods. Therefore, the present invention utilizes a baculovirus expression system to produce a recombinant E2 glycoprotein derived from classical swine fever virus from insect cells, and tests the immunity of the recombinant E2 glycoprotein produced in pigs to evaluate Its availability as a secondary unit vaccine and its use for detecting pigs infected with wild virus strains. When a vertebrate is stimulated by an antigen, the B lymphocyte is induced to differentiate into a plasma cell capable of secreting a specific antibody via an immune reaction, and a plasma cell population (proliferation) proliferated by a B lymphocyte is secreted. The ability of an antibody, but because an antigen molecule usually has many epitopes, and at least one antibody can be induced in each decision, and the antigens in contact with the animal are numerous, so in the traditional antiserum, there are many Different antibodies are called multi-source antibodies. For the study and medical needs, it is necessary to obtain antibodies that recognize a single epitope, and thus develop single-source antibody technology. Since the lifespan of plasma cells is very short and cannot be maintained in vitro by artificial culture methods, the myeloma cell line derived from BALB/c mice can grow in vitro for a long time, so KShler and Milstein (1 975) For the first time, myeloma cells are fused with plasma cells to obtain cells that can be cultured in vitro for a long time and have a secretory function, called a hybridoma cell. The fusion tumor cells are further screened to obtain a cell line having a specific antibody secreted, and the same

/V 1328039 The secreted antibody is a monoclonal antibody. In recent years, many researchers have successfully produced recombinant single-source antibodies using the myeloma expression system and used them for diagnostic and therapeutic applications. In the process of cell fusion, not all plasma cells and myeloma cells can successfully form fusion tumor cells, so it is still necessary to inhibit the growth of unfused myeloma cells with a culture solution containing HAT. Normal cells generally synthesize nucleic acids in de novo synthesis (de wovo synthes ss). When this pathway is blocked by a factor such as aminopterin, the cells can be taken from the salvage pathway. T) and hypoxanthine (H) to synthesize DNA to constitute a nucleic acid. However, myeloma cell lines used for fusion, such as NS-1, cannot utilize chest and hypoxanthine due to lack of both thoracic (TK) and hypoxanthine-guanine phosphoribose transfer (HGPRT) enzymes. NS-1 cannot grow in the presence of aminopterin; although normal cells (such as B lymphocytes) can continue to grow through the rescue metabolic pathway, spleen cells will naturally die after one week of in vitro culture, so in HAT medium, only The fusion cell of NS_1 and spleen cells successfully grows in vitro, and because it has the genes of TK and HGPRT, it can rescue the metabolic pathway for nucleic acid synthesis when the aminopterin inhibits de novo synthesis. Since the fusion tumor cells can be cultured in vitro for a long period of time, and stably produce a single antibody, and the single-source antibody has high antigen specificity, it has been widely used in the diagnosis and treatment of diseases in recent years. In clinical applications of human medicine, single-source antibodies can be used for the diagnosis or treatment of tumors, cardiovascular diseases, viral infections, and inflammatory disorders. In animal medicine, single-source antibodies are most commonly used for antigen or antibody detection of infectious agents. In terms of academic research, single-source antibodies can be applied to the purification of specific proteins, the study of protein structure and properties, and the analysis and typing of antigenic characteristics of pathogens. For example, Paton et al. (Vet Res 26: 92-109, 1995) identified 76 different anti-1328039 cells from 66 peii/Wruies single-source antibodies. Original Type β The present invention also preliminarily produced a single-source antibody that is resistant to the formation of the lio2 glycoprotein of the liomodimer structure. According to the results of viral valence measurement, the single-source antibody TY 125 can recognize the epitope on the E2 glycoprotein, and should have high retention among different molecular types of swine fever. Thus, the single-source antibody _ TY125 of the present invention is extremely useful for detecting a detection reagent for the presence of classical swine fever virus. The following examples are intended to describe the invention in more detail and are not to be construed as limiting the scope of the invention. Example 1. Preparation of swine fever E2 glycoprotein single-source antibody NS-1 myeloma cells stored in liquid nitrogen were thawed one month before the scheduled cell fusion assay. In the first step, the cell cryotube was taken out from the liquid nitrogen tube, placed in a 37 ° C water bath, the cells were quickly dissolved, and cultured in 10 mL of RPMI-1640 containing 15% fetal bovine serum (FBS; HyClone®). After liquid suspension, the supernatant was removed by centrifugation at 800 rpm for 5 minutes, and the cells were cultured in 10 mL of RPMI-1640 containing 15% fetal bovine serum in a 25 cm2 φ cell culture flask at 37 ° C with 5% C02. Incubate in a constant temperature incubator. After the cells are up to 80% full, the cells are subcultured to a 75 cm2 cell culture flask and started at 1:1 days before the fusion, maintaining the optimal activity of the cells. And try to keep the cell density at 5χ105 cells/mL. After immunizing BALB/cByj mice with CSFV-G1E2 prepared in Example 3, - seven days after the second immunization, specific antibodies against swine fever virus in the serum of the mouse can be detected, which can identify the infection. S-59 swine fever virus strain of PK-15 cells, and virions with obvious green fluorescence under IFA staining. Take immunized mice and sterilize their spleen cells in a sterile manner. The spleen of each mouse The cell lavage fluid contains about 1~2xl 08 spleen cells. The spleen 1332539, spleen cells were fused with NS-1 cells, and then packed into 10 wells and 96-well microplates, and cultured in HAT-RPMI/FBS culture medium for 7 to 10 days in an incubator, and then observed cell growth. As a result, growth of 1 to 2 cell lines was observed in almost every well. Fourteen days after the fusion, the cell culture medium of 10 96-well microplates was initially screened by enzyme-binding immunosorbent assay, and 1/2 of the culture solution was replaced. A total of 960 samples were screened, and a total of 140 samples showed a positive reaction with a positive rate of approximately 14.58%. Then, 18 days after the fusion, the 140 samples were screened by IFA. A total of 8 samples were still positive, and the positive rate was about 5-73%. The 8 positive cell lines (numbered as 〇〇7, 051, 071, 〇85, 090, 117, 125 and 134, respectively) were sequentially expanded to 24 and 12-well cell culture plates, and after cell fusion On the 26th day, the cell culture medium was taken, and the staining of the swine fever virus spot was confirmed again by IFA. As a result, only the culture solution of the number 125 cell line was positive in all the samples. Thus, the fusion cell strain No. 125 was subjected to monoculture by the limiting dilution method. In the experiment, it was found that the growth of individual cells was observed about 7 to 10 days after the limiting dilution of the fusion tumor cells, and the culture medium of all the cell strains was observed to have obvious green fluorescence in the IFA screening results. The plaque is colored. Therefore, it is speculated that the fusion tumor cell line No. 125 may itself be a single cell-derived cell line. After two consecutive monocultures, it was confirmed that a fusion tumor cell was obtained and named as TY125 cell line. Example 2. Characterization of E2 single-source antibody TY 125 To further understand the single-source antibody TY 125 for different molecular typing The identification ability of the swine fever virus strain, 500-fold dilution of the single-source antibody TY125 produced by ascites, and the use of IAPICC as a stain for detecting the cost of swine fever virus, and further performing the IFA measurement with Pno. The detection results of the method are compared with each other (see Figure 1 for the results). The following different molecular typing types of CSFV strains were used in this experiment, including LPC and S-59 of the first type, TD/96/TWN of the second type, and 38/ of the third type. The strains of KS/93/TWN, 1328039, 94.4/IL/94/TWN were subjected to antibody titer measurement. PK-15 cells were cultured in 96-well microplates in DMEM containing 5% FBS. Each well was inoculated with 50 μί of a suspension containing 0.5~lx10 cells and placed in a 5% C02 incubator at 37 °C. Cultivate overnight. The virus solution to be tested is serially diluted 1 〇 in serum-free DMEM, and the dilution factor is from 10 〃 to 1 (Γ6, and each diluted virus solution is inoculated into a 96-well micro-flat plate, each Add 50 μί of virus dilution to one well and repeat for 4 times each dilution. Place the 96-well culture dish in a 37-inch 5% C02 incubator for 3 days, pour the culture solution, and then rinse with PB ST. Three times, it was dried in an oven at 37 ° C, and subjected to subsequent dyeing. Two kinds of antigenic discs were prepared by the above method, and one of the trays was diluted 100 times. Serum antibody Ριιο. 13, for indirect immunofluorescence staining, secondary antibody using rabbit anti-porcine IgG FITC-labeled antibody (Sigma), and another plate of E2 single-source antibody (TY 125) produced by ascites, after 500-fold dilution Indirect alkaline phosphatase immunocytochemistry (IAPICC) staining was performed to compare whether the viral titers measured by the two immunostaining methods were consistent. Indirect alkaline phosphatase immunocytochemical staining was similar to indirect immunofluorescence staining, only two Sub-antibody changed to goat The antibody was labeled with anti-mouse IgG alkaline phosphatase and stained with NBT/BCIP receptor (Sigma) for about 20 minutes at room temperature. The results are shown in Table 1. I328D39, Table 1. TY125 single Viral valence determination of ΡΚ-15 cells infected with different molecular typing porcine scorpion virus strains by source antibody and Ραα13 multi-source antibody TCIDjo/itiL CSFV disease Pno.13 ΤΥ125 Type I LPC 2x106 9.28χ105 S-59 4·3χ106 2x106 Type TD/96/TWN 9.28χ102 9.28x102 0406/CH/01/TWN 4·3χ103 4.3χ103 Type m 38/KS/93/TWN 2.94χ104 6.32χ104 94.4/IL/94/TWN 6.32χ103 6.32χ102

The results of the force test for the first type of swine fever virus strain were higher in Pno. 13 and about twice as high as the viral power measured by TY125. The results of the determination of the second type of swine fever virus strain showed that both antibodies measured the same price. However, for the results of the third type of swine fever virus strain, there is a big difference. The 38/KS/93/TWN virus price measured by TY125 is about 2 times higher than the power price measured by Pno.13. However, for the 94.4/IL/94/TWN virus price measurement, the opposite result is obtained, and the power price measured by Pno.13 is higher, and the viral power price measured by TY125 is 10 times higher; Pno.13 is a highly immune serum of pigs obtained by experimental infection with CSFV 94.4/IL/94/TWN strain, so Pno.13 is highly sensitive to the recognition ability of this strain. The single-source antibody TY1 25 is molecularly classified to the second type of TD/96/TWN and 0406/CH/01/TWN swine fever virus strains and the multi-source antibody Pno produced by the third type virus. 13 is quite. According to Huang et al. (Golden City, Deng Mingzhong, Huang Tianxiang, Zhong Minghua, Lin Shizhen. Abstract of the Association of Taiwan Animal Husbandry and Veterinary Association Annual Meeting. 50, 2 00 1) Needle I328D39. Epidemiology of swine fever virus in Taiwan from 199 to 2001 The analysis results show that the current isolates in Taiwan are mainly type II swine fever virus. Therefore, the single-source antibody TY 125 can be used for the detection of swine fever virus when it is used for virus isolation and screening. To further confirm the identification of the single-source antibody TY 125 on the swine fever virus protein, the experiment will be carried out by PK-15 cell proliferation of CSFV PT/99/TWN and 0406/CH/01/TWN strains, with sucrose. Purification by gradient-cushion semi-purification, and the semi-purified virus solution was subjected to protein electrophoresis under reducing and non-reducing conditions, and then transferred to ImmobilonTM-NC Transfer Membranes, and SHI125 produced by WH303 and cell culture method was used as Primary antibody for Western blot analysis. Results The single-source antibodies WH3 03 and TY125 can clearly identify the E2 glycoprotein with a molecular weight of about 55 kDa in the viral proteins of the swine fever virus PT/9 9/TWN and 04 0 6/CH/01/TWN strain. The E2 protein homodimer and monomer structures with molecular weights of 105 kDa and 54 kDa were identified in the non-reducing conditions. TY125 is shown to be a specific single-source antibody against the porcine E2 glycoprotein (Figure 1). Example 3. Preparation and antigenicity analysis of recombinant E2 glycoprotein of type 1 swine fever virus The construction kit used in this experiment was pENTR Directi'onal TOPO*9 Cloning Kits (InvitrogenTM) and BaculoDirectTM Baculovirus Expression System (Invitrogen). TM), the construction method is carried out according to the manufacturer's recommended steps. In this experiment, the first type of swine fever virus was used as the standard selection strain of the subunit E2 gene, and the first type of swine fever virus cDNA which has been constructed in the laboratory was used as a template, and P/«polymerase (combinbinant) (MBI) , Fermentas) blunt-end polymerase chain reaction (PCR) of E2 gene, primer selection: 1328,039, select strains 1 and 6 with M13 Forward (-20) and M13 Reverse primer, the full-length sequence of the target gene E2 Bidirectional sequencing and alignment. The sequencing results showed that the E2 gene sequence of strain No. 6 was identical to the cDNA sequence used in the experiment. The recombinant vector of strain No. 6 was extracted and dissolved in DDW, and the carrier concentration was 392.6 iig/pL by photoelectric colorimetry. Therefore, 1 pL of the recombinant vector solution was mixed with BaculoDirectTM Linear DNA (containing 300 ng). And added LR Clonase® at 25 for 18 hours, allowing the E2 gene to be converted to a specific position of baculovirus DN A via LR recombination reaction. The primary recombinant virus was purified by limiting dilution and point hybridization and screened twice to obtain a recombinant baculovirus strain: g CSFV-G1E2 baculovirus. The recombinant baculovirus was inoculated into Sf9 cells, and after 72 hours of culture at 27t, the virus was isolated and co-proliferated to obtain three generations of recombinant baculoviruses such as sputum, sputum and sputum. The recombinant baculovirus-infected insect cells and their culture solutions were taken, and the molecular weight and antigenicity of the recombinant protein were analyzed by protein colloidal electrophoresis (SDS-PAGE) and Western blotting.

After extracting the recombinant protein, the extracted protein product was confirmed by protein colloidal electrophoresis and western blotting under reduced and non-reducing conditions. However, on the protein colloid electrophoresis film, the stained bands of the recombinant protein were not apparent. Therefore, the protein on the film was transferred to ImmobilonTM-NC Transfer Membranes, and the single-source antibody WH303 was used for Western blotting. WH303 recognizes the CSFV-G1E2 recombinant protein extracted from the cells and the culture medium, and estimates the molecular weight through the immage system. The results show that the molecular weight of the CSFV-G1E2 recombinant protein extracted from the culture solution is about 56 kDa, but not reduced. Under conditions, this recombinant protein can form a homodimer protein structure with a molecular weight of approximately 115 kDa. The CSFV-G1E2 recombinant protein extracted from the cell has a larger molecular weight of about 59 kDa and can form a homodimer protein structure with a molecular weight of about 117 kDa. In addition, an E2 protein product with a molecular weight of about 42 kDa can be seen. 2 ). The results showed that the CSFV-G1E2 recombinant protein can be successfully expressed by baculovirus and still has its antigenicity, and the recombinant protein extracted from cells and culture medium, 1328.039, can form the homodimer protein structure under non-reducing conditions. In addition, the CSFV-G1E2 recombinant protein can be successfully secreted into the culture medium by the action of the signal sequence and cleavage after being expressed in Sf9 cells. The molecular weight of CSFV-G1E2 recombinant protein predicted by DNASTAR software should be 43.1 kDa. If the signal sequence is added, the molecular weight will increase to 46.3 kDa, but the actual performance of recombinant E2 protein will increase from the predicted 43.1 and 46.3 kDa. 56 and 59 kDa. From this result, it is speculated that when the CSFV-G1E2 protein is expressed by Sf9 cells, it should undergo some degree of post-translational modification.

To further understand the antigenicity of the CSFV-G1E2 recombinant protein, Sf9 cells (about 0.1 MOI) were infected with CSFV-G1E2 baculovirus in the experiment, and cultured at 27 ° C for 4 days, the cells were directly fixed on the culture plate, and then Staining was performed by the IFA and IAPICC methods using the single-source antibodies WH303, TY125 and the multi-source antibody Pno.13, respectively. The results showed that the single-source antibody WH3 03, TY125 and the multi-source antibody Pno. 13 can recognize the CSFV-G1E2 recombinant protein expressed in the cytoplasm of Sf9 by the IFA and IAPICC methods (Fig. 3). When the CSFV-G1E2 protein is expressed in Sf9 cells, it has antigenicity. In addition, the extracted CSFV-G1E2 recombinant protein was subjected to protein electrophoresis in reducing and non-reducing conditions, and its antigenic analysis was carried out by Western blotting. As a result, the 08?¥-61 £2 protein extracted from the cells and the culture medium was recognized by the single-source antibody WH3 03 in the reducing condition (see Fig. 4). The reactivity of the recombinant E2 protein of Figure 5 with single-source and multi-source antibodies showed that the pigs had high immune serum (multi-source antibody) Ρηο.13, Ρπο.68 and PHct, but only weakly identified intracellular extraction. The recombinant protein does not recognize the recombinant protein in the culture solution. The single-source antibody TY125 and the multi-source antibodies PM10-1 and E101 were unable to recognize the CSFV-G1E2 recombinant protein under reducing conditions. However, in the non-reducing conditions, the single-source antibody WH303 and the multi-source antibodies PM10-1, E101, Ρηο·13, 卩11〇.68 and ? 11(^ can identify the CSFV-G1E2 recombinant protein which forms the structure of ho mo dimer and monomer protein, indicating that the recombinant E2 glycoprotein is similar to the swine fever virus in the natural state. The single-source antibody TY125 can only be identified to form homodimer. Structure 1328039,

I I CSFV-G1E2 recombinant protein. Therefore, the results of the Western blotting method show that the homodimer and monomer protein structures formed by the CSFV-G1E2 protein can be recognized by high-immunity sera of pigs of various origins, including inoculation of different molecular types of swine fever virus or immune products. After the E2 subunit vaccine, the prepared pigs were highly immune to serum. However, the linear form of the protein formed by the reduction of the disulfide bond of the CSFV-G1E2 protein by P-mercaptoethanol is not easily recognized by the high immune serum of pigs. This shows that the structure of the recombinant E2 protein formed by the disulfide bond has a decisive influence on its maintenance of the antigenicity of the CSFV E2 protein.

In order to try to understand whether CSFV-G1E2 recombinant protein can induce the production of neutralizing antibodies with neutralizing classical swine fever virus, after initial confirmation of its high antigenicity, it is expressed in Sf9 cells in large quantities and extracted from the culture medium. The recombinant protein was concentrated by UltracelTM Low Binding Regenerated Cellulose (300, 00 MWCO) to serve as an immunogen for animal immunization experiments. The recombinant E2 protein prepared in this way does not contain fetal bovine serum in its culture solution. In the mouse immunization experiment, the recombinant protein concentrate is concentrated before immunization (the total protein concentration is about 518.92 m/mL, Immage system estimates that recombinant E2 protein accounts for about 20.1% of total protein, and the concentration of recombinant E2 protein is about 104.3 pg/mL. It is diluted to the desired concentration with TNM-FH, and then with adjuvant 1^〇1^11丨<; 16 * 11 ^ 1113 mixed in a 1:1 volume, as the mouse immunogen. Mice were immunized twice with different doses of recombinant protein (4, 8 and 16 pg/mouse), and blood was collected every two weeks after immunization, and the serum neutralizing antibody titer was carried out with S-5 9 strain. test. It was found that this recombinant protein can successfully induce mice to produce antibodies with the ability to neutralize swine fever virus (Fig. 6), but the highest dose (16 pg/mouse) of mice can be compared with the antibody titer. The reaction, and 56 days after immunization, the neutralizing antibody was found to be as high as 32 and 128 times. In the vaginal control group, there was no sputum neutralizing antibody ascending response. In the pig immunization experiment, the extracted CSFV-G1E2 recombinant protein was concentrated, and the total protein concentration was determined to be about 7307.74 mg/mL, while the immunized 132.39. system estimated that the recombinant E2 protein accounted for about 11% to 4% of the total protein. The concentration of the CSFV-G1E2 recombinant protein was approximately 857.02 pg/mL. The recombinant protein concentrate was diluted to the desired concentration with TNM-FH before immunization, and then mixed with the adjuvant Montanide® IMS 1113 in a volume of 1:1, and immunized intramuscularly. The pigs did not show any adverse reactions after immunization. Pigs 1 to 4 in group A were basal-immunized with concentrated and then diluted recombinant E2 protein, and the immunization dose was 100 pg/pig. Two weeks after the basal immunization, neither the serum neutralizing antibody titer test nor the CHEKiT® CSF-SERO ELISA detected the antibody ascending response (Fig. 7) = therefore, in the case of booster immunity,

The pigs numbered A-1 and A-2 were changed to the unconcentrated CSFV-G1E2 protein (containing 5% fetal bovine serum), and the first reinforcement was performed three weeks after the basic immunization. One week after the booster immunization, the swine fever specific neutralizing antibody can be measured in the serum of the pig, and the antibody titer is as high as 512 and 128 times, respectively, and three weeks after the booster immunization, the neutralizing antibody Still showing a sustained upward response. Pigs still vaccinated with concentrated CSFV-G1E2 protein (A-3, A-4) have lower antibody responses than A-1 and A-2, but the antibody titers can still be as high as 128 and 32. Times. This result shows that the pig's neutralizing antibody power will produce a rapid and stable ascending response from a week after the booster immunization, and the unconcentrated CSFV-G1E2 protein can induce good pigs. Neutralizing antibody response. In addition, the serum neutralizing antibody titer of the pigs showed a tendency to rise gradually with time during the test period, and the 42-day basal immunity of the pigs increased to 512, respectively. 25, 64, 25 and 25 6 times. The results showed that recombinant E2 protein could induce the production of antibodies against swine fever virus in pigs, and the rise of hog-specific antibodies was also detected in pig serum by CHEKiT® CSF-SERO ELISA. The measured ELISA force price and the neutralizing antibody force price showed a parallel trend. Example 4. Preparation and antigenicity analysis of recombinant E2 glycoprotein of CSFV type 2a isolate A baculovirus expression system was used in a method similar to that described in Example 3 >5 - 1328,039. The insect cell expresses and secretes the recombinant swine fever virus E2 glycoprotein derived from the swine fever virus type II 2a isolate and performs antigenic analysis. The constructing kit used in this experiment is pENTR Directional TOPO® Cloning Kits (InvitrogenTM). With the BaculoDirectTM Baculovirus Expression System • (InvitrogenTM), the construction method is carried out in accordance with the manufacturer's recommended procedures. The swine fever virus strain used in this experiment is a swine fever virus strain 96TD which belongs to the second type 2a isolated from the wild in Taiwan, and reverse transcribed its RNA into cDNA using reverse transcriptase, and then uses this cDNA as the subunit E2 gene. The template, the amplification of the E2 gene by Taq polymerase, the primer for the polymerization enzyme chain reaction (PCR) selected for this experiment is:

CSFV-G2E2A- F3- 5'- AAG GAA AAA AGC GGC CGC CCC CTT CAC CAT GGC ATT TCT CAT CTG CTT 3* (48 mer)

CSFV-G2E2A-R3- 5'- TTG GCG CGC CCA CCC TTA AAT TCG GCG AAG TAG-3 ’ (33mer)

The CSFV-G2E2A-F3 and CSFV-G2E2A-R3 primers were designed according to the sequence of 23 53 ～ 2373 bp and 3442 ～ 3465 bp of the 96TDE2 strain sequence, respectively, and 2353~2 442 bp was the gene sequence for translating the E2 protein signal sequence. In addition, the primer was used to create an AscI restriction enzyme cleavage site and a TOPO® cloning site (C ACC) and a Kozak transcription initiation sequence (ACC ATG G) containing ATG at the 5' end of the PCR product; It contains a Notl restriction enzyme cleavage site. The detailed steps of PCR were as follows: 1 μΐ^ purified 96TD strain cDNA was used as a template, followed by introduction of primer (10 μΜ) 1 μΙ each, 5 μί dNTPs (2.5 mM), 5 μί 10× PCR buffer, 1 μΙ> Taq Polymerase, 36pLSDDW. The mixture was heated at 95 ° C for 2 minutes and then at 94 ° C for 30 seconds, 57 ° C for 30 seconds, and 72 ° C for 30 seconds for 30 cycles. After the reaction, electrophoresis was carried out on a 2% agar gel at a voltage of 100 volts. If the analysis results are consistent with the expected product size, the amplified E2 gene PCR product is further recovered by QIAguickTM PCR Purification Kit, and then the PCR product and the pENTR/D TOPO vector are cleaved with AscI and Notl restriction enzymes to obtain a PCR product. Body = 3 : 1 1328039. After mixing the molar ratio, add 2 μί 10 times the ligation buffer and 1 μί 4 DNA ligase to make the reaction volume 20 μί, constant temperature at 16 ° C. The ligation reaction was carried out for 16 hours in an incubator. The ligation reaction product was then transformed into One Shot® TOP10 Competent cells by heat shock and cultured and screened on LB plates containing 100 pg/ml. On the next day, 15 colonies were randomly selected, and the recombinant transfer vector was selected by plastid extraction and PCR reaction. Finally, the plastids of strains Nos. 6, 8, 22, 23 and 29 were selected for PCR and their product size was analyzed by electrophoresis. It is expected that 1,120 bp is consistent.

The strains 6 and 8 were selected by M13 Forward (-20) and M13 Reverse primers for bidirectional sequencing and alignment of the full-length sequence of the target gene E2. The sequencing results showed that the E2 gene sequence of strains 6 and 8 and the E2 gene sequence of the 96 TD strain cDNA sequence Nos. 6 and 8 used in the experiment and the 96 TD strain E2 cDNA sequence selected in the experiment were 4 nucleotide sequences differing, located on the 96TDE2 sequence, 2,613 (t-c), 2,736 (a->t), 2,763 (c-»t) and 3,165 (a-g) nucleosides The acid has a gene similarity of 99.6%. The translated amino acid is only the nucleotide at position 3, 165, which is converted from the original Lysine (Lys; K) to Arginine (Agr; R), and the similarity of the amino acid sequence is 99.9 ° / ^ The recombinant plastid containing TDE2 was subjected to LR recombination reaction in the manner described in Example 3, and mixed with LR Clonase® at 25 ° C for 18 hours to convert the E2 gene into a rod by LR recombination reaction. The specific location of the viral DNA. The primary recombinant virus was further purified by limiting dilution and point hybridization and screened twice to obtain a recombinant baculovirus strain: CSFV-G2E2A baculovirus. This recombinant baculovirus was inoculated into Sf9 cells, and after 72 hours of incubation at 27 ° C, the virus was isolated and co-proliferated to obtain three generations of recombinant baculoviruses such as PI, PII and PIII. The recombinant baculovirus-infected insect cells and their culture solutions were subjected to protein colloidal electrophoresis (SDS-PAGE) and Western blotting to analyze the molecular weight of the recombinant protein and the antigenicity of 乂 1328039. After extracting the recombinant protein, the extracted protein product was confirmed by protein colloidal electrophoresis and western blotting under reduced and non-reducing conditions. On the protein colloidal electrophoresis film, the dyed bands of the recombinant protein were not clearly displayed. Therefore, the protein on the film was transferred to the ImmobilonTM-NC Transfer Membranes membrane, and then the western blotting method was performed using the single source antibody WH3 03. . WH303 recognizes the CSFV-G2E2A recombinant protein extracted from the cells and the culture medium, and estimates the molecular weight by the immage system. The results show that the molecular weight of the TDE2 recombinant protein extracted from the culture medium is about 56 kDa, while in the non-reducing condition. The recombinant protein then forms a homodimer protein structure with a molecular weight of approximately 115 kDa. The results show that the CSFV-G2E2A recombinant protein can be successfully expressed by baculovirus and still has its antigenicity, and the recombinant protein extracted from the cells and the culture medium can be converted into the homodimer protein structure under non-reducing conditions. In addition, after expression of the CSFV-G2E2A recombinant protein in Sf9 cells, it can be successfully secreted into the culture solution by the action and cleavage of a signal sequence.

The molecular weight of CSFV-G2E2A recombinant protein predicted by DNASTAR software should be 43.1 kDa. If the signal sequence is added, the molecular weight will increase to 46.3 kDa, but the actual performance of recombinant E2 protein will increase from 43.1 and 46.3 kDa. 56 and 59 kDa. From this result, it is speculated that when the CSFV-G2E2 protein is expressed by Sf9 cells, it should undergo some degree of post-translational modification. To further understand the antigenicity of the CSFV-G2E2A recombinant protein, Sf9 cells (about 1 MOI) were infected with RBV-CSFV-G2E2A baculovirus in the experiment, and cultured at 27 ° C for 7 days, the cells were directly fixed on the culture plate. Then, the single-source antibody WH3 03 was used to stain with the multi-source antibody (including wheat stalk CSFV, PT strain CSFV, 94_4 mixed wheat sorghum CSFV challenge high serum), and IFA and IAPICC methods were used. The results showed that both the single-source antibody WH303 and the multi-source antibody were able to recognize the CSFV-G2E2A recombinant protein expressed in the cytoplasm of Sf9 by the IFA and IAPICC methods. When the CSFV-G2E2A protein is expressed in Sf9 cells, it has its antigen 1328,039. In addition, the recombinant protein of CSFV-G2E2A in the cell culture medium was collected, subjected to protein electrophoresis under non-reducing conditions, and subjected to antigenic analysis by Western blotting. Infected pigs by single-source antibody WH303 (Fig. 8) and TY125 (Fig. 9), and by type 1 (Fig. 10), type 2 (Fig. 11) and type 3 (Fig. 12) The results of detection of the porcine scorpion virus multi-source antibody porcine serum showed that the CSFV-G2E2A glycoprotein of the present invention can also be recognized by the single-source and multi-source antibodies in a non-reduced state. Further, when the same experiment was carried out under reducing conditions, it was found that similar to the recombinant protein produced in Example 3, only the single-source antibody φ WH303 could weakly recognize the recombinant protein, and TY125 and porcine polyclonal antibody were not recognized. The analysis by Western blotting method showed that the second type (2a) swine fever virus E2 protein was similar in antigenicity to the recombinant glycoprotein of CSFV-G1E2, that is, it could be single-source antibody WH3 03, TY 125 The pigs were identified against the first type of swine fever virus, the pig against the second type of swine fever virus and the pig against the multi-type serum antibody of the third type swine fever virus. Based on the results of the above examples, the single-source antibody TY 125 produced by the present invention has been confirmed to recognize three different molecular types of swine fever virus strains infected with PK-15 cells, and thus is extremely useful for preparing samples for detection. Whether there is industrial availability of diagnostic reagents for swine fever virus. Moreover, the single-source antibody TY1 25 can only recognize the homodimer structure of the E2 glycoprotein, and according to the study of the invention, the homodimer protein structure is closely related to the in vivo immunity of the classical swine fever virus E2 glycoprotein, and thus the single obtained by the present invention The source antibody TY 125 also has the potential to screen for highly immunological swine fever 'toxic E2 glycoproteins and fragments thereof. The recombinant E2 glycoprotein expressed by the insect cell infected by the recombinant baculovirus according to the method of the present invention can be identified by the Western blot method under non-reducing conditions, and can be recognized by the high immune serum of pigs infected with different molecular typing of swine fever virus. It shows that it has very similar antigenicity to the natural swine fever virus E2 glycoprotein, and it is also very useful for the production of 1328039. The preparation of the broad-spectrum subunit identification vaccine and the diagnosis for pigs infected with classical swine fever virus Industrial utilization of reagents. Therefore, the present invention utilizes the high degree of natural law, which can achieve the intended purpose of the present invention, and the present invention is a design that has never been seen before, and has a very practical effect. , 提起 file an invention application in accordance with the law, and please give a patent as soon as possible, to the sense of virtue. 1328039, [Simple diagram of the diagram] Figure 1 shows cell supernatants infected with pig-stained virus strains C Lanes 1 and 4: PT/99/TWN; Lanes 2 and 5: 0406/CH/01/TWN) and Infected cell supernatants (Lanes 3 and 6) under reducing conditions (A) or under non-reducing conditions (B) with anti-CSFV single-source antibodies WH3 03 (Lands 1, 2 and 3) and TY125 (Lands 4, 5 and 6) The results of the Western blot analysis. Fig. 2 shows the results of Western blot analysis using WH3 03 for culture supernatants (A) and lysates (B) of Sf9 cells infected with CSFV-G1E2 baculovirus under electrophoresis conditions under reducing conditions.

Figure 3 shows the detection of the recombinant protein CSFV-G1E2 produced by CSFV-G1E2 baculovirus-infected Sf9 cells by IAPICC (A) and IFA (B). Among them, Sf9 cells were immunofluorescently stained with WH303 (A1 and Bl), Pno.13 (A2 and B2) or TY125CA3 and B3 after fixation. Figure 4 shows the culture supernatant (Lane 1) and lysate (Lane 2) of recombinant baculovirus-infected Sf9 cells separated by electrophoresis under reducing conditions (A) or under non-reducing conditions (B). The results of Western blot analysis using WH3 03. Lane 3: Cell lysate of uninfected Sf9 cells. Figure 5 shows the anti-reactivity of recombinant E2 protein with individual single-source and multi-source antibodies. Among them, Lane 1, 4, 7, 10, 13 and 16 were applied to the culture supernatant of recombinant baculovirus-infected Sf9 cells; and Laiie 2, 5, 8, 11, 14 and 17 were reconstituted. The lysate of the baculovirus-infected Sf9 cells was subjected to 12.5% SDS-PAGE electrophoresis under reducing conditions (A) or under non-reducing conditions (B) to separate the port 18: uninfected Sf9 cells. • Figure 6 shows the neutralizing antibody response in BALB/c mice after vaccination.

Dpv: The number of days after vaccination. Figure 7 shows the antibody titer in pig serum obtained from recombinant E2 protein vaccination by serum neutralizing antibody test (A) and CHKiT®1 CSF-SERO ELISA (B). Dpv: The number of days after vaccination. 1328.039 Figure 8 shows the complete CSFV-G2E2A glycoprotein (Lane CSFV-G2E2A, molecular weight about 55 kDa) and CGFV-G2E2B glycoprotein with different degrees of glycosylation (Lane 2) detected by the single-source antibody WH3 03. , CSFV-G2E2B, molecular weight of about 27kDa) Western blot analysis results. _ Figure 9 shows the complete CSFV-G2E2A glycoprotein (Lane 1, CSFV-G2E2A, molecular weight about 55 kDa) and CSFV-G2E2B glycoprotein with different degrees of glycosylation (Lane 2) detected by the single-source antibody TY125. , CSFV-G2E2B, molecular weight of about 27 kDa) Western blot analysis results = Figure 10 shows the complete CSFV detection of non-reducing state by multivalent antiserum against serotonin immunized with first swine fever virus -G2E2A glycoprotein (Lane 1, CSFV-G2E2A, molecular weight about 55 kDa) and different degrees of glycosylation 05 households ¥-62£26 glycoprotein (1^1^2, 08卩¥-〇2 ugly 28, molecular weight Approximately 27 1 £: 〇 3) The result of the Western blot analysis. Figure 11 shows the complete CSFV-G2E2A glycoprotein (Lane 1, CSFV-G2E2A, molecular weight about 55 kDa) and different glycosyl groups detected by multivalent antiserum antibodies obtained from the second type of classical swine fever virus infection. The degree of the degree of 卩 〇 〇 〇 丑 ugly 2 ugly glycoprotein (1 ^ 116 2, € 5? ¥ - 〇 2 ugly 28, molecular weight of about 27 1 £ ^) Western blot analysis.

Figure 12 shows the complete CSFV-G2E2A glycoprotein (Lane 1, CSFV-G2E2A, molecular weight of about 55 kDa) and different glycosyl groups detected in a non-reducing state by a multivalent antiserum antibody obtained by immunization with a third type of swine fever virus. The degree of the Western blotting analysis of the degree of the succulent degree of the succulent 26-glycoprotein (1^116 2,08?¥-〇2£23, molecular weight of about 27 15^).

Claims (1)

1328039 • X. Patent application scope: 1. A single-source antibody TY125' against a recombinant swine fever virus E2 glycoprotein (CSFV-G1E2 recombinant protein) characterized by binding only to an E2 glycoprotein forming a homodimer structure' and is registered under the accession number BCRC 9602405 Produced by a fusion tumor cell line. 2. According to the single source antibody of claim 1 of the patent application, it can recognize LPC at the same time. Different molecular typing types of swine fever virus strains such as S-59, TD/96/TWN, 0406/CH/01/TWN, 38/KS/93/TWN 94.4/IL/94/TWN. 3. A diagnostic reagent for detecting the presence or absence of swine fever virus in a sample, characterized by comprising a single-source antibody according to item 1 of the scope of the patent application. 4. A diagnostic reagent for detecting whether a pig is infected with classical swine fever virus, comprising: a recombinant swine fever virus E2 glycoprotein derived from a classical swine fever virus (CSFV) type I LPC isolate, and The swine fever virus E2 glycoprotein has the amino acid sequence as set forth in SEQ ID NO. 5. The diagnostic reagent according to claim 4, wherein the recombinant classical swine fever virus E2 glycoprotein is produced in an insect cell expression system. 6. The diagnostic reagent according to claim 4, further comprising a pig Prion Efns (E0) glycoprotein. 7. A porcine subunit identification vaccine characterized by comprising a recombinant swine fever virus E2 glycoprotein derived from classical swine fever virus (CSFV) 33 1328039 type I LPC isolated strain and a veterinary acceptable adjuvant. Or an excipient wherein the porcine mad virus E2 glycoprotein has an amino acid sequence as set forth in SEQ ID NO. 8. The swine madness subunit identification vaccine according to claim 7 of the patent application scope, wherein the recombinant hog cholera virus E2 glycoprotein is produced in an insect cell expression system* and forms a homodimer protein structure under non-reducing conditions and presents A pig immune response similar to that of a wild-type swine fever virus strain. 34 1328039 \ \1 B. Μ :4 k 111 k ww 1328039 1328039 1328039 1328039 M i,-· Μ ΠΊ ΠΊ:> ΡΜ10-1 1.10] IVll; ΡΜ1Π-1 [;!0! Μ 10 1112 Ι:&gt 14 15 16 17 1S Μ 10 1112 13 14 15 1Λ 17 ]Χ ΚΜ _ _ .4 k 1328039 8 7 6 mouth A: 4/zg 〇B: 8 β% △ C: 16 /zg XD: control △ Vaccination 1 Vaccination 2 ▼ Itl· ▼ Δ Q Δ 务 28 Δ □ Q 42 厶 □ □ ο ^— —1 56 (dPv) Figure 6 8 1328039 A· (aoo s NS Oooooooo 64208642 lx 1x 11 lx(%) A-l, A-2 A-3, A-4 0 14 28 42 (φν) Figure 7 8 7 1328039 1328039 1328039 % 1328039 1328039