TW201116627A - Recombined swine fever virus E2 glycoprotein, monoclonal antibody thereof, application in diagnosis agent and sub-unit vaccine - Google Patents

Abstract

The invention relates to utilize a baculovirus express system in an insect cell to produce a recombined E2 glycoprotein originated from an isolated strain of type II swine fever virus of classical swine fever virus (CSFV). The produced recombined E2 glycoprotein is characterized in that the immune responses generated in pigs are similar to that infected by wild swine fever virus. The invention also discloses a specific monoclonal antibody TY125 prepared from the recombined E2 glycoprotein, which can simultaneously recognize swine fever virus stains of different molecule subtypes, such as LPC, S-59, TD/96/TWN, 0406/CH/01/TWN, 38/KS/93/TWN and 94.4/IL/94/TWN. The invention also relates to the applications of using the produced recombined E2 glycoprotein and monoclonal antibody thereof for preparing the swine fever diagnosis agents sub-unit identification vaccines.

Description

201116627 IV. Designated representative map: (1) The representative representative of the case is: (1). (2) A brief description of the symbol of the representative figure:

5. If there is a chemical formula in this case, please disclose the chemical formula that best shows the characteristics of the invention: VI. Description of the invention: 201116627 [Technical field of the invention] The present invention utilizes a baculovirus expression system to produce insect-derived cells derived from swine fever virus. The E2 glycoprotein was recombined and the immunity of the recombinant E2 glycoprotein produced in pigs was tested to assess its availability as a secondary unit vaccine and for the detection of pigs infected with wild virus strains. [Prior Art] [Background of the Invention] Hog cholera (HC) is currently officially named and widely used internationally as a disease name of classical swine fever (CSF), which is a highly contagious and highly lethal pig. Viral disease. 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, 159-172, 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 a major animal disease of List A and Class A. 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 a strict culling policy to achieve the goal of clearing swine fever (Edwards et al., Vet Microbiol 73: 1 03- 1 1 9,2000 ) 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, and the LPC organ is fully applied from the field. After the tissue culture vaccine was used as a control measure for swine fever, the pig plague situation in the province gradually improved. For the prevention of swine fever, only live-attenuated vaccines were available in the early days, including LPC swine fever vaccine, Japanese guinea pig ascending-negative (GPE) strain and Thiverval strain. Among them, 〇?£_ and Thiverval strains were domesticated by ALD and Alfort successively by cells. The currently widely used live poison reduction 201116627 toxic vaccine is the LPC swine fever vaccine/which provides very good immunoprotective efficacy for 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 Qirschot, JT, Ve.t Microbiol 73:103..- 1.19, 2003), and The antibody reaction caused by the stimuli can not be distinguished from the antibodies infected by the wild virus, thus increasing the difficulty in extinguishing the swine fever. Therefore, although the LPC vaccine is highly safe and protective, there are still many scholars working on the development of marker vaccines. * The three envelope proteins of the swine fever virus were E2 before 1994 (gp44/48). ), E3 (gp3 3) and El (gP55), and then officially changed their name to Efns (E0, gp44/48), E1 (gp33) and E2 (gp55) ( Ramenapf et al. J. Virol. 67: 3288-3294 «1 993; and Stark et al., Virology 1 74: 286-289, 1 990). Of the three glycoproteins of classical swine fever virus, the E2 glycoprotein is the first major envelope protein to be discovered and studied more (Hulst et al., J Virol 67: 5435-5442, 1993; Moormann et al., Virology, 177: 18). 4- 198,19 90 ). Its importance is due to the fact that E2 protein is the most important component of the swine fever virus surface and the main antigen that triggers the immune response in pigs infected with swine fever virus (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 efficacy (Hulst et al., supra; KSnig et al, J Virol 69: 6479-6486, 1 995). Since the E2 glycoprotein of classical swine fever virus has been considered 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, so the protein provided in 201116627 will have a eukaryotic expression environment (0reilly et al./in: baculovirus expression vector, laboratory manual, Oxford University Publishing , New York, 27-29, 1994). Since the successful establishment of the baculovirus expression system, it has been widely used in the expression of many viruses and eukaryotic genes, including the envelope protein of hepatitis C virus (HCV) (Httssy et al., Virus Res 45: 45-57). , 1996), nucleocapsid protein of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) (Denac et al, J Virol Methods 65: 169-181, 1997), structural and non-structural proteins of classical swine fever virus (Hulst et al., supra) Kiinig et al., supra; Steffens et al., Gen Virol 80: 2583-25 90, 1 999), human matrix metalloproteinase-9 (MMP-9) (Sadatmansoori et al., Protein Express Purif 23: 447- 45 2,2001) with triglyceride hydrolyzing enzyme (hTGH) (Alam et al, Protein Express Purif 24: 33-42 · 2002), etc., and by analyzing the characteristics of recombinant proteins to further understand the virus or human protein Function, or application to the development of vaccines and ELIS A detection reagents. In this experiment, the baculovirus expression system was used to carry out the colonization of the E2 protein of the swine fever virus strain, and it is expected to develop a swine fever labeling vaccine with protective benefits. Pigs infected with wild-type infections can be detected by the enzyme-binding immunosorbent assay (ELISA) for the detection of swine fever E2 or Vs antibodies (Floegel-Niesmann > G., Vet Microbiol 83: 121-136, 2001; Moormann) Et al., Vet Microbiol 73: 209-219, 2000), thus facilitating the removal of swine fever. However, in the current immunization group with E2 subunit vaccine, the timing of the production of the ^5 antibody is difficult to control due to the pig infected with the wild virus, and the ELIS A test kit cannot detect the pig in the wild virus. Providing sufficient sensitivity and specificity (van Oirschot, as described above), there is still much room for effort in the detection kits that work with the E2 marker vaccine. The single-source antibody to swine fever was first published in 1986 by Wens voort et al. At the time, 13 single-source antibodies were prepared (Wensvoort et al., Vet 201116627).

Microbiol 12: 101-108, 1 986 ). 'The successful preparation of swine fever single-source antibody has greatly contributed to the study of swine fever virus. For example, the structural region of the swine fever virus glycoprotein E2 was confirmed by analysis using these 13 single-source antibodies (van Rijn et al., Vet Microbiol 33: 221-230, 1992: van Rijn et al.: Human., J Gen Virol, 74: 2053-2060, 1.993; and Wensvoort'. J Gen Virol 70: 2865-2876, 1989). After that, the single-source antibody of swine fever has been continuously prepared by scholars from various countries and used to identify the antigenicity analysis of the swine fever virus glycoproteins E0 and E2 (Weiland et al., J Virol 64: 3563-3569, 1990; Weiland et al. Human, 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). In clinical applications, swine fever single-source antibodies can also be further applied to the development of antigen capture assays for the detection of swine fever virus antigens (Colijn et al, Vet Microbiol 59: 1 5-25, 1997); or development of composite capture-blocking Type enzymes combined with immunosorbent assay (CTB ELISA) to detect specific antibodies against swine fever with greater sensitivity and specificity (Clavijo et al, Vet Microbiol 60: 155-168' 1998; Wensvoort et al, J Gen Virol 70: 2865-28 76, 1 98 8) provides many useful tools for the diagnosis and monitoring of swine fever. SUMMARY OF THE INVENTION [Invention] The present invention relates to the production of recombinant pigs derived from a classical swine fever virus (CSFV) type II field isolate using an baculovirus expression system in insect cells. The prion E2 glycoprotein is produced, 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 epidemic 201116627 seedling is characterized by the ability to protect pigs against different molecular types of pig bacilli. In one embodiment, the recombinant E2 glycoprotein CSFV-G1E2 produced according to 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 under non-reducing 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-strain are prepared and have molecular weights of about 55 kDa and 27 kDa, respectively, and are non-reduced. The 1101110(^11^1* protein structure can also be formed under conditions. In one aspect, the invention also relates to a single-source antibody TY 125 which can specifically recognize the E2 glycoprotein which is a homodimer protein structure. In a specific aspect The single-source antibody TY125 recognizes different molecular types such as LPC, S-5 9, TD/96/TWN, 0406/CH/01/TWN, 38/KS/93/TWN, and 94.4/IL/94/TWN. 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 TY1 25» and is used for detecting whether a pig is infected with swine fever A diagnostic agent for viral infection, characterized by comprising a recombinant CSFV E2 glycoprotein prepared according to the method of the present invention.

[Detailed Description of the Invention] The pig was first thought to be caused by Βμζ·//μ·ϊ cAo/erasuifl (hog cholera bacillus), and was confirmed by de Schweinitz and . Dorset in 1904 by a viral strain. Classical swine fever virus (CSFV) was classified as PeW ί virus in the Virology family. In recent years, due to molecular biology research, the genetic composition and viral replication of PeW/Wrwi virus scorpion are similar to those of FMv/WriVw virus, so they were classified at the 5th International Virus Conference in 1991. In the F/flWv/rWare virus family, 201116627 is the same as the bovine viral diarrhea virus (BVDV) and the 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. The viral typing method can show the genetic correlation between different swine 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 A suitable control strategy. The appropriate genomic location can be selected as the alignment of the gene sequences to establish the phylogenetic trees of the swine fever virus, further explaining the variation of the virus and the world distribution. The regions generally used as gene sequence comparisons include conservative regions and variable regions. In the early comparison, the nucleic acid sequences of E2 and NS5B were used to compare the nucleic acid sequences of different virus isolates. The results of the comparison were the most similar. Where the E2 gene sequence is aligned, the swine fever virus can be divided into two groups (group s). 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 European isolates. The strain source system is represented by Alfort strain. According to the infrastructure of this classification system, Paton et al. separately added a 5' non-translated region (5'-NTR) for comparison, and a partial gene with E2 and NS5B, or After the three-stage sequence is combined for further analysis, the swine fever virus can be mainly divided into three types. The first type is a long-standing isolate, 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 popular isolate in Europe and other countries, 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 be divided into four subtypes: 3.1, 3.2, 3.3 201116627 and 3.4. In recent years, g-study scholars have used 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 1960 and 1970, all of which belonged to the first type, but from 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 1997, the 2.1 subtype has even become the mainstream of viral genotypes in cases of swine fever in European countries. As for the isolates of Taiwan before the 1990s, their genotypes can be located and classified as type 3 (or native) and should have existed in Taiwan before the era of data. However, according to the molecular epidemiological analysis of the swine fever virus in Taiwan from 199 to 200, the genotype of the swine fever virus isolate from Taiwan after 1996 was completely replaced by the 2.1 subtype swine fever virus that was invaded by the country. . The old names of the three envelope proteins of the swine fever virus before E1 were E2 (gp44/48), E3 (gp3 3) and El (gp55), and they were officially renamed E&quot in the order of the genome. ;15 (E0; gp44/48), E1 (gp33), and E2 (gp55). The protein precursor of the porcine prion is translated in the cytoplasm, after which the glycoprotein portion enters the endoplasmic reticultum. (endoplasmic reticultun. lume.n), at the same time, the cellular signalase will be located in the nucleus. The protein is cleaved with the end of the endogenous signal sequence of the C-terminus of the E2 glycoprotein to form a protein complex of Ε0-ΕE2; then the end of the endogenous signal sequence of the C-terminus of the El glycoprotein is cleaved to produce a mature E2 glycoprotein. Finally, the proteinase cleaves the E0-E1 protein complex to form the brewed proteins E0 and E1. Since both the C-terminus of glycoprotein E1 and E2 have a hydrophobic amino acid sequence, it is thought to form a transmembrane region (TMR) of the protein, thereby immobilizing the protein on the envelope of the virus; Protein E0 does not have a TMR and therefore may be linked to the viral envelope by covalent bonding. In addition, the sugar egg 9 m 201116627 white El TMR 尙 can be used as a signal sequence of the E2 protein, and has the function of transferring the E2 protein to the lumen of the endoplasmic reticulum. The glycoproteins of the three envelope glycoproteins in purified viral particles or infected host cells, even expressed in cells by recombinant vaccinia.virus, are mostly in the form of dimer-bonded dimers. . In addition to E0 and E2, in addition to forming a Ε0-Ε0, E2-E2 homopolymer with a size of about 100 kDa, E2 also forms an E.1-E2 heterogeneous polymerization with E1 of about 75 kDa. Heterodimer ° Among the three glycoproteins of classical swine fever virus, E2 is the first major envelope protein that was first discovered and studied. Its importance is due to the fact that E2 protein is the most important component on 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 alone after immunizing E2 glycoprotein can produce sufficient immunoprotective efficacy. In addition, glycoprotein E2 is also thought to be involved in the ability of the swine fever virus to infect the host cell. Early researchers have used 13 porcine singly-derived antibodies to classify the epitopes of E2 glycoproteins (antigenic determinant or epitope) into four domains: A, B, C, and D: A, B, and C is a structural region with neutralizing ability, and the A structural region is considered to have mobility retention between different classical swine fever virus strains. These structural regions are located at the N-terminus of glycoprotein E2 and constitute two independent antigenic structural units: one of the antigenic structural units consists of B and C structural regions (unti B/C), and the other contains the structure of the A site. Area. The B/C structure unit is connected to the 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 peW/v/rMs. Immunization of the B/C or A structural units of the E2 glycoprotein alone allows the pig to produce sufficient neutralizing antibodies to combat the lethal dose of swine prion. 10 201116627 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 be quite high in the future. Humoral immune response. In general, in the acute sputum infection period, the presence of neutralizing antibodies should not be detected in the blood of pigs. This may cause a rapid decrease in the number of B lymphocytes in blood and lymphoid tissues due to infection with swine fever. Some viruses are involved in the proliferation and replication of lymphoid tissue centers and destroy surrounding lymphocytes. As for the moderately and weakly toxic swine fever virus, the host can induce different degrees of antibody response during the long course of the disease. However, if the sow is infected with the attenuated strain of swine fever virus before the 85th day of pregnancy, most of the piglets born will not have antibiotic resistance in the future because the immune system of most of the fetuses is not developed and is immune to tolerance. Antibody production of the swine fever virus. Infected animals produce antibodies against structural proteins, Ε2 and the non-structural protein NS3. Among them, NS3 protein is different between them, and has similar high retention, and can cause cross-reaction of antibodies between CSFV, BVDV and BDV. As for the E2 glycoprotein of swine mad virus, it is a viral envelope protein which mainly causes the production of neutralizing antibodies in pigs, and the antibodies induced by NS3 have low neutralizing ability to viruses. In addition, due to the large 'variability between glycoprotein Efns and ugly 2, most of the swine fever antibody test kits were mainly based on the detection of 疸 疸 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 Between 1950 and 1960, microbiologists used electron microscopy to observe baculovirus and investigate its pathogenic sequence. From the late 1960s to the early 1970s, cell lines of insect cells were developed to enable baculovirus to be used in cell lines. The Environmental Protection Agency first applied baculovirus to biological pesticides in 1975. Since then, many scientists have studied the baculovirus and further developed the baculovirus into a carrier of eukaryotic expression. In the early stage of the establishment of the baculovirus expression system, the proportion of the recombinant virus in the 201116627 generation virus was very low, which caused great difficulty in screening. In the early days, most of them were replaced by foreign genes; genes were used to regulate the expression of recombinant protein genes using their promoters. And because the wild type baculovirus forms a nuclear polyhedron when the cells are subcultured, it exhibits occlusion-positive (〇cc + ; K phenotype; but the recombinant baculovirus lacking the pdA gene cannot form a nuclear polyhedron, Therefore, in cell culture, another additive advantage of Occ·virus according to its occlusion-negative (occlusion phenotype) is that it cannot cause natural infection between insect larvae, so the environment can be excluded. Safety concerns. However, the occT phenotype is not easy to observe in cell culture. Therefore, the /acZ gene can be added to the transfer vector, and the foreign gene can be simultaneously transformed into the gene of the recombinant virus, and then selected by X-gal. Blue plaque recombinant virus to increase the sensitivity of recombinant virus screening. In recent years, researchers have used baculovirus to produce virus-like particles (VPLs), which are used in development. Vaccines, and have been shown to induce protective immune responses, can also further explore the viral assembly process. In addition, VPLs also have development as a gene The potential of the gene delivery system tool • In addition, the baculovirus expression system is also used in eukaryotic virion display applications, the principle of which is related to the mechanism of phage display. Similarly, the protein to be expressed is fused to the baculovirus envelope protein gp67, so that the foreign protein can be correctly expressed on the surface of the baculovirus, and in this way, the unknown protein can be explored between the cells and the cells. The function is one of the important methods for studying the structure of the protein and its actual regulatory function. 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 recombinant preparation. E2 glycoprotein is immunogenic in pigs to assess its availability as a secondary unit vaccine and for the detection of pigs infected with wild virus strains. 0 12 201116627 When vertebrates are stimulated by antigen, they are exempted The reaction induces B lymphocyte differentiation into a plasma cell with the ability to secrete specific antibodies, consisting of one The plasma cell population derived from the differentiation of B lymphocytes has the ability to secrete an antibody, but since an antigen molecule usually has many epitopes, at least one antibody can be induced in each decision, plus Animals have a wide variety of antigens, so 'in traditional antiserum, there are many different antibodies, called multi-source antibodies. For the sake of research and medical needs, it is necessary to obtain antibodies that recognize a single epitope, and thus develop Single-source antibody technology] Because the life span 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. For the first time, K0hler and Milstein (1 975) fused myeloma cells with plasma cells to obtain cells that can be cultured in vitro and secreted, called hybridoma cells. After further screening of the fusion tumor cells, a cell strain secreting a specific antibody is obtained, and the antibody secreted 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 purposes. - In the process of cell fusion, not all plasma cells and myeloma cells can successfully form fusion tumor cells, so the growth of unfused myeloma cells must still be inhibited by the culture solution containing HAT. Normal cells generally synthesize nucleic acids by de novo synthesis (ί/e « ovo synthesis). When this pathway is blocked by certain factors (such as aminopterin), 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 thoracic (TK) and hypoxanthine-guanine phosphoribose transfer (HGPRT) enzymes. NS-1 is stored in aminopterin

13 I ϋ J 201116627 Can't grow underneath; although normal cells (such as sputum 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 NS-1 and The fusion tumor cells successfully spleen cells can maintain growth in vitro, and because they have the genes of TK and HGPRT, they 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-source 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: 92-1 09, 995) identified 76 different jes/ZvirMses isolated from anti-sea animals and pigs using 76 pesiivirwei single-source antibodies. Antigen typing. The present invention also preliminarily produces a single-source antibody against the formation of a homodimer-structured E2 glycoprotein. According to the results of the viral valence assay, the single-source antibody TY125 recognizes the epitope at the E2 glycoprotein, which is different. Molecular typing of swine fever virus strains should be highly reserving. Thus, the single-source antibody TY 125 of the present invention is extremely useful for detecting the detection reagent for detecting the presence of classical swine fever virus. [Embodiment] The following examples are intended to describe the purpose of the present invention in more detail and should not be considered. It is intended to limit the scope of the invention. Example 1. Preparation of Hog Cholera E2 glycoprotein single-source antibody 201116627 NS-1 myeloma cells preserved in liquid nitrogen were thawed one month prior to the scheduled cell fusion assay. In the first step, the cell cryotube is taken out from the liquid nitrogen tube, placed in a 37 ° C water bath to rapidly dissolve the cells, and 10 mL of RPMI containing 15% fetal bovine serum (fetal, .bovine serum, FBS; HyClone®). -1640 culture medium was suspended, centrifuged at 800 rpm for 5 minutes, and then the supernatant was removed, and the cells were further cultured in a 25 cm 2 cell culture flask with 10 mL of RPMI-1640 containing 15% fetal bovine serum, and placed at 37 ° C. Incubate in a 5% C02 incubator. After the cells are up to 80% full, the cells are subcultured to a 75 cm2 cell culture flask and started 3 days before the fusion, subcultured 1:1 daily to maintain the most cells. Good activity, and try to maintain the cell density at 5 χ 105 cells/mL » After immunizing BALB/cByj mice with CSFV-G1E2 prepared in Example 3, mouse serum can be detected seven days after the second immunization. It has a specific antibody against swine fever virus, which can identify S-59 swine fever virus strain infected with PK-15 cells, and can display virions with obvious green fluorescence under IFA staining. The immunized mice were lavaged in a sterile manner, and each mouse spleen cell lavage solution contained about 1 to 2 x 108 spleen cells. The spleen cells were fused with NS-1 cells, and then packed in 10 wells and 96-well microplates, and cultured in HAT-RPMI/FBS medium for 7 to 1 day, and then observed for 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, and a total of 8 samples were still positive, and the positive rate was about 5.71%. 8The 8 positive cell lines (numbered 007, 051, 071, 085, 090, 117, 125 and 134, respectively) were sequentially expanded to 24 and 12-well cell culture plates, and after the cell fusion of 201116627 On the 26th day, the cell culture medium was taken, and the staining of the swine fever virus plaque was confirmed again by IFA. As a result, only the culture solution of the number 125 cell strain was still positive in all the samples. - The fusion cell line No. 125 was then 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 strain. After two consecutive monocultures, it was confirmed that a fusion tumor cell was obtained and named as TY 125 cell strain. • Example 2. Characterization of E2 single-source antibody TY125 To further understand the ability of single-source antibody TY 125 to identify different molecularly typed swine prion strains, the single-source antibody TY1 25 produced by ascites was diluted 500-fold. The IAPICC was used as a stain for detecting the cost of the swine fever virus, and the results of the assay were further compared with those of the Pno. 13 IFA method (see Figure 1 for the results). The following different molecularly typed swine fever virus 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. For strains such as KS/93/TWN and 94.4/IL/94/TWN, the antibody was measured. PK-15 cells were cultured in 96-well microplates in DMEM containing 5% FBS, and each well was inoculated with 50 μΐ^ suspension containing 0.5~lx10 cells and placed in a 5% 002 constant temperature incubator at 37 °C. Cultivate overnight. The virus solution to be tested was serially diluted 10-fold in serum-free DMEM, and the dilution factor was from 10.1 to 1 (Γ6, and each diluted virus solution was inoculated into a 96-well micro-flat plate, each well. Add 50 μί of virus dilution and repeat 4 times for each dilution. Place the 96-well culture plate in a 5% C02 incubator for 3 days at 37 °C, pour the culture solution, and rinse with PBST. Three times, it was dried in an oven at 37 ° C and subjected to subsequent dyeing. The various strains of the swine fever virus strain were prepared by the above method, and the two plates of antigen 16 201116627 were prepared, one of which was diluted 100 times. Indirect immunofluorescence staining of the serum antibody Pno. 13, secondary antibody using rabbit anti-porcine IgG FITC labeling antibody (Sigma), and another E2 single source antibody (TY125) produced by ascites, after 500-fold dilution Indirect 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. Sub-antibody changed to goat anti- The murine IgG alkaline phosphatase was labeled with the antibody and stained with NBT/BCIP receptor (Sigma) for about 20 minutes at room temperature after induction. The results are shown in Table 1 below. Table 1. TY125 single-source antibody and Viral value of Pno.13 multi-source antibody against PK-15 cells infected with different molecular typing CSFV strains TCIDso/niL CSFV strain Pno.13 ΤΥ125 Type I LPC 2xl06 9.28χ105 S-59 4·3χ106 2χ106 Type TD/96/TWN 9.28x102 9.28x102 0406/CH/01/TWN 4.3χ103 4.3x103 Type 38/KS/93/TWN 2.94x104 6.32χ104 94.4/IL/94/TWN 6.32x103 6.32χ102

The results of the force test for the first type of swine fever virus strains are both higher than the power price measured by Pno.13, which is about twice the viral power value measured by TY125 - for the second type of swine fever virus strain. The results of the assay showed that both antibodies measured the same strength. However, for the results of the third type of swine mad virus strain, there is a big difference. The 38/KS/93/TWN 17 201116627 virus price measured by TY125 is higher than the Pno. 1 3 measured. About 2 times; however, the virus price test for 94.4/IL/94/TWN has the opposite result, and the price measured by Pno. 13 is higher, and the virus price is higher than that measured by TY1 25 10 times; Pno. 13 is highly sensitive to the identification ability of the virus strain because Pno.13 is a highly immune serum of pigs obtained by experimental infection of CSF and V. 94.4/IL/94/TWN strain. However, the single-source antibody TY12 5 is classified into the TD/9 6/TWN of the second type and the recognition ability of the swine fever virus strain of the second type with the infection of the third type virus. The resulting multi-source antibody Pno. 13 is comparable. According to Huang et al. (Golden City, Deng Mingzhong, Huang Tianxiang, Zhong Minghua, Lin Shizhen. Abstract of the Joint Annual Meeting of the Taiwan Society of Animal Husbandry and Veterinary Medicine. 50, 2001) The results of the epidemiological analysis of swine mad virus in Taiwan from 993 to 2001 show that The isolate of Taiwanese field is mainly type II swine fever virus, so the single-source antibody TY1 25 can be used for the detection of swine fever virus when it is used for virus isolation and screening. To further confirm the identification position of the single-source antibody TY1 25 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 in the original and non-reducing conditions, and then transferred to ImmobilonTM-NC Transfer Membranes, and then produced by using WH303 and cell culture method. TY125 was used as a primary antibody for Western blot analysis. Results The single-source antibodies WH303 and TY 125 can clearly identify the E2 glycoprotein with a molecular weight of about 55 kDa in the viral proteins of the swine fever virus PT/99/TWN and 0406/CH/01/TWN strain, and can also identify non-reducing. In the conditions, the molecular weights were 105 kDa and 54 kDa of the E2 protein homodimer and monomer structure, respectively. TY125 is shown to be a specific single-source antibody against the swine fever virus E2 glycoprotein (Fig. 1). Example 3. Preparation and anti-recombination of recombinant E2 glycoprotein of type 1 swine fever virus 18 201116627 Originality analysis The construction kits used in this experiment were pENT'R Directional TOPO® Cloning Kits (InvitrogenTM) and BaculoDirectTM Baculovirus Expression System ( Invitrog.enTM), construction method: follow the manufacturer's recommended steps. ...: This experiment uses the first type of swine fever virus as the standard selection strain of the subunit E2 gene, and uses the first type of swine fever virus cD.NA which has been constructed in the laboratory as a template to P/w. Recombinant (MBI, Fermentas) for the blunt-end polymerase chain of the E2 gene (PCR), the selection of the scorpion: CSFV-G1E2-: 5' especially ACCl ATGIGCATTT CTC ATC TGC TTG GTA|~ 3, CSFV-G1E2-R1: 5'~|AAATTC TGC GAA GTA ATC TGA GTG|~3' CSFV-G1E2-F1 and CSFV-G1E2-R1 primers are based on the first type of swine fever virus (GenBank accession number: AF352565) Preface! J 2351 ~ 2371 bp and 3440 ~ 3463 bp were designed, of which 235 1~2440 bp is the gene sequence for translating the E2 protein signal sequence. In addition, the primers were used to create the TOPO® selection site at the 5' end of the PCR product (|δ A_CCh and the Kozak transcription initiation sequence containing ATG (ACC ATG GV PCR detailed steps are as follows: Take 1 μί of purified cDNA) For the template, then add the above primer (10 μΜ) 1 pL, 5 μί dNTPs (2.5 mM), 5 pL 10 蓓 PCR buffer, 1 pL P/«polymerase, 36 SDDW. Heat at 95 °C for 3 minutes. The reaction was further carried out for 35 cycles at 94 ° C for 30 seconds, 57 ° C for 30 seconds, and 72 ° C for 1 minute. After the reaction, electrophoresis was carried out on a 2% agar gel at a voltage of 100 volts. The E2 囟 PCR product was further recovered by QIAguickTM PCR Purification Kit and stored at -20 °C for use.: Construction of the rod-shaped 19 201116627 virus E2 gene transfer vector using pENTR Directional TOPO® Cloning Kits. The previously prepared and purified E2 gene was mixed with the TOPO® cloning reaction solution and allowed to stand at room temperature (22-23 ° C) for 15 minutes to allow the E2 gene to bind to the pENTR/D-TOPO® vector. The reaction product is further reacted by heat shock The cells were introduced into One Shot18 TOPIO Competent cells and cultured and screened with LB-kana plates. On the next day, a number of colonies with resistance to kanamycin were observed on the culture medium. Therefore, 24 colonies were randomly selected from the culture medium and PCR was used to carry out the weight. · Initial screening of the vector transfer vector; the BacE2-Fl and BacE2-Rl primers were used to increase the E2 gene in the plastids of the 24 strains, and after the PCR product was analyzed by electrophoresis, only the No. 20 strain had no expected product. Strips, while the product bands of strains 5 and 10 were less obvious. The remaining strains showed obvious PCR product bands, and the product size was as expected, which was 1,120 bp. Select strains 1 and 6 to M13 Forward (- 20) and M13 Reverse primer, the bidirectional sequence and alignment of the full-length sequence of the target gene E2. The sequencing results showed that the E2 gene sequence of strain 6 was identical to the cDNA sequence used in the experiment. The carrier was dissolved in DDW, and the carrier concentration was 392.6 ng/pL as measured by photoelectric colorimetry. Therefore, 1 μί of the recombinant vector solution was mixed with BaculoDirectTM Linear DNA (containing 300 ng). And added at 25 ° for sensing LRClonase® C 18 h, the LR recombination reaction via the E2 gene, the conversion of baculovirus DNA to a specific location. The primary recombinant virus was purified by continuous dilution and point hybridization. After two selections, a recombinant baculovirus strain was obtained: CSFV-G1E2 baculovirus. This recombinant baculovirus was inoculated to 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 P I, sputum and sputum. 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 and antigenicity of the recombinant protein. After extracting the recombinant protein, the extracted protein product was confirmed by the egg colloidal electrophoresis and the Western blotting method under the conditions of reducing and non-reducing conditions. However, on the protein colloidal electrophoresis film, the stained band of the recombinant protein was not apparently displayed. Therefore, the protein on the film was transferred to Imm〇bil〇nTM-NC TransferMem.branes, and the single source antibody WH303 was used for western ink. Dot staining. WH3 03 can identify the CSFV-G1E2 recombinant protein extracted from the cells and the culture medium, and estimate 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. Under non-reducing conditions, the recombinant protein can form a homodimer protein structure with a molecular weight of about 115 kDa. The CSFV-G1E2 recombinant protein has a larger molecular weight of about 59 kDa and a molecular weight of about 117. The homodimer protein structure of kDa, in addition, can be seen as an E2 protein product with a molecular weight of approximately 42 kDa (Fig. 2). The results show that the CSFV-G1E2 recombinant protein can be successfully expressed by the baculovirus and still has its antigenicity, and the recombinant protein extracted from the cells and the culture medium 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 4 3.1 k'D a. If the signal sequence is added, the molecular weight will increase to 46.3 kDa, but the actual expression of recombinant E2 protein will be predicted by the molecular weight. 43.1 and 46'.3 kDa increased to 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 WH303, TY125 and the multi-source antibody Ρη〇·13 can recognize the CSFV-G1E2 recombinant protein expressed in the Sf9 cell 21 201116627 by the IFA and IAPICC methods (Fig. 3). It is shown that the CSFV-G1E2 protein has its antigenicity when expressed in Sf9 cells. In addition, the extracted CSFV-G IE2 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 CSFV-G1E2 protein extracted from the cells and the culture medium can be recognized by the single-source antibody WH303 under reducing conditions (see Fig. 4). The reactivity of the recombinant E2 protein of Figure 5 with single-source and multi-source also showed that the pigs had high immune serum (multi-source antibody) Pno.13, Pno.6 8 and PHct, but only weakly identified cells. The recombinant protein is extracted internally, and the recombinant protein in the culture solution cannot be identified. The single-source antibody TY125 and the multi-source antibodies PM10-1 and E101 could not 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, Pno.13, Pno.68 and PH ct can recognize the CSFV-G1E2 recombinant protein forming the homodimer and m ο η omer protein structures. , indicating that the recombinant E2 glycoprotein is similar to the swine fever virus in its natural state. The single-source antibody TY1 25 showed that only the CSFV-G1E2 recombinant protein forming the homodimer structure was recognized. Therefore, the results of the above western blot method show that the homodimer and monomer protein structures formed by CSFV-G1E2 protein can be recognized by a variety of different sources - pig high immune serum, including inoculation with different molecular types of swine fever virus or immunization. After commercialization of the E2 subunit vaccine, the prepared pigs were highly immunized, 'serum. However, the linear protein form formed by the reduction of the disulfide bond of CSFV-G1E2 protein by β-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 as the antigenicity of the classical swine fever virus 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. Recombinant protein, followed by UltracelTM Low Binding m 22 201116627

Regenerated Cellulose (30,000 MWCO) is concentrated in volume as the immunogen for animal immunization experiments. The recombinant E2 protein was prepared in this manner, and fetal bovine serum was not added to the culture solution. In the mouse immunization experiment, the recombinant protein concentrate was pre-immune (the total protein concentration was about 5.18.92 111§/111[, and 111111^§6 8>^6111 estimated that the recombinant E2 protein accounted for about 20.1 of the total protein. %, converted to recombinant E2 protein concentration of about 104.3 pg / inL) diluted to the required concentration with TNM-FH, and then with adjuvant] ^ 〇 1 ^ & 11 丨 (1 ^ 11 ^ 8 1113 to 1:1 Volume-mixed, used as the immunogen of mice. The mice were immunized twice with the same dose of recombinant protein (4, 8 and 16 pg/mouse), and blood was taken every two weeks after immunization, and S- The 59 strain was subjected to a serum neutralizing antibody titer test. It was found that this recombinant protein can successfully induce antibodies against mice that have the ability to neutralize swine fever virus (Fig. 6), but the highest dose of immunization (16 μg/mo use) The mouse, in which the antibody valence can show a more obvious response, and 56 days after immunization, the neutralizing antibody titer can be as high as 32 and 128 times. In the negative control group, there is no mad mad neutralizing antibody. Ascension reaction. In the pig immunization experiment, the extracted CSFV-G1E2 recombinant protein is concentrated and the total protein is determined. The concentration is about 75 1 7.74 mg/mL, and the recombinant E2 protein is estimated to account for 11.4% of the total protein by the immage system. The concentration of the recombinant protein is about 857.02 4/1111^. The recombinant protein concentrate was diluted to the desired concentration with TNM-FH, and then mixed with adjuvant 1^〇111&11丨(16@11^1113 in a 1:1 volume to immunize by intramuscular injection. The pigs did not show any adverse reactions. Groups 1 to 4 pigs in group A were basal immunized with concentrated and then diluted recombinant E2 protein, and the immunization dose was 1〇〇pg/pig. Two weeks after the basic 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 immunization, the number A-1 was The pigs of A-2 were changed to the unconcentrated CSFV-G1E2 protein (containing 5% fetal bovine serum), and the first arrest was performed three weeks after the immunization of the base 23 I Si 201116627. One week after the boosting immunization In pig serum, the specific response of the swine fever specific neutralizing antibody can be measured, and the antibody titers are as high as 5 12 and 12 respectively. Eight times, and three weeks after the booster immunization, the neutralizing antibody still showed a continuous ascending response, while the antibody was still reacted with concentrated CSFV-G1E2 protein for porcine immunization (Α-3, Α-4). Although it is lower than Α-1 and Α-2, the antibody price can still be as high as 128 and 32 times. This result shows that the pigs are only one week after the tonifying immunity. A rapid and stable ascending response is obtained, and the unconcentrated CSFV-G1E2 protein induces a good neutralizing antibody response in pigs. 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-old antibody titer of the 1st to 4th pigs rose to 512, respectively. 256, 64 and 25 6 times. The results showed that the recombinant Ε2 protein did induce the production of antibodies against the ability of pig scorpion virus to neutralize, and CHEKiT® CSF-SERO ELIS A also detected the ascending of porcine mad antibodies in pig serum. And the measured ELIS A 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 II 2a isolate, class: using the method described in Example 3, using a baculovirus expression system to express insect cells and The recombinant swine 't-virus E2 glycoprotein derived from the swine fever virus 113⁄2 2a isolate was secreted and subjected to antigenic analysis. The constructs used in this experiment were pENTR Directional TOPO® Cloning Kits' (InvitrogenTM) and BaculoDirectTM Baculovirus. Expression System (InvitrogenTM), the construction method is carried out according to the manufacturer's recommended steps. The swine fever virus strain used in this experiment is a pig mad virus strain 96TD belonging to the second type 2a isolated from the wild in Taiwan. The RNA is reverse transcribed into cDNA by reverse transcriptase, and this cDNA is used as a model of the subunit E2 gene. 24 t Si 201116627 version, the amplification of the E2 gene by Taq polymerase, the primer of the polymerase chain reaction (PCR) selected by this experiment is: CSFV-G2E2A- F3- 55- 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) where CSFV-G2E2A-F3 and CSFV-G2E2A The -R3 arch I was designed according to the sequence of 2353 ~ 23 73 bp and 3442 ~ 3465 bp of the 96TDE2 strain sequence, wherein 2353~2442 bp is the gene sequence for translating the signal sequence of the E2 protein. In addition, the primer is in the PCR product. The 5' end creates a Kozak transcription initiation sequence (ACC ATG G) containing the AscI restriction enzyme cleavage site and the TOPO® cloning site (C ACC) and ATG: and the Notl restriction enzyme cleavage site at the 3' end. The detailed steps are as follows: Take 1 μΐ^ purified 96TD strain cDN A is used as a template, followed by introduction of primer (10 μΜ) 1 μιη, 5 pL dNTPs (2.5 mM) * 5 μιη 10 times PCR buffer, 1 gL Taq polymerase, 36 μί SDDW » first heated at 95 ° C for 2 minutes, The reaction was further carried out for 30 cycles at 94 ° C for 30 seconds, 57 ° C for 30 seconds, and 72 ° C for 30 seconds. After the reaction, electrophoresis was carried out on a 2% agar gel at a voltage of 100 volts. If the expected product size is consistent, the amplified E2 gene PCR product is further recovered by QIAguickTM PCR Purification Kit, and then the PCR product is cleaved with the pENTR/D TOPO vector using AscI and Notl restriction enzymes to obtain a PCR product: plastid=3: 1 molar ratio is mixed, then add 2 fiL of 10 times the ligation buffer and 1 μΐ of T4 DNA ligase, the reaction volume is 20 jiL, and the reaction is carried out in a 16t incubator for 16 hours. Join reaction. 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 Mg/ml. On the next day, 15 colonies were randomly selected, and the recombinant transfer vector was selected by plastid extraction and PCR reaction. The cells were selected for the plastids of No. 6, 8, 22, 23 and 29, and the products were analyzed by electrophoresis. The size is in accordance with the expected 1,120 bp. Select strains 6 and 8 with M13 Forward (-20) and M13 Reverse primers, and perform bidirectional sequencing and alignment of the full-length sequence of the target gene E2. The sequencing results show that the E2 gene sequences of strains 6 and 8 are in the experiment. The cDNA sequence of the 96TD strain used in the 6th and 8th strains of the Ε2 gene sequence and the 96 TD strain E2 cDNA sequence selected in the experiment have 4 nucleotide sequences. The difference is 2,613 on the 96TDE2 sequence ( T-»c), 2,736 (a-»t), 2,763 (c->t) and 3,165 (a-g) nucleotides have a gene similarity of 99.6%. The translated amino acid has only the nucleotide at position 3, 165, which is converted from the original 1^811^(1^3;1〇 to Arginine(Agr; R), and the amino acid sequence similarity is 99.9%. The constructed recombinant TDE2-containing plasmid 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 recombine the E2 gene via LR. Switch to the specific position of baculovirus DNA. The primary recombinant virus was purified by limiting dilution method and dot hybridization reaction and screened twice to obtain a recombinant baculovirus strain: CSFV-G2E2A baculovirus. The cells were inoculated into Sf9-cells and cultured at 27 ° C for 72 hours, and the virus was isolated and co-proliferated to obtain three generations of recombinant baculoviruses such as PI, PII and sputum. The recombinant baculovirus-infected insect cells and their culture solutions were taken as protein colloids. The molecular weight and antigenicity of the recombinant protein were analyzed by electrophoresis (SDS-PAGE) and Western blotting. After extracting the recombinant protein, the extracted protein was confirmed by protein colloidal electrophoresis and Western blotting under reducing and non-reducing conditions. The product was not apparently stained with the recombinant protein on the protein colloidal electrophoresis film. Therefore, the protein on the film was transferred to the ImmobilonTM-NC Transfer Membranes membrane, and then the single-source antibody WH303 was used for West 26 m 201116627. Ink dot staining. 'WH.303 can identify the recombinant protein of CSFV-G2E2A extracted from cells and culture medium, and estimate the molecular weight of the recombinant protein by immage system. The results show that the molecular weight of TDE2 recombinant protein extracted from the culture solution is about It is 56 kDa, and under non-reducing conditions, this recombinant protein can form a homodimer protein structure with a V-subamount of about 115 kDa. The results show that the CSFV-G2E2A recombinant protein can be successfully expressed by baculovirus and still have its The antigenicity, and the recombinant egg white extracted from the cells and the culture medium can form the homodimer protein structure under non-reducing conditions. In addition, the CSFV-G2E2A recombinant protein can be expressed in Sf9 cells by The signal sequence was successfully secreted into the culture medium by the action of the signal sequence. The molecular weight of the recombinant protein CSFV-G2E2A was predicted by DNASTAR software. For 43.1 kDa, the molecular weight is increased to 46.3 kDa with the addition of the signal sequence, but the actual performance of the recombinant E2 protein increases from the predicted 43.1 and 46.3 kDa to 56 and 59 kDa. From this result, it is speculated that CSFV- When the 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 to the culture plate. In addition, the single-source antibody WH3 03 and the multi-source antibody (including the wheat sorghum strain CSFV 'PT 8 && 丨 11 € 3 卩 ¥, 94.4 mixed with the wheat cultivar € 8 乂 乂 after the challenge high serum), respectively Dyeing was performed by IFA and IAPICC methods. The results showed that both the single-source antibody WH303 and the multi-source antibody can recognize the CSFV-G2E2A recombinant protein expressed in the cytoplasm of Sf9 by IFA and IAPICC methods. It is shown that the CSFV-G2E2A protein has its antigenicity when expressed in Sf9 cells. In addition, the recombinant protein of CSFV-G2E2A in the cell culture medium is collected, subjected to protein electrophoresis in non-reducing conditions, and then subjected to Western blotting. Sexual analysis. Infected pigs by single-source antibody WH303 (Fig. 8) and TY125 27 201116627 (Fig. 9), and molecular typing using type 1 (Fig. 10), second (Fig. 11) and type 3 (Fig. 12), respectively. 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 the recombinant protein produced in Example 3 was similar, and only the single-source antibody WH303 could weakly recognize the recombinant protein, and TY 125 and the porcine polyclonal antibody were not recognized. The results of the Western blotting method show that the second type (2a) of the swine fever virus E2 protein is similar in antigenicity to the recombinant glycoprotein of CSFV-G1E2, that is, it can be used as a single source antibody WH303, TY125. 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 typing 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 live immunity of the classical swine fever virus E2 glycoprotein, and thus the present invention has a single The source antibody TY125 also has the potential to screen for highly immunological antibodies to the prion E2 glycoprotein and its fragments. 'Recombinant E2 glycoprotein expressed by insect cells infected with recombinant baculovirus according to the method of the present invention, and can be infected with different molecular typing pig swine fever virus high immune serum by Western blotting method under non-Liaoyuan conditions. It is identified as having a very similar antigenicity to the natural swine fever virus E2 glycoprotein, and is also very useful for the preparation of a broad-spectrum subunit identification vaccine and for diagnosis of 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. The present invention is a design that has never been seen before, and has a very practical effect, so the above creation has met the height of the invention patent. The requirements for creation, 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. 201116627 [Simplified Schematic] Figure 1 shows cell supernatants infected with classical swine fever virus strains (Lanes 1 and 4 : PT/99/TWN; Lanes 2 and 5: 0406/CH/01/TWN) and uninfected Cell supernatants (Lanes 3 and 6) under reducing conditions (A) or under non-reducing conditions (B) with anti-CSFV single-source antibodies WH303 (Lands 1, 2 and 3) and TY 125 (Lands 4, 5) And 6) the results of the Western blot analysis. · Figure 2 shows the results of Western blot analysis using WH303 for culture supernatant (A) and lysate (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 TY125 (A3 and B3) after fixation. Figure 4 shows Sf9 cells infected with recombinant baculovirus. The culture supernatant (Lane 1) and the lysate (Lane 2) were separated by electrophoresis under reducing conditions (A) or under non-reducing conditions (B), and the results of Western blot analysis were performed with WH303. Lane 3: Cell lysate of uninfected Sf9 cells. Figure 5 shows the 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 Lane 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). Lane 18: cytosolic product of uninfected Sf9 cells. Figure 6 shows the neutralizing antibody response of BALB/c mice after vaccination. Dpv: The number of days after vaccination. Figure 7 shows the detection of antibodies in pig serum from recombinant E2 protein vaccination by serum neutralizing antibody test (A) and CHKiT® m 30 201116627 ·» >. CSF-SERO ELISA (B) Price. Dpv: The number of days after vaccination. Figure 8 shows the CSFV-G2E2B glycoprotein (Lane 2, CSFV) detected by the single-source antibody WH303 in the non-reduced state of the intact CSFV-G2E2A glycoprotein (Lane 1, CSFV-G2E2A, molecular weight about 55 kDa) and different degrees of glycosylation. -G2E2B, molecular weight of about 27 kDa) Western blot analysis results. Figure 9 shows the CSFV-G2E2B glycoprotein (Lane 2, a complete CSFV-G2E2A glycoprotein (Lane 1, CSFV-G2E2A, molecular weight about 55 kDa) and a different degree of glycosylation detected by the single-source antibody TY1 25 in a non-reduced state. The results of Western blot analysis of CSFV-G2E2B, molecular weight of approximately 27 kDa). Figure 10 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 first type of swine fever virus. The result of Western blot analysis of the degree of CSFV-G2E2B glycoprotein (Lane 2, CSFV-G2E2B, molecular weight of about 27 kDa). 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 result of Western blot analysis of the degree of CSFV-G2E2B glycoprotein (Lane 2, CSFV-G2E2B, molecular weight of about 27 kDa). Figure 12 shows the complete CSFV-G2E2A glycoprotein (Lane 1, CSFV-G2E2A, molecular weight 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 result of Western blot analysis of the degree of CSFV-G2E2B glycoprotein (Lane 2, CSFV-G2E2B, molecular weight of about 27 kDa). [Major component symbol description] m 31 201116627 Sequence Listing <110> Maoxing Biotechnology Co., Ltd. <120> Recombinant swine fever virus E2 glycoprotein, single-source antibody and application thereof to diagnostic reagents and subunit vaccines (2) <160>2 <170> Patentln Version 2.0

<210> 1 <211>406 <212> PRT

<213>Swine fever virus (CSFV) type I Brescia isolated strain <400>1

Ala Ser Thr Thr Ala Phe Leu lie Cys Leu lie Lys Val Leu Arg Gly Gin Val Val Gin Gly 15 10 15 20

Val lie Trp Leu Leu Leu Val Thr Gly Ala Gin Gly Arg Leu Ala Cys Lys Glu Asp His Arg 25 30 35 40

Tyr Ala lie Ser Thr Thr Asn Glu lie Gly Leu His Gly Ala Glu Gly Leu Thr Thr Thr Trp 45 50 55 60

Lys Glu Tyr Asn His Asn Leu Gin Leu Asp Asp Gly Thr Val Lys Ala lie Cys Met Ala Gly 65 70 75 80

Ser Phe Lys Val Thr Ala Leu Asn Val Val Ser Arg Arg Tyr Leu Ala Ser Leu His Lys Asp 85 90 95 100 105

Ala Leu Pro Thr Ser Val Thr Phe Glu Leu Leu Phe Asp Gly Thr Ser Pro Leu Thr Glu Glu \ 110 115 120 125

Met Gly Asp Asp Phe Gly Phe Gly Leu Cys Pro Tyr Asp Thr Ser Pro Val Val Lys Gly Lys \ 130 135 140 145

Tyr Asn Thr Thr Leu Leu Asn Gly Ser Ala Phe Tyr Leu Val Cys Pro lie Gly Trp Thr Gly 150 155 160 165

Val lie Glu Cys Thr Ala Val Ser Pro Thr Thr Leu Arg Thr Glu Val Val Lys Thr Phe Arg 170 175 180 185

Arg Glu Lys Pro Phe Pro Tyr Arg Arg Asp Cys Val Thr Thr Thr Val Glu Asn Glu Asp Leu 190 195 200 205 210

Phe Tyr Cys Lys Trp Gly Gly Asn Tip Thr Cys Val Lys Gly Glu Pro Val Thr Tyr Thr Gly 215 220 225 230

Gly Pro Val Lys Gin Cys Arg Trp Cys Gly Phe Asp Phe Asn Glu Pro Asp Gly Leu Pro 235 240 245 250

His Tyr Pro lie Gly Lys Cys lie Leu Ala Asn Glu Thr Gly Tyr Arg lie Val Asp Ser Thr Asp 255 260 265 270

Cys Asn Arg Asp Gly Val Val lie Ser Thr Glu Gly Ser His Glu Cys Leu lie Gly Asn Thr 275 280 285 290

Thr Val Lys Val His Ala Leu Asp Glu Arg Leu Gly Pro Met Pro Cys Arg Pro Lys Glu lie 201116627 295 300 305 310 315

Val Ser Ser Ala Gly Pro Val Arg Lys Thr Ser Cys Thr Phe Asn Tyr Ala Lys Thr Leu Arg 320 325 330 335

Asn Arg Tyr Tyr Glu Pro Arg Asp Ser Tyr Phe Gin Gin Tyr Met Leu Lys Gly Glu Tyr Gin 340 345 350 355

Tyr Trp Phe Asp Leu Asp Val Thr Asp Arg His Ser Asp Tyr Phe Ala Glu Phe lie Val Leu 360 365 370 375

Val Val Val Ala Leu Leu Gly Gly Aig Tyr Val Leu Tip Leu lie Val Thr Tyr lie Val Leu Thr 380 385 390 395 400

Glu Gin Leu Ala Ala Gly 405

<210>2 <211>406 <212> PRT

<213> Swine fever virus (CSFV) type II TD isolated strain <400> 2

Ala Ser Thr Thr Ala Phe Leu lie Cys Leu lie Lys lie Leu Arg Gly Gin Val Val Gin Gly lie 1 5 10 15 20 lie Trp Leu Leu Leu Val Thr Gly Ala Gin Gly Arg Leu Ser Cys Lys Glu Asp His Arg Tyr 25 30 35 40

Ala lie Ser Ser Thr Asn Glu lie Gly Pro Leu Gly Ala Glu Gly Leu Thr Thr Thr Trp Lys 45 50 55 60

Glu Tyr Asn His Gly Leu Gin Leu Asp Asp Gly Thr Val Arg Ala lie Cys lie Ala Gly Ser 65 70 75 80 85

Phe Lys Val Thr Ala Leu Asn Val Val Ser Arg Arg Tyr Leu Ala Ser Leu His Lys Arg Ala 90 95 100 105

Leu Pro Thr Ser Val Thr Phe Glu Leu Leu Phe Asp Gly Thr Ser Pro Ala lie Glu Glu Met 110 115 120 125

Gly Asp Asp Phe Gly Phe Gly Leu Cys Pro Phe Asp Thr Thr Pro Val Val Lys Gly Lys Tyr 130 135 140 145

Asn Thr Thr Leu Leu Asn Gly Ser Ala Phe Tyr Leu Val Cys Pro lie Gly Tip Thr Gly Val 150 155 160 165 lie Glu Cys Thr Ala Val Ser Pro Thr Thr Leu Arg Thr Glu Val Val Lys Thr Phe Lys Arg 170 175 180 185 190

Glu Lys Pro Phe Pro His Arg Val Asp Cys Val Thr Thr lie Val Glu Lys Glu Asp Leu Phe 195 . 200 205 210

Tyr Cys Lys Leu Gly Gly Asn Trp Thr Cys Val Lys Gly Asn Pro Val Thr Tyr Thr Gly Gly 215 220 225 230

Gin Val Arg Gin Cys Arg Trp Cys Gly Phe Asp Phe Lys Glu Pro Asp Gly Leu Pro His Tyr 235 240 245 250

Pro lie Gly Lys Cys lie Leu Thr Asn Glu Thr Gly Tyr Arg Val Val Asp Ser Pro Asp Cys 255 260 265 270

Asn Arg Asp Gly Val Val lie Ser Thr Glu Gly Glii His Glu Cys Leu lie Gly Asn Thr Thr 275 280 285 290 295 201116627

Val Lys Val His Ala Leu Asp Gly Arg Leu Ala Pro Met Pro Cys Arg Pro Lys Glu lie Val 300 305 310 315

Ser Ser Ala Gly Pro Val Arg Lys Thr Ser Cys Thr Phe Asn Tyr Thr Lys Thr Leu Arg Asn 320 325 330 335

Lys Tyr Tyr Glu Pro Arg Asp Ser Tyr Phe Gin Gin Tyr Met Leu Lys Gly Glu Tyr Gin Tyr 340 345 350 355

Trp Phe Asp Leu Asp Val Thr Asp His His Thr Asp Tyr Phe Ala Glu Phe Val Val Leu Val 360 365 370 375

Val Val Ala Leu Leu Gly Gly Arg Tyr Val Leu Trp Leu lie Val Thr Tyr lie Val Leu Thr 380 385 390 395 400

Glu Gin Leu Ala Ala Gly

Claims (1)

201116627 VII. Patent application scope: 1. A diagnostic reagent for detecting whether a pig is infected with classical swine fever virus, characterized in that it comprises a recombinant pig derived from classical swine fever virus (CSFV) type II TD isolated strain. A prion E2 glycoprotein, and the classical swine fever E2 glycoprotein has the amino acid sequence as set forth in SEQ ID NO. 2. The diagnostic reagent according to item 2 of the patent application, wherein the heavy group of classical swine fever virus E2 glycoprotein is produced in an insect cell expression system. 3. The diagnostic reagent according to item 2 of the patent application further comprises • the swine fever virus Erns (E0) glycoprotein. 4. A swine fever subunit identification vaccine characterized by comprising a recombinant swine fever virus E2 glycoprotein derived from classical swine fever virus (CSFV) type II TD isolate and a veterinary acceptable adjuvant or form The porcine scorpion virus E2 glycoprotein has the amino acid sequence as set forth in SEQ ID NO: 5. The porcine sputum subunit identification vaccine according to item 4 of the patent application scope, wherein the recombinant porcine virus E2 glycoprotein It is produced in an insect cell expression system and forms a homodimer protein structure under non-reducing conditions and exhibits a swine fever immunity similar to that of a wild-type swine fever virus strain.